Pioneering role of RNA in the early evolution of life (2024)

The catalytic, regulatory and structural properties of RNA, combined with their extraordinary ubiquity in cellular processes, are consistent with the proposal that this molecule played a much more conspicuous role in heredity and metabolism during the early stages of biological evolution. This review explores the pivotal role of RNA in the earliest life forms and its relevance in modern biological systems. It examines current models that study the early evolution of life, providing insights into the primordial RNA world and its legacy in contemporary biology.

Keywords:
RNA world; ribozyme; origin of life; RNA viruses; viroids

Mentioning heredity today almost automatically brings DNA to mind. Historically, the determination that DNA was the molecule responsible for heredity can be traced to Griffith’s experiments in 1928Griffith F (1928) The significance of Pneumococcal types. J Hyg 27:113-159. (Griffith, 1928), the Avery-MacLeod-McCarty experiment in 1944Avery OT, MacLeod CM and McCarty M (1944) Studies on the chemical nature of the substance inducing transformation of Pneumococcal types. J Exp Med 79:137-158. (Avery et al., 1944), and the Hershey-Chase experiment in 1952Hershey AD and Chase M (1952) Independent functions of viral protein and nucleic acid in growth of bacteriophage. J Gen Physiol 36:39-56. (Hershey and Chase, 1952). A year later, the discovery of the double helix structure by Watson and Crick established the framework for the central dogma of molecular biology, defining the flow of genetic information from DNA to RNA to protein, placing DNA at the highest and most prestigious position in biochemistry (Watson and Crick, 1953aWatson JD and Crick FHC (1953a) Molecular structure of nucleic acids: A structure for Deoxyribose Nucleic Acid. Nature 171:737-738., bWatson JD and Crick FHC (1953b) Genetical implications of the structure of deoxyribose nucleic acid. Nature 171:964-967.; Crick, 1958Crick FH (1958) On protein synthesis. In: Sanders FK (ed.). Symposia of the Society for Experimental Biology, Number XII: The Biological Replication of Macromolecules. Cambridge University Press. pp 138-163.; 1970Crick F (1970) Central dogma of molecular biology. Nature 227:561-3.; Cobb, 2017Cobb M (2017) 60 years ago, Francis Crick changed the logic of biology. PLoS Biol. 15:e2003243.). However, this narrative underwent a significant paradigm shift with a series of findings that included the discovery of messenger RNA (mRNA) in the 1960s (Brenner et al., 1961Brenner S, Jacob F and Meselson M (1961) An unstable intermediate carrying information from genes to ribosomes for protein synthesis. Nature 190:576-581.; Gros et al., 1961Gros F, Hiatt H, Gilbert W, Kurland CG, Risebrough RW and Watson JD (1961) Unstable ribonucleic acid revealed by pulse labelling of Escherichia Coli. Nature 190:581-585.), as well as its proposal as a genetic regulator (Jacob and Monod, 1961Jacob F and Monod J (1961) Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol 3:318-356.), the discovery of catalytic RNA molecules (ribozymes) in the 1980s (Kruger et al., 1982Kruger K, Grabowski PJ, Zaug AJ, Sands J, Gottschling DE and Cech TR (1982) Self-splicing RNA: Autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell 31:147-157.; Guerrier-Takada et al., 1983Guerrier-Takada C, Gardiner K, Marsh T, Pace N and Altman S (1983) The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 35:849-857.), the development of RNA in vitro evolution techniques in the 1990s (Ellington and Szostak, 1990Ellington AD and Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346:818-822.; Tuerk and Gold, 1990Tuerk C and Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505-510.), and the description of a long list of regulatory non-coding RNAs (ncRNA) over the last decades (Fu, 2014Fu X-D (2014) Non-coding RNA: A new frontier in regulatory biology. Natl Sci Rev 1:190-204.; Delihas, 2015Delihas N (2015) Discovery and characterization of the first non-coding RNA that regulates gene expression, micF RNA: A historical perspective. World J Biol Chem 6:272-280.). These milestones have elevated RNA to a much more important level, recognizing its crucial roles in cellular processes, genetic regulation, and evolutionary biology.

Both DNA and RNA are nucleotide polymers, where each nucleotide consists of three fundamental components: a nitrogenous base (also known as a nucleobase), a pentose sugar, and a phosphate group. The nucleobases are derived from heterocyclic compounds, specifically purines (adenine and guanine) and pyrimidines (cytosine and thymine in DNA, or uracil in RNA). Nucleic acids have two types of pentose sugars (in beta-furanose form), which are covalently joined to a nucleobase by an N-β-glycosyl bond (at N-1 in pyrimidines and N-9 in purines). RNA contains D-ribose, whereas DNA features 2’-deoxy-D-ribose (Yoffe et al., 2008Yoffe AM, Prinsen P, Gopal A, Knobler CM, Gelbart WM and Ben-Shaul A (2008) Predicting the sizes of large RNA molecules. Proc Natl Acad Sci U S A 105:16153-16158.). Nucleotides are covalently linked together through phosphate-group bridges via a phosphodiester linkage, in which the 5’-phosphate group of a nucleotide is joined to the 3’-hydroxyl group of the next. This structure results in a phosphate backbone, whose ionization confers a negative charge to both DNA and RNA.

There are two main differences that distinguish DNA and RNA. The first one lies in their chemical components, while the second involves the three-dimensional structure they adopt in space. In the case of the chemical components, DNA contains thymine, whereas RNA contains uracil (Leslie and Grover, 2022Leslie E and Grover N (2022) RNA: Composition and base pairing. In: Grover, N (eds) Fundamentals of RNA structure and function learning materials in biosciences. Springer, Cham, 1-19.). Although occasionally, DNA may have uracil (Sire et al., 2008Sire J, Quérat G, Esnault C and Priet S (2008) Uracil within DNA: An actor of antiviral immunity. Retrovirology 5:45. ), and RNA may have thymine in the form of ribothymidine (Clark et al., 2019Clark DP (2019) Chapter 13 - Protein synthesis. In: Clark DP, Pazdernik NJ and McGehee MR (eds) Molecular Biology, 3rd edition, Elsevier, pp 397-444.). Compared to thymine, uracil has no methyl group in the C5’ carbon, making it less stable and susceptible to mutations (Vértessy and Tóth, 2009Vértessy BG and Tóth J (2009) Keeping uracil out of DNA: physiological role, structure and catalytic mechanism of dUTPases. Acc Chem Res, 42:97-106.; Roberts and LaBonte, 2023Roberts J and LaBonte M (2023) The importance of the fifth nucleotide in DNA: Uracil. In: Sett A (ed) Oligonucleotides - Overview and Applications. IntechOpen.). However, the main difference in components is the pentose sugar, which defines the identity of each nucleic acid. As mentioned above, RNA contains a D-ribose, with a hydroxyl (OH-) group in the C2’ position. This chemical group is tremendously reactive compared to the hydrogen in 2’-deoxy-D-ribose. In fact, the 2’ hydroxyl group endows RNA with the ability to catalyze phosphoryl transfer reactions, particularly transesterification or hydrolysis of phosphate esters (Figure 1 A ). These reactions involve the nucleophilic attack of the 2′-oxygen towards the neighboring phosphate atom, this phosphoester transfer generates a pentacoordinated phosphorus species that is not stable, resulting in the formation of a 2′,3′-cyclic phosphate compound and the liberation of a second product featuring a 5′-hydroxyl group (Emilsson et al., 2003Emilsson GM, Nakamura S, Roth A and Breaker RR (2003) Ribozyme speed limits. RNA 9:907-918.). While transesterification is important for RNA catalytic activities involving self-cleavage and splicing (Lilley, 2003Lilley DMJ (2003) The origins of RNA catalysis in ribozymes. Trends Biochem Sci 28:495-501.; 2011Lilley DM (2011) Mechanisms of RNA catalysis. Phil Trans R Soc B 366:2910-2917.), it can also affect the stability of RNA, especially at high pH, where spontaneous degradation occurs (Adams et al., 1992Adams RLP, Knowler JT and Leader DP (1992) The structure of the nucleic acids. In: The Biochemistry of the Nucleic Acids. Springer, Dordrecht. pp 675.; Emilsson et al., 2003Emilsson GM, Nakamura S, Roth A and Breaker RR (2003) Ribozyme speed limits. RNA 9:907-918.).

Regarding their three-dimensional structure, DNA consists of two regular and stable helical chains wound around the same axis, forming a double helix that is held together by hydrogen bonds. Hydrogen bonding occurs in the same RNA chain, generating sequence-dependent secondary structure elements that are important in RNA function and stability. Some of these elements include stems, helices, bulges, and hairpin loops (Nowakowski and Tinoco, 1997Nowakowski J and Tinoco I (1997) RNA structure and stability. Sem Virol 8:153-165.; Anderson-Lee et al., 2016Anderson-Lee J, Fisker E, Kosaraju V, Wu M, Kong J, Lee J, Lee M, Zada M, Treuille A and Das R (2016) Principles for predicting RNA secondary structure design difficulty. J Mol Biol 428:748-757.). Using these motifs, as well as its interactions with ions, RNA can adopt stable and complex three-dimensional structures, such as helical duplexes (Matsumoto and Sugimoto, 2021Matsumoto S and Sugimoto N (2021) New insights into the functions of nucleic acids controlled by cellular microenvironments. Top Curr Chem (Cham) 379:17.), major and minor groove triplexes (Devi et al., 2015Devi G, Zhou Y, Zhong Z, Toh DF and Chen G (2015) RNA triplexes: from structural principles to biological and biotech applications. Wiley Interdiscip Rev RNA 6:111-128.), A-minor motifs (Baulin, 2021Baulin EF (2021) Features and functions of the A-Minor motif, the most common motif in RNA structure. Biochemistry (Mosc) 86:952-961.), triple-stranded structures (Brown, 2020Brown JA (2020) Unraveling the structure and biological functions of RNA triple helices. Wiley Interdiscip Rev RNA 11:e1598.), quadruplexes (Xu and Komiyama, 2023Xu Y and Komiyama M (2023) G-Quadruplexes in human telomere: structures, properties, and applications. Molecules 29:174.; Zareie et al., 2024Zareie AR, Dabral P and Verma SC (2024) G-Quadruplexes in the regulation of viral gene expressions and their impacts on controlling infection. Pathogens 213:60.), among many others (Butcher and Pyle, 2011Butcher SE and Pyle AM (2011) The molecular interactions that stabilize RNA tertiary structure: RNA motifs, patterns, and networks. Acc Chem Res 44:1302-11.). All the functions that RNA performs are closely linked to its ability to form these three-dimensional structures, including intricate quaternary complexes like the ribosome (an RNA heterotrimer) (Butcher and Pyle, 2011; Jones and Ferré-D’Amaré, 2015Jones CP and Ferré-D’Amaré AR (2015) RNA quaternary structure and global symmetry. Trends Biochem Sci 40:211-20.; Ganser et al., 2019Ganser LR, Kelly ML, Herschlag D and Al-Hashimi HM (2019) The roles of structural dynamics in the cellular functions of RNAs. Nat Rev Mol Cell Biol 20:474-489.).

Another important aspect to consider when discussing RNA structure and functionality is that several chemical modifications are often mistakenly identified as RNA adducts-and therefore as chemical damage-rather than as necessary modifications for the correct folding, regulation, and function of RNA. For example, 1, N ⁶ -dimethyladenosine (m¹,⁶A), is a modified nucleobase present in some mammalian transfer RNAs (tRNAs) that regulates processes related to cancer development (You et al., 2022You XJ, Zhang S, Chen JJ, Tang F, He J, Wang J, Chu-Bo Q, Yu-Qi F and Bi-Feng Y (2022) Formation and removal of 1, N 6-dimethyladenosine in mammalian transfer RNA. Nucleic Acids Res 50:9858-9872.). Another interesting RNA modification is N ¹ -methyladenosine (m¹A), which is present in mRNAs and regulates their structure and translation efficiency under stress conditions (Qi et al., 2023Qi Z, Zhang C, Jian H, Hou M, Lou Y, Kang Y, Wang W, Lv Y, Shang S, Wang C, et al (2023) N1-Methyladenosine modification of mRNA regulates neuronal gene expression and oxygen glucose deprivation/reoxygenation induction. Cell Death Discov 9:59.). These are just a few examples of the fascinating diversity of RNA beyond the canonical nucleobases, which inevitably lead to questions about the potential role of modified RNA molecules in early evolution.

Although some researchers began to recognize the importance and antiquity of RNA molecules during the first half of the 20th century, after the emergence of molecular biology, most of the research efforts in biochemistry focused on understanding gene and enzyme functions, placing DNA and proteins as the most important molecules in the cell. It seemed that RNA was simply a mere intermediary between these two macromolecules. Nevertheless, as mentioned previously, the paradigm changed when the diverse roles of RNA molecules in crucial cellular processes such as DNA replication, gene expression, and regulation started to be discovered (Darnell, 2011Darnell J (2011) RNA: Life ‘s indispensable molecule. 1st edition. Cold Spring Harbor Laboratory Press, New York, 416 pp.).

The discovery that the infectivity of the tobacco mosaic virus resides in its RNA opened doors to speculation about its role in the early evolution of life. Notable researchers such as John B. S. Haldane, Jean L. A. Brachet, Andrey N. Belozerzky, and John D. Bernal concluded that RNA likely preceded DNA as the genetic molecule during the early stages of cellular evolution (Lazcano, 2016Lazcano A (2016) The RNA world: Piecing together the historical development of a hypothesis. Mètode Revista de Difusió de La Investigació, 6:167-173.). Nevertheless, as discussed in later sections, the ability of RNA to store genetic information in some viruses does not necessarily imply that viruses are ancestral entities. Instead, it enriches its biological importance and emphasizes its versatility and adaptability.

The same year that Watson and Crick elucidated the double helix structure of DNA, Stanley Miller reported the first synthesis of organic molecules under conditions resembling the atmosphere of the early Earth (Miller, 1953Miller SL (1953) Production of amino acids under possible primitive earth conditions. Science 117:528-9.). This synthesis was accomplished by applying an electric discharge to a combination of hydrogen, methane, ammonia, and water. The conditions were based on Harold Urey’s calculations (1952Urey HC (1952) The planets: Their origin and development. Yale University Press. pp. 245.) and simulated a reducing atmosphere, an idea originally proposed by Oparin (Oparin, 1938Oparin AI (1938) The origin of life, Sergius M, translation with annotations. Macmillan, New York. pp 270.). In his experiment, Miller reported amino acids such as glycine, α-alanine, β-alanine, α-amino-n-butyric acid, plus hydroxy acids, and urea (Miller, 1953Miller SL (1953) Production of amino acids under possible primitive earth conditions. Science 117:528-9.). However, none of the nucleic acid components were obtained in his experiment. It was not until the early 1960s that Joan Oró synthesized adenine from the polymerization of hydrogen cyanide (Oró, 1960Oró J (1960) Synthesis of adenine from ammonium cyanide. Biochem Biophys Res Commun 2:407-412.; Oró and Kimball, 1961Oró J and Kimball AP (1961) Synthesis of purines under possible primitive earth conditions. I. Adenine from hydrogen cyanide. Arch Biochem Biophys 94:217-227.). This finding was crucial in demonstrating that RNA components could be synthesized under possible early Earth conditions. Since then, significant efforts in prebiotic chemistry have focused on synthesizing the building blocks of RNA. Notable achievements include the synthesis of activated pyrimidine nucleotides from glycolaldehyde, cyanamide, and phosphate (Powner et al., 2009Powner MW, Gerland B and Sutherland JD (2009) Synthesis of activated pyrimidine ribonucleotides in prebiotically plausible conditions. Nature 459:239-242.), and the synthesis of purine nucleotides from formamidopyrimidines (Becker et al., 2016Becker S, Thoma I, Deutsch A, Gehrke T, Mayer P, Zipse H and Carell T (2016) A high-yielding, strictly regioselective prebiotic purine nucleoside formation pathway. Science 352:833-836.). The simultaneous prebiotic synthesis of pyrimidine and purine nucleosides (mono- and diphosphates) from cyanoacetylene under wet-dry cycles has been reported recently (Becker et al., 2019Becker S, Feldmann J, Wiedemann S, Okamura H, Schneider C, Iwan K, Crisp A, Rossa M, Amatov T and Carell T (2019) Unified prebiotically plausible synthesis of pyrimidine and purine RNA ribonucleotides. Science 366:76-82.). Remarkably, hydrogen cyanide and formamide, produced through electric discharges and laser-driven plasma impacts in a reducing atmosphere of NH₃, CO, and H₂O, can generate RNA nucleobases when exposed to UV light at high temperatures (Ferus et al., 2017Ferus M, Pietrucci F, Saitta AM, Knížek A, Kubelík P, Ivanek O, Shestivska V and Civiš S (2017) Formation of nucleobases in a Miller-Urey reducing atmosphere. Proc Natl Acad Sci U S A 114:4306-4311.). Similarly, experiments using a reducing atmosphere and borosilicate yielded a complete set of biological nucleobases, including RNA nucleobases (Criado-Reyes et al., 2021Criado-Reyes J, Bizzarri BM, García-Ruiz JM, Saladino R and Di Mauro E (2021) The role of borosilicate glass in Miller-Urey experiment. Sci Rep 11:21009.). Furthermore, the discovery of both pyrimidine and purine nucleobases in carbonaceous chondrites (Botta and Bada, 2002Botta O and Bada JL (2002) Extraterrestrial organic compounds in meteorites. Surv Geophys 23:411-467.; Rios and Tor, 2013Rios AC and Tor Y (2013) On the origin of the canonical nucleobases: An assessment of selection pressures across chemical and early biological evolution. Isr J Chem 53:469-483.; Oba et al., 2022Oba Y, Takano Y, Furukawa Y, Koga T, Glavin DP, Dworkin JP and Naraoka H (2022) Identifying the wide diversity of extraterrestrial purine and pyrimidine nucleobases in carbonaceous meteorites. Nat Commun 13:2008.; Krishnamurthy et al., 2022Krishnamurthy R, Goldman AD, Liberles DA, Rogers KL and Tor Y (2022) Nucleobases in meteorites to nucleobases in RNA and DNA? J Mol Evol 90:328-331.) implies that these molecules were available in the solar system and were likely delivered to early Earth during meteorite collisions.

This body of research is essential for understanding the prebiotic era, marked by the synthesis and accumulation of organic compounds, including RNA nucleobases. During this period, chemical evolution facilitated the transformation of simple molecules into complex organic compounds through prebiotic reactions (Oró, 1983Oró J (1983) Chemical evolution and the origin of life. Adv Space Res 3:77-94.; Jia et al., 2021Jia TZ, Caudan M and Mamajanov I (2021) Origin of Species before Origin of Life: The role of speciation in chemical evolution. Life (Basel) 11:154.). This process provided the necessary precursors for the emergence of RNA as an early genetic polymer, and for crucial molecules, such as the proposed RNA-dependent RNA polymerase ribozyme, capable of catalyzing its own replication and the replication of other ribozymes (Figure 1 B ) (Joyce, 2002Joyce G (2002) The antiquity of RNA-based evolution. Nature 418:214-221.; Joyce and Szostak, 2018Joyce GF and Szostak JW (2018) Protocells and RNA self-replication. Cold Spring Harb Perspect Biol 10:a034801.; Papastavrou et al., 2024Papastavrou N, Horning DP, Joyce GF (2024) RNA-catalyzed evolution of catalytic RNA. Proc Natl Acad Sci U S A 121:e2321592121.).

Although there are still many unsolved issues related to the synthesis and stability of RNA components and RNA molecules (as discussed in later sections), the evidence discussed in this review offers supports to the proposal of a pre-DNA era where RNA played a central role in the earliest cells, a concept known as the RNA world hypothesis.

Many proposals on the origin of life are based on the hypothesis that, following a period of prebiotic synthesis and accumulation of organic compounds, RNA molecules played a conspicuous role in both the replication of genetic material and catalysis, an epoch commonly known as the RNA world (Gilbert, 1986Gilbert W (1986) Origin of life: The RNA world. Nature 319:618-618.; Joyce, 2002Joyce G (2002) The antiquity of RNA-based evolution. Nature 418:214-221.). In broad terms, the concept of the RNA world refers to a set of models that seek to explain a hypothetical period in the early evolution of life on Earth, where ribonucleic acid molecules played a central role. The presence of biological entities endowed with RNA as genetic material, the astonishing in vitro expansion of the catalytic repertoire of RNA molecules, and the manifold roles that RNA molecules and ribonucleotides play in extant cells, provide a strong support to the RNA world hypothesis (Chen and Li, 2007Chen X and Li N (2007) Ribozyme catalysis of metabolism in the RNA world. Chem Biodivers 4:633-655.; Lazcano, 2012Lazcano A (2012) The biochemical roots of the RNA world: From zymonucleic acid to ribozymes. Hist. Philos. Life Sci 34:407-423., 2014Lazcano, A (2014). “The RNA world: stepping out of the shadows”, In: Trueba G (ed) Why does evolution matter? The importance of understanding evolution, Cambridge Scholars Publishing, 101-119.; Vázquez-Salazar and Lazcano, 2018Vázquez-Salazar A and Lazcano A (2018) Early Life: Embracing the RNA world. Curr Biol 28:R220-R222.; Hernández-Morales et al., 2019Hernández-Morales R, Becerra A and Lazcano A (2019) Alarmones as vestiges of a bygone RNA world. J Mol Evol 87:37-51.). However, as discussed by Robertson and Joyce (2012Robertson MP and Joyce GF (2012) The origins of the RNA world. Cold Spring Harb Perspect Biol , 4:a003608.), the RNA implies different premises for different researchers, with each holding their own interpretation of what it represents, thus creating not one but several RNA worlds. It is worth noting that Thomas Cech has already introduced and discussed the concept of ‘RNA worlds’ (Cech, 2012Cech TR (2012) The RNA worlds in context. Cold Spring Harb Perspect Biol 4:a006742.). Cech uses this plural form to refer essentially to three distinct RNA worlds: the primordial RNA world, the contemporary RNA world, and the world of RNA technology and medical applications. The latter two refer to the contemporary era which, given the recent discoveries in catalytic RNA, mRNA, and regulatory non-coding RNA, is an RNA-dominated world.

In this review, the RNA world is considered a stage in the early evolution of life where genetic information continuity was ensured by RNA self-replication. These primitive biological systems (ribocells) depended on RNA and its interactions with metal ions, minerals, and a broad catalog of organic molecules to carry out the chemical reactions necessary for maintaining their metabolism. This catalog may have included compounds capable of forming membranes, ribonucleotides, amino acids synthesized on primitive Earth or delivered by meteorites, and even small peptides, all of which could have contributed to shaping the chemical environment in which life evolved (Jadhav and Yarus, 2002Jadhav VR and Yarus M (2002) Coenzymes as coribozymes. Biochimie 84:877-888.; Hsiao et al., 2013Hsiao C, Chou IC, Okafor CD, Bowman JC, O’Neill EB, Athavale SS, Petrov AS, Hud NV, Wartell RM, Harvey SC and Williams LD (2013) RNA with iron(II) as a cofactor catalyses electron transfer. Nat Chem 5:525-528.; Wieczorek et al., 2013Wieczorek R, Dörr M, Chotera A, Luisi PL and Monnard P (2013) Formation of RNA phosphodiester bond by bistidine‐containing bipeptides. ChemBioChem 14:217-223.; Vázquez-Salazar and Lazcano, 2018Vázquez-Salazar A and Lazcano A (2018) Early Life: Embracing the RNA world. Curr Biol 28:R220-R222.; Müller et al., 2022Müller F, Escobar L, Xu F, Węgrzyn E, Nainytė M, Amatov T, Chan CY, Pichler A and Carell T (2022) A prebiotically plausible scenario of an RNA-peptide world. Nature 605:279-284. ).

The independent works of Carl Woese (1967Woese CR (1967) The genetic code: The molecular basis for genetic expression. Harper & Row, New York, pp 200.), Francis Crick (1968Crick FH (1968) The origin of the genetic code. J Mol Biol 38:367-379.), and Leslie Orgel (1968Orgel LE (1968) Evolution of the genetic apparatus. J Mol Biol 38:381-393.) in the late 1960s are often acknowledged as the seminal basis for formulating the RNA world hypothesis. The conclusions reached by these three authors are similar, suggesting that the earliest organisms on Earth used RNA as a genetic polymer and, possibly, as a catalyst before ribosomal protein synthesis. Yet, even before these bold proposals, biochemists such as Alexander Rich (1962Rich A (1962) On the problems of evolution and biochemical information transfer. In: Kasha M and Pullman B (eds) Horizons in Biochemistry Academic Press; New York, NY, USA: 1962 pp 103-126.), Philip Handler (1963Handler P (1963) Evolution of the coenzymes. In: Oparin A (ed) Proceedings of the Fifth International Congress of Biochemistry, Vol. III. Biochemistry. Pergamon Press/Macmillan Company, 149-157.), and Robert Eakin (1963Eakin RE (1963) An approach to the evolution of metabolism. Proc Natl Acad Sci U S A 49:360-366.) recognized the importance of ribonucleic acid and its ribonucleotide derivatives in primitive stages of metabolism, proposing that modern biochemistry is the result of evolution from an ancestral state where RNA and modified ribonucleotides were the protagonists.

The discovery of ribozymes and the consolidation of an evolutionary hypothesis

In the early 1980s, Thomas R. Cech, from the University of Colorado Boulder, was investigating the in vitro splicing mechanism of one of the precursors of ribosomal RNA (rRNA) in the protist Tetrahymena (Cech et al., 1981Cech TR, Zaug AJ and Grabowski PJ (1981) In vitro splicing of the ribosomal RNA precursor of Tetrahymena: involvement of a guanosine nucleotide in the excision of the intervening sequence. Cell 27:487-496.). The initial hypothesis of his group was that a protein was catalyzing the editing of rRNA. However, they discovered that the folded structure of the RNA in the intron, as well as the involvement of a guanosine nucleotide and Mg²⁺ were sufficient to carry out the excision activity (Kruger et al., 1982Kruger K, Grabowski PJ, Zaug AJ, Sands J, Gottschling DE and Cech TR (1982) Self-splicing RNA: Autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell 31:147-157.). Cech and his colleagues categorized these self-splicing RNA molecules as group I introns, and coined the term ribozyme to define RNAs that are catalytically active.

Meanwhile, at Yale University, the group led by Sydney Altman was investigating the maturation mechanism of transfer RNA in bacterial organisms (Guerrier-Takada et al., 1983Guerrier-Takada C, Gardiner K, Marsh T, Pace N and Altman S (1983) The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 35:849-857.). In this maturation process, the tRNA molecule undergoes cleavage at its 5’ end site, which was known to be catalyzed by the RNAse P, a ribonucleoprotein (RNP) complex whose protein portion was postulated to be catalytic. However, Altman and his colleagues demonstrated that the nucleic acid portion of the RNAse P was responsible for catalysis.

These groundbreaking discoveries provided crucial evidence for understanding how RNA could have performed essential biochemical functions independently of proteins in early stages of life.

The resurgence of the RNA world

In the foreword to the first edition of the book The RNA World (Gesteland and Atkins, 1993Gesteland R and Atkins J (Eds) (1993) The RNA world: The nature of modern RNA suggests a prebiotic RNA world. 1st. edition. Cold Spring Harbor Laboratory Press. New York, 630 pp. ), Francis Crick wrote: “This hypothesis of an RNA world without protein was largely forgotten but has now become fashionable again because of the remarkable discoveries by Altman and by Cech.” Why was the proposal forgotten? Orgel and Crick (1993Orgel LE and Crick FH (1993) Anticipating an RNA world. Some past speculations on the origin of life: where are they today? FASEB J 7:238-239.) answered this question by arguing that neither of them thought that RNA would continue to play a decisive role in the modern cell. Nevertheless, the finding of catalytic RNA molecules provided, almost inadvertently, the experimental evidence needed to bolster the theoretical proposals of Woese, Rich, Crick, and Orgel, and all the (r)evolutionary biochemists before them.

In addition to the ribozymes described by Cech and Altman, several naturally occurring ribozymes have been discovered. In 1986, a new type of self-splicing introns, termed group II introns, was independently reported by two groups (Peebles et al., 1986Peebles CL, Perlman PS, Mecklenburg KL, Petrillo ML, Tabor JH, Jarrell KA and Cheng HL (1986) A self-splicing RNA excises an intron lariat. Cell 44:213-223.; van der Veen et al., 1986van der Veen R, Arnberg AC, van der Horst G, Bonen L, Tabak HF and Grivell LA (1986) Excised group II introns in yeast mitochondria are lariats and can be formed by self-splicing in vitro. Cell 44:225-234.). These introns are less prevalent than group I and are commonly found in organellar and bacterial genomes. In the same year, other catalytic RNAs were discovered in plant pathogens with small circular RNA (circRNA) genomes. These ribozymes, named hammerhead (Hutchins et al., 1986Hutchins CJ, Rathjen PD, Forster AC and Symons RH (1986) Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Res 14:3627-3640.; Prody et al., 1986Prody GA, Bakos JT, Buzayan JM, Schneider IR and Bruening G (1986) Autolytic processing of dimeric plant virus satellite RNA. Science 231:1577-1580.) and hairpin (Buzayan et al., 1986Buzayan J, Gerlach W and Bruening G (1986) Non-enzymatic cleavage and ligation of RNAs complementary to a plant virus satellite RNA. Nature 323:349-353.), catalyze self-cleavage via transesterification reactions (de la Peña et al., 2017de la Peña M, García-Robles I and Cervera A (2017) The hammerhead ribozyme: A long history for a short RNA. Molecules (Basel) 22:78.).

Other examples of naturally occurring ribozymes include the hepatitis delta virus (HDV) ribozyme, found in 1988, which catalyzes site-specific self-cleavage with a double-nested pseudoknot fold (Kuo et al., 1988Kuo MY, Sharmeen L, Dinter-Gottlieb G and Taylor J (1988) Characterization of self-cleaving RNA sequences on the genome and antigenome of human hepatitis delta virus. J Virol 62:4439-4444.). HDV-like ribozymes, identified in 2006, are structurally related to HDV ribozymes and include sequences from mammalian CPEB3, retrotransposons, and bacteria (Salehi-Ashtiani et al., 2006Salehi-Ashtiani K, Lupták A, Litovchick A and Szostak JW (2006) A genomewide search for ribozymes reveals an HDV-like sequence in the human CPEB3 gene. Science 313:1788-1792.). Other notable small ribozymes include the Varkud satellite (VS) ribozyme, the largest known self-cleaving ribozyme, discovered in 1990 (Saville and Collins, 1990Saville BJ and Collins RA (1990) A site-specific self-cleavage reaction performed by a novel RNA in Neurospora mitochondria. Cell 61:685-696.), and the glmS riboswitch, a self-cleaving ribozyme activated by glucosamine-6-phosphate, so far the only example of a natural ribozyme that uses a small organic cofactor for catalysis (Winkler et al., 2004Winkler WC, Nahvi A, Roth A, Collins JA and Breaker RR (2004) Control of gene expression by a natural metabolite-responsive ribozyme. Nature 428:281-286.; Ferré-D’Amaré, 2010Ferré-D’Amaré AR (2010) The glmS ribozyme: Use of a small molecule coenzyme by a gene-regulatory RNA. Q Rev Biophys 43:423-447.). Recent discoveries include the twister ribozyme (Roth et al., 2014Roth A, Weinberg Z, Chen AG, Kim PB, Ames TD and Breaker RR (2014) A widespread self-cleaving ribozyme class is revealed by bioinformatics. Nat Chem Biol 10:56-60. ), the twister-sister, hatchet, and pistol ribozymes (Weinberg et al., 2015Weinberg Z, Kim PB, Chen TH, Li S, Harris KA, Lünse CE and Breaker RR (2015) New classes of self-cleaving ribozymes revealed by comparative genomics analysis. Nat Chem Biol 11:606-610.), and the hovlinc ribozyme found in human very long intergenic noncoding (vlinc) RNAs (Chen et al., 2021Chen Y, Qi F, Gao F, Cao H, Xu D, Salehi-Ashtiani K and Kapranov P (2021) Hovlinc is a recently evolved class of ribozyme found in human lncRNA. Nat Chem Biol 17:601-607.), all of which are nucleolytic ribozymes.

Besides naturally occurring ribozymes (products of biological evolution), various laboratories worldwide have successfully selected an extensive catalog of artificial ribozymes. These laboratory ribozymes (results of in vitro evolution experiments) display a variety of catalytic activities not found in the repertoire of biological RNAs (Wilson and Szostak, 1999Wilson DS and Szostak JW (1999) In vitro selection of functional nucleic acids. Annu Rev Biochem 68:611-647.; Martin et al., 2015Martin LL, Unrau PJ and Müller UF (2015) RNA synthesis by in vitro selected ribozymes for recreating an RNA world. Life (Basel) 5:247-268.).

One recent example of in vitro selected ribozymes is the lineage of RNA-dependent RNA polymerase ribozymes, which descended from the class I ligase (Bartel and Szostak, 1993Bartel DP and Szostak JW (1993) Isolation of new ribozymes from a large pool of random sequences. Science 261:1411-1418.; Ekland et al., 1995Ekland EH, Szostak JW and Bartel DP (1995) Structurally complex and highly active RNA ligases derived from random RNA sequences. Science 269:364-370.) and are capable of amplifying RNA (Horning and Joyce, 2016Horning DP and Joyce GF (2016) Amplification of RNA by an RNA polymerase ribozyme. Proc Natl Acad Sci U S A 113:9786-9791.). Continuous work on the in vitro evolution of the polymerase ribozyme made it capable of copying and amplifying multiple different RNA templates, albeit with modest fidelity (Tjhung et al., 2020Tjhung KF, Shokhirev MN, Horning DP and Joyce GF (2020) An RNA polymerase ribozyme that synthesizes its own ancestor. Proc Natl Acad Sci U S A 117:2906-2913.). Building on this, Papastavrou et al. (2024Papastavrou N, Horning DP, Joyce GF (2024) RNA-catalyzed evolution of catalytic RNA. Proc Natl Acad Sci U S A 121:e2321592121.) further evolved the ribozyme, demonstrating that it not only replicates RNA sequences for other ribozymes with high fidelity but also evolves over time, producing new variants with increased evolutionary fitness. This process mimics natural selection at the molecular level, providing evidence that early RNA molecules could undergo Darwinian evolution.

Collectively, both natural and artificial ribozymes can be classified under the six classes of catalytic activities present in protein enzymes: hydrolases, oxidoreductases, lyases, transferases, ligases, and isomerases (Hernández-Morales et al., 2019Hernández-Morales R, Becerra A and Lazcano A (2019) Alarmones as vestiges of a bygone RNA world. J Mol Evol 87:37-51.; Müller et al., 2016Müller S, Appel B, Balke D, Hieronymus R and Nübel C (2016). Thirty-five years of research into ribozymes and nucleic acid catalysis: where do we stand today?. F1000Research 5:F1000 Faculty Rev-1511.; Wilson and Lilley, 2021Wilson TJ and Lilley DMJ (2021) The potential versatility of RNA catalysis. WIREs RNA, 12:e1651.; Deng et al., 2023Deng J, Shi Y, Peng X, He Y, Chen X, Li M, Lin X, Liao W, Huang Y, Jiang T, Lilley DMJ, Miao Z and Huang L (2023) Ribocentre: a database of ribozymes. Nucleic Acids Res 51:D262-D268.; Fine and Pearlman, 2023Fine JL and Pearlman RE (2023) On the origin of life: An RNA-focused synthesis and narrative. RNA 29:1085-1098.). A diverse catalog of ribozymes enhances the appeal of the RNA world hypothesis, allowing the envision of a scenario where a ribocell, devoid of protein enzymes, could sustain a protometabolism.

RNA plays a wide variety of roles in the modern cell. For instance, it is involved in genome replication, transcription, protein translation, regulation of gene expression, and metabolism control (Cech and Steitz, 2014Cech TR and Steitz JA (2014) The noncoding RNA revolution-trashing old rules to forge new ones. Cell 157:77-94. ). It has been proposed that some of these functions could have originated in the RNA world. Among these, perhaps the most surprising is protein synthesis, performed in all living cells by the ribosome (cf. Noller, 2012Noller HF (2012) Evolution of protein synthesis from an RNA world. Cold Spring Harb Perspect Biol 4:a003681.). This ribonucleoprotein complex represents one of the most ancient molecular machineries of the cell (Petrov et al., 2014Petrov AS, Bernier CR, Hsiao C, Norris AM, Kovacs NA, Waterbury CC, Stepanov VG, Harvey SC, Fox GE, Wartell RM, Hud NV and Williams LD (2014) Evolution of the ribosome at atomic resolution. Proc Natl Acad Sci U S A 111:10251-10256.).

It was precisely the interest in protein synthesis that led to the early recognition of the significant role played by RNA in this process, as experimental measurements showed a correlation between the amount of RNA in cells and their protein synthesis capacity (Caspersson, 1941Caspersson T (1941) Studien über den Eiweißumsatz der Zelle. Naturwissenschaften 29:33-43.; Brachet; 1942Brachet J (1942) La localisation des acides pentosenucle ́iques dans les tissus animaux et les oeufs d’Amphibiens en voie de de ́veloppement. Arch Biol 53:207-257.). However, it was not until the biochemical description of the microsomal fraction of the cytosol (Claude, 1946Claude A (1946) Fractionation of mammalian liver cells by differential centrifugation: II experimental procedures and results. J Exp Med 84:61-89.) that an association between RNA and proteins in the translation process was established (Keller et al., 1954Keller EB, Zamecnik PC and Loftfield RB (1954) The role of microsomes in the incorporation of amino acids into proteins. J Histochem Cytochem 2:378-386.). In 1954, George Palade used electron microscopy to morphologically characterize granules composed of RNA and proteins (Palade, 1955Palade GE (1955) A small particulate component of the cytoplasm. J Biophys Biochem Cy 1:59-68.), which were later named ribosomes (Hartman, 1959Hartman PE (1959) Microsomal particles and protein synthesis papers presented at the first symposium of the Biophysical Society, at the Massachusetts Institute of Technology, Cambridge, February 5, 6 and 8, 1958 Richard B Roberts, Q Rev Biol, 34:87.). Since then, the evolution of the translation apparatus has captured the attention of countless scientists (Woese, 1965Woese CR (1965) On the evolution of the genetic code. Proc Natl Acad Sci U S A 54:1546-1552.; Crick, 1968Crick FH (1968) The origin of the genetic code. J Mol Biol 38:367-379.; Orgel, 1968Orgel LE (1968) Evolution of the genetic apparatus. J Mol Biol 38:381-393.).

Biochemical approaches aimed to understand the peptide bond formation pointed towards a possible involvement of ribosomal RNA in this process (Noller and Chaires, 1972Noller HF and Chaires JB (1972) Functional modification of 16S ribosomal RNA by kethoxal. Proc Natl Acad Sci U S A 69:3115-3118.). However, it was the pioneering work of Ada Yonath (Yonath et al., 1980Yonath AE, Mussig J, Tesche B, Lorenz S, Erdmann VA and Wittmann HG (1980) Crystallization of the large ribosomal subunits from Bacillus stearothermophilus. Biochem Int 1:428-435.) that standardized the preparation and availability of quality ribosome crystals, enabling the molecular-level scrutiny of the RNA-protein interactions that constitute this ribonucleoprotein complex (Ban et al., 1999Ban N, Nissen P, Hansen J, Capel M, Moore PB and Steitz TA (1999) Placement of protein and RNA structures into a 5 A-resolution map of the 50S ribosomal subunit. Nature 400:841-847.; 2000Ban N, Nissen P, Hansen J, Moore PB and Steitz TA (2000) The complete atomic structure of the large ribosomal subunit at 24 A resolution. Science 289:905-920.; Cate et al., 1999Cate JH, Yusupov MM, Yusupova GZ, Earnest TN and Noller HF (1999) X-ray crystal structures of 70S ribosome functional complexes. Science 285:2095-2104.; Clemons et al., 1999Clemons WM, May Jr JL, Wimberly BT, McCutcheon JP, Capel MS and Ramakrishnan V (1999) Structure of a bacterial 30S ribosomal subunit at 55 A resolution. Nature, 400:833-840.; Nissen et al., 2000Nissen P, Hansen J, Ban N, Moore PB and Steitz TA (2000) The structural basis of ribosome activity in peptide bond synthesis. Science 289:920-930.; Schluenzen et al., 2000Schluenzen F, Tocilj A, Zarivach R, Harms J, Gluehmann M, Janell D, Bashan A, Bartels H, Agmon I, Franceschi F and Yonath A (2000) Structure of functionally activated small ribosomal subunit at 33 angstroms resolution. Cell 102:615-623.; Wimberly et al., 2000Wimberly BT, Brodersen DE, Clemons WM Jr, Morgan-Warren RJ, Carter AP, Vonrhein C, Hartsch T and Ramakrishnan V (2000) Structure of the 30S ribosomal subunit. Nature 407:327-339.). The most significant conclusion eventually reached is the recognition that the catalytic center of the ribosome, where the peptide bond forms and which is highly conserved across all domains of life, is entirely constituted by RNA (cf. Moore and Steitz, 2011Moore PB and Steitz TA (2011) The roles of RNA in the synthesis of protein. Cold Spring Harb Perspect Biol 3:a003780.); that is, the ribosome is a ribozyme (Cech, 2000Cech TR (2000) Structural biology: The ribosome is a ribozyme. Science 289:878-879.). Evolutionarily, this evidence suggests that protein synthesis originated in an RNA world as a result of a very early process (Fox, 2010Fox GE (2010) Origin and evolution of the ribosome. Cold Spring Harb Perspect Biol, 2:a003483.).

With the study of the role of RNA in the evolution of life, it was also proposed that certain molecules present in modern metabolism are, in fact, molecular fossils of catalysts dating back to the RNA world. Although this idea had already been contemplated in the 1960s by Handler (1963Handler P (1963) Evolution of the coenzymes. In: Oparin A (ed) Proceedings of the Fifth International Congress of Biochemistry, Vol. III. Biochemistry. Pergamon Press/Macmillan Company, 149-157.), Eakin (1963Eakin RE (1963) An approach to the evolution of metabolism. Proc Natl Acad Sci U S A 49:360-366.), and Orgel (1968Orgel LE (1968) Evolution of the genetic apparatus. J Mol Biol 38:381-393.), it was not until 1976 that Harold White developed a comprehensive hypothesis on the evolution of contemporary metabolism from an ancestral one (White, 1976White III HB (1976) Coenzymes as fossils of an earlier metabolic state. J Mol Evol 7:101-104.).

Similar to Handler and Eakin, White was interested in the chemical structure of coenzymes, which play a crucial role in the catalytic functionality of a large number of enzymes (White, 1976White III HB (1976) Coenzymes as fossils of an earlier metabolic state. J Mol Evol 7:101-104.; 1982White III HB (1982) Evolution of coenzymes and the origin of pyrimidine nucleotides, In: Everse J, Anderson MB and You K (eds) The pyrimidine coenzymes. New York, Academic Press, 1-17.). White observed that several coenzymes possess a ribonucleotide motif in their chemical structures, which could be attributed to an early diversification process in the catalytic capabilities of RNA. Considering the presence of coenzymes in living organisms, the dependency of enzymes on these molecules, and the fact that many coenzymes are ribonucleotides or derivatives thereof, White proposed that these molecules are the molecular fossils of an ancestral metabolic state (White, 1976White III HB (1976) Coenzymes as fossils of an earlier metabolic state. J Mol Evol 7:101-104.; 1982White III HB (1982) Evolution of coenzymes and the origin of pyrimidine nucleotides, In: Everse J, Anderson MB and You K (eds) The pyrimidine coenzymes. New York, Academic Press, 1-17.).

White proposed a scheme where modern metabolism is the result of an evolutionary process where protein enzymes were preceded by enzymes made from nucleic acids (White, 1976White III HB (1976) Coenzymes as fossils of an earlier metabolic state. J Mol Evol 7:101-104.; 1982White III HB (1982) Evolution of coenzymes and the origin of pyrimidine nucleotides, In: Everse J, Anderson MB and You K (eds) The pyrimidine coenzymes. New York, Academic Press, 1-17.). Particularly, the ribonucleotides present in the catalytic sites of ribozymes were the portions conserved through evolution, found in contemporary metabolism as coenzymes of a ribonucleotide nature. This evolutionary reasoning was supported by experimental results demonstrating that some coenzymes, particularly thiamine (Mizuhara and Handler, 1954Mizuhara S and Handler P (1954) Mechanism of thiamine-catalyzed reactions. J Am Chem Soc 76:571-573.), could catalyze reactions by themselves similar to those catalyzed in the enzymatic systems where they participate (White, 1976White III HB (1976) Coenzymes as fossils of an earlier metabolic state. J Mol Evol 7:101-104.).

The RNA world might have also driven the evolution of molecules crucial for chemical signaling and environmental sensing. These molecules, known as alarmones, include ribonucleotides and their derivatives. Alarmones remain essential in modern cells and are synthesized under stress conditions (Stephens et al., 1975Stephens JC, Artz SW and Ames BN (1975) Guanosine 5’-diphosphate 3’-diphosphate (ppGpp): positive effector for histidine operon transcription and general signal for amino-acid deficiency. Proc Natl Acad Sci U S A 72:4389-93. ), triggering a stringent response (Stent and Brenner, 1961Stent GS and Brenner S (1961) A genetic locus for the regulation of ribonucleic acid synthesis. Proc Natl Acad Sci U S A 47:2005-14. ) that affects major cellular processes such as genome replication, gene expression, and metabolism (Nelson and Breaker, 2017Nelson JW and Breaker RR (2017) The lost language of the RNA world. Sci Signal 10:eaam8812.; Hernández-Morales et al., 2019Hernández-Morales R, Becerra A and Lazcano A (2019) Alarmones as vestiges of a bygone RNA world. J Mol Evol 87:37-51.). The widespread distribution of the enzymes that biosynthesize alarmones suggests that these molecules were present in ancestral populations before the divergence of the Archaea, Bacteria, and Eukarya domains (Lazcano et al., 2011Lazcano A, Becerra A and Delaye L (2011) Alarmones. In: Margulis L, Asikainen CA and Krumbie WE (ed) Chimeras and consciousness: Evolution of the sensory self. The MIT Press, Massachusetts Institute of Technology, pp 35-43.; Hernández-Morales et al., 2019Hernández-Morales R, Becerra A and Lazcano A (2019) Alarmones as vestiges of a bygone RNA world. J Mol Evol 87:37-51.). The ribonucleotide structure of alarmones, their extensive biological distribution, functional role in highly conserved cellular processes, presence in meteorites and prebiotic experiments, and the possibility of synthesis by ribozymes all support the proposal that these modified nucleotides are molecular fossils from the RNA world. During this period of evolution, RNA molecules, ribonucleotides, and their derivatives were essential not only for catalysis and genetic information transfer but also for chemical signaling and metabolite sensing (Hernández-Morales et al., 2019Hernández-Morales R, Becerra A and Lazcano A (2019) Alarmones as vestiges of a bygone RNA world. J Mol Evol 87:37-51.).

In addition to its well-known role in protein synthesis (rRNA, tRNA, mRNA), RNA also serves various non-coding functions. Eukaryotes contain a plethora of non-coding RNAs, some of which are also present in prokaryotes (Figure 2). Furthermore, some of these RNAs are believed to have originated and played significant roles during the RNA world, as discussed below.

Long non-coding RNAs (lncRNAs) are RNA molecules normally exceeding 200 bases in length. Similar to other ncRNAs, lncRNAs lack protein-coding capacity, and their structure is characterized by modularity and an abundance of sequence repeats (Mattick et al., 2023Mattick JS, Amaral PP, Carninci P, et al. (2023) Long non-coding RNAs: Definitions, functions, challenges and recommendations. Nat Rev Mol Cell Biol 24:430-447.). They are broadly classified into five categories based on the locations of their transcripts: 1) intergenic, 2) intronic, 3) sense, 4) antisense, and 5) bidirectional (Ma et al., 2013Ma L, Bajic VB and Zhang Z (2013) On the classification of long non-coding RNAs. RNA Biol 10:925-933.). lncRNAs play crucial roles in cellular functions, such as recruiting RNA-protein complexes to target genes. They act as decoys by binding and sequestering regulatory proteins away from their target DNA sequences (Mattick et al., 2023Mattick JS, Amaral PP, Carninci P, et al. (2023) Long non-coding RNAs: Definitions, functions, challenges and recommendations. Nat Rev Mol Cell Biol 24:430-447.). Additionally, lncRNAs significantly contribute to the regulation of gene expression by modulating chromatin function and influencing the assembly and functioning of nuclear components, including nuclear bodies (Statello et al., 2021Statello L, Guo CJ, Chen LL and Huarte M (2021) Gene regulation by long non-coding RNAs and its biological functions. Nat Rev Mol Cell Biol 22:96-118.).

Small nuclear RNAs (snRNAs) bind to proteins in the cellular nucleus, forming complexes known as small nuclear ribonucleoproteins (snRNPs), which collaborate with other snRNAs to facilitate the maturation of mRNAs before transporting them out of the nucleus (Karijolich and Yu, 2010Karijolich J and Yu YT (2010) Spliceosomal snRNA modifications and their function. RNA Biol 7:192-204.).

Similar to snRNAs, small nucleolar RNAs (snoRNAs) are RNA molecules residing in the nuclei of eukaryotic cells, specifically within the nucleolus, mainly encoded by intronic regions of both protein-coding and non-protein coding genes (Huang et al., 2022Huang Z, Du Y, Wen J, Lu B and Zhao Y (2022) snoRNAs: Functions and mechanisms in biological processes, and roles in tumor pathophysiology. Cell Death Discov 8:259.). These snoRNAs catalyze the 2′-O-methylation and pseudouridylation of rRNAs (Badis et al., 2003Badis G, Fromont-Racine M and Jacquier A (2003) A snoRNA that guides the two most conserved pseudouridine modifications within rRNA confers a growth advantage in yeast. RNA 9:771-9.; Monaco et al., 2018Monaco PL, Marcel V, Diaz JJ and Catez F (2018) 2’-O-Methylation of ribosomal RNA: Towards an epitranscriptomic control of translation? Biomolecules 8:106.), both critical post-transcriptional modifications that influence the stability, structure, and consequently the proper functioning of ribosomes (Kiss, 2002Kiss T (2002) Small Nucleolar RNAs. Cell 109:145-148.). Although mainly found in eukaryotes, there are reports of similar molecules in Archaea (Omer et al., 2000Omer AD, Lowe TM, Russell AG, Ebhardt H, Eddy SR and Dennis PP (2000) hom*ologs of small nucleolar RNAs in Archaea. Science 288:517-522. ).

A subtype of snoRNAs, small Cajal RNAs (scaRNAs), are localized within Cajal bodies, nuclear organelles responsible for the biogenesis of small nuclear ribonucleoproteins (snRNPs) (Huang et al., 2022Huang Z, Du Y, Wen J, Lu B and Zhao Y (2022) snoRNAs: Functions and mechanisms in biological processes, and roles in tumor pathophysiology. Cell Death Discov 8:259.). It has been observed that scaRNAs are responsible for the 2′-O-methylation and pseudouridylation of RNA pol II-specific U1, U2, U4, and U5 spliceosomal snRNAs (Darzacq et al., 2002Darzacq X, Jády BE, Verheggen C, Kiss AM, Bertrand E and Kiss T (2002) Cajal body-specific small nuclear RNAs: a novel class of 2’-O-methylation and pseudouridylation guide RNAs. EMBO J 21:2746-2756.).

The idea that snoRNAs might be ancient remnants from the RNA world aligns with the hypothesis that early protein-coding gene exons evolved around snoRNA sequences after the emergence of templated protein synthesis (Hoeppner and Poole, 2012Hoeppner MP and Poole AM (2012) Comparative genomics of eukaryotic small nucleolar RNAs reveals deep evolutionary ancestry amidst ongoing intragenomic mobility. BMC Evol Biol 12:183.). However, while there is a strong argument for an RNA-world origin of snoRNAs, it is equally plausible that these RNAs have a more recent origin, evolving with the complex regulatory needs of eukaryotic cells. Further research into snoRNAs may shed light on their role in the molecular evolution of life, considering both ancient and contemporary origins (Hoeppner and Poole, 2012Hoeppner MP and Poole AM (2012) Comparative genomics of eukaryotic small nucleolar RNAs reveals deep evolutionary ancestry amidst ongoing intragenomic mobility. BMC Evol Biol 12:183.).

MicroRNAs (miRNAs), which are 21-23 nucleotides in size, are associated with silencing and managing gene expression through post-transcriptional regulation. miRNAs interact with a seed region, a 2 to 7 nucleotide sequence, in the 3’ untranslated region (UTR) of mRNAs, leading to translational repression through mRNA degradation (Lu and Rothenberg, 2018Lu TX and Rothenberg ME (2018) MicroRNA. J Allergy Clin Immunol 141:1202-1207.).

Another category of well-known RNAs is small interfering RNAs (siRNAs). These RNA molecules typically range from 21 to 23 base pairs in length. Existing as duplexes, siRNAs can downregulate gene expression. This suppression is achieved through the binding of the entire siRNA duplex to specific mRNA sequences by complementarity (Lam et al., 2015Lam JK, Chow MY, Zhang Y and Leung SW (2015) siRNA versus miRNA as therapeutics for gene silencing molecular therapy. Nucleic Acids 4:e252.). Both miRNAs and siRNAs are small RNA-mediated regulation mechanisms (Lam et al., 2015Lam JK, Chow MY, Zhang Y and Leung SW (2015) siRNA versus miRNA as therapeutics for gene silencing molecular therapy. Nucleic Acids 4:e252.). In an RNA world, similar molecules would have enabled primitive RNA to adapt to environmental changes and rapidly regulate expression mechanisms in response to external stimuli. This adaptation would have laid the evolutionary foundations for the development of increasingly complex gene regulation systems.

Circular RNAs (circRNAs) are covalently closed-loop RNA molecules formed through back-splicing, where an upstream 3’ splice site is joined to a downstream 5’ splice site, resulting in a circular structure (Awasthi et al., 2018Awasthi R, Singh AK, Mishra G, Maurya A, Chellappan DK, Gupta G, Hansbro PM and Dua K (2018) An overview of circular RNAs. Adv Exp Med Biol 1087:3-14.). It has been shown that circRNAs act as miRNA and protein sponges, play a role in gene regulation, serve as transcriptional regulators, and act as scaffolds for proteins (Huang, et al., 2020Huang A, Zheng H, Wu Z, Chen M and Huang Y (2020) Circular RNA-protein interactions: functions, mechanisms, and identification. Theranostics 10:3503-3517.). These circRNAs vary in length from 100 base pairs to 4 kb, depending partly on the length and number of exons they contain (Verduci et al., 2021Verduci L, Tarcitano E, Strano S, Yarden Y and Blandino G (2021) CircRNAs: Role in human diseases and potential use as biomarkers. Cell Death Dis 12:468.). Their circular structure makes circRNAs less susceptible to degradation compared to linear RNAs, which have free ends. This ensures complete replication without the need for specific initiation or termination signals. Such stability and efficiency would have been advantageous in an RNA world, where circRNAs are proposed to have played a crucial role in the transition from RNA to DNA (Diener, 1989Diener TO (1989) Circular RNAs: Relics of precellular evolution? Proc Natl Acad Sci U S A 86:9370-9374.; Soslau, 2018Soslau G (2018) Circular RNA (CircRNA) Was an important bridge in the switch from the RNA world to the DNA world. J Theor Biol 447:32-40.).

Enhancer RNAs (eRNAs) are transcribed from enhancer regions of the genome. These eRNAs promote the recruitment of transcription factors and RNA polymerase II to target genes. Some studies indicate that eRNAs participate in cellular processes such as cell differentiation and development (Arnold et al., 2020Arnold PR, Wells AD and Li XC (2020) Diversity and emerging roles of enhancer RNA in regulation of gene expression and cell fate. Front Cell Dev Biol 7:377. ). Generally, this type of non-coding RNA is less than 150 nucleotides long, but some eRNAs can be as long as 4 kb (Sartorelli and Lauberth, 2020Sartorelli V and Lauberth SM (2020) Enhancer RNAs are an important regulatory layer of the epigenome. Nat Struct Mol Biol 27:521-528.).

Another class of non-coding RNA is piwi-interacting RNAs (piRNAs), which engage with a family of proteins known as Argonaute/Piwi. These molecules play an important role in safeguarding germline cells by preventing the insertion of transposable elements. piRNAs typically range from 21 to 35 base pairs in length and exhibit 2’-O-methyl-modified 3’ ends; this modification involves the addition of a methyl group to the 2’ hydroxyl (-OH) of the ribose moiety. Such modification extends the lifetime of alternative RNA conformational states and imparts resistance to nucleases, contributing to the overall stability and functionality of piRNAs in their protective role within germline cells (Tóth et al., 2016Tóth KF, Pezic D, Stuwe E and Webster A (2016) The piRNA pathway guards the germline genome against transposable elements. Adv Exp Med Biol 886:51-77.).

Riboswitches are another type of non-coding RNA, characterized as structured domains located within the 5’ untranslated region (UTR) of mRNAs. They consist of two distinct functional domains: an aptamer domain and an expression platform (Garst et al., 2011Garst AD, Edwards AL and Batey RT (2011) Riboswitches: Structures and mechanisms. Cold Spring Harb Perspect Biol 3:a003533.). These particular RNAs can switch gene expression on and off using a mechanism involving the binding of small target molecules, including metabolites (Kavita and Breaker, 2023Kavita, K and Breaker RR (2023) Discovering riboswitches: The past and the future. Trends Biochem Sci 48:119-141.). Riboswitches also exhibit regulatory control over the transcription of non-coding RNAs and the sequestration of the ribosome binding site (Breaker, 2018Breaker RR (2018) Riboswitches and translation control. Cold Spring Harb Perspect Biol 10:a032797.; Kavita and Breaker, 2023). Riboswitches are believed to be remnants of ancient regulatory mechanisms due to their ability to regulate gene expression independently of proteins, their distribution across all domains of life, and the fact that many riboswitches interact with RNA-derived molecules, including organic cofactors and alarmones. In this manner, riboswitches likely served sensing and regulatory functions in the RNA world, similar to their roles in present-day organisms (Breaker, 2012Breaker RR (2012) Riboswitches and the RNA world. Cold Spring Harb Perspect Biol 4:a003566.; Pavlova et al., 2019Pavlova N, Kaloudas D and Penchovsky R (2019) Riboswitch distribution, structure, and function in bacteria. Gene 708:38-48.; Salvail and Breaker, 2023Salvail H and Breaker RR (2023) Riboswitches. Curr Biol 33:R343-R348.).

Other non-coding RNAs include extracellular RNAs (exRNAs), which function as signaling molecules between cells. These molecules travel via exosomes or within microvesicles and can perform long-distance regulation (Wu et al., 2022Wu D, Tao T, Eshraghian EA, Lin P, Li Z and Zhu X (2022) Extracellular RNA as a kind of communication molecule and emerging cancer biomarker. Front Oncol 12:960072.). Enhancer-associated lncRNAs (elncRNAs) are involved in the regulation of gene expression by facilitating interactions between enhancers and promoters and by recruiting transcription factors to the promoters of target genes (Hou et al., 2019Hou Y, Zhang R and Sun X (2019) Enhancer lncRNAs influence chromatin interactions in different ways. Front Genet 10:936.). Long intergenic non-coding RNAs (lincRNAs) are independently transcribed (without directly depending on the transcription of protein-coding genes or other nearby genetic elements) molecules exceeding 200 nucleotides in length, formed from transcribed regions situated between protein-coding genes (intergenic regions). These RNAs are involved in the regulation of gene expression through different mechanisms. They can directly affect nuclear architecture, sequester intracellular molecules, or promote their function, among other roles (Ransohoff et al., 2018Ransohoff JD, Wei Y and Khavari PA (2018) The functions and unique features of long intergenic non-coding RNA. Nat Rev Mol Cell Biol 19:143-157.).

The diversity of RNA functions is still being unraveled, with new discoveries constantly emerging. As we continue to explore the different functions of RNA in prokaryotes, eukaryotes, in vitro and in silico systems, we can expect to gain a deeper understanding of the intricate mechanisms underlying cellular processes where RNA plays a crucial role.

Although RNA molecules exhibit catalytic, structural, and regulatory properties and are ubiquitous in cellular processes, strongly suggesting they played a major role in the early evolution of life, the RNA world hypothesis still faces significant challenges that require thorough exploration (Bernhardt, 2012Bernhardt HS (2012) The RNA world hypothesis: the worst theory of the early evolution of life (except for all the others)(a). Biol Direct, 7:23.; Le Vay and Mutschler, 2019Le Vay K and Mutschler H (2019) The difficult case of an RNA-only origin of life. Emerg Top Life Sci 3:469-475.).

Prebiotic synthesis of RNA monomers

A major hurdle for the RNA world proposal is the prebiotic synthesis of RNA monomers. Although ribonucleotides have been produced abiotically using various subsystems, common intermediates, and byproducts to link them (Powner et al., 2009Powner MW, Gerland B and Sutherland JD (2009) Synthesis of activated pyrimidine ribonucleotides in prebiotically plausible conditions. Nature 459:239-242.; Powner and Sutherland, 2011Powner MW and Sutherland JD (2011) Prebiotic chemistry: A new modus operandi. Philos Trans R Soc Lond B Biol Sci 366:2870-2877. ; Sutherland, 2016Sutherland JD (2016) The origin of life--Out of the blue. Angew Chem Int Ed Engl 55:104-21.), several obstacles persist. For example, there are issues related to the conditions and environments necessary for producing intermediates and byproducts, the availability of certain metals or high concentrations of compounds on early Earth, and the numerous iterations required to achieve the desired molecules (Lazcano, 2018Lazcano A (2018) Prebiotic evolution and self-assembly of nucleic acids. ACS Nano 12:9643-9647.).

Stability of RNA and its components

The susceptibility of RNA and its components to degradation in a dynamic environment influenced by various chemical and physical factors is a proposed counterargument against their presence on primordial Earth (Bernhardt, 2012Bernhardt HS (2012) The RNA world hypothesis: the worst theory of the early evolution of life (except for all the others)(a). Biol Direct, 7:23.; Xu et al., 2020Xu J, Chmela V, Green NJ, Russell DA, Janicki MJ, Góra RW, Szabla R, Bond AD and Sutherland JD (2020) Selective prebiotic formation of RNA pyrimidine and DNA purine nucleosides. Nature 582:60-66.). It is widely recognized that ribose, which can be formed abiotically through the formose reaction (Butlerow, 1861Butlerow A (1861) Bildung einer zuckerartigen Substanz durch Synthese. Justus Liebigs Ann Chem 120:295-298. ; Zafar and Senad, 2012Zafar I and Senad N (2012) The formose reaction: A tool to produce synthetic carbohydrates within a regenerative life support system. Curr Org Chem 16:769-88.; Delidovich et al., 2014Delidovich IV, Simonov AN, Taran OP and Parmon VN (2014) Catalytic formation of monosaccharides: from the formose reaction towards selective synthesis. Chem Sus Chem 7:1833-1846.; Omran et al., 2020Omran A, Menor-Salvan C, Springsteen G and Pasek M (2020) The messy alkaline formose reaction and its link to metabolism. Life (Basel) 10:125.), is unstable in strong acid or basic environments (Larralde et al., 1995Larralde R, Robertson MP and Miller SL (1995) Rates of decomposition of ribose and other sugars: Implications for chemical evolution. Proc Natl Acad Sci U S A 92:8158-8160.). Even under neutral conditions its half-life is very short, leading to high rates of decomposition and the low availability of this sugar in a primitive environment (Larralde et al., 1995).

Ultraviolet (UV) irradiation is another important factor that likely played a dual role in the prebiotic era (Cnossen et al., 2007Cnossen I, Sanz‐Forcada J, Favata F, Witasse O, Zegers T and Arnold NF (2007) Habitat of early life: Solar X‐ray and UV radiation at Earth’s surface 4-3.5 billion years ago. J Geophys Res Planets 112:2006JE002784.), acting as an energy source while also causing molecular damage, thus representing a significant selective pressure in the prebiotic environment (Sagan, 1973Sagan C (1973) Ultraviolet selection pressure on the earliest organisms. J Theor Biol 39:195-200.; Ranjan and Sasselov, 2016Ranjan S and Sasselov DD (2016) Influence of the UV environment on the synthesis of prebiotic molecules. Astrobiology 16:68-88.). Single nucleobases absorb more UV light than single or double strands of nucleic acids, with single strands absorbing more than double strands (Vorlíčková et al., 2005Vorlíčková M, Kypr J and Sklenář V (2005) Nucleic Acids | Spectroscopic Methods. In: Worsfold P, Poole C and Townshend A (eds) Encyclopedia of Analytical Science. 2nd edition. Elsevier, pp 391-399.; Winkler et al., 2020Winkler M, Giuliano BM and Caselli P (2020) UV resistance of nucleosides-An experimental approach. ACS Earth Space Chem 4:2320-2326.). Consequently, this makes the spontaneous synthesis of RNA difficult, as nucleobases can be degraded before they form RNA strands. However, once RNA strands are formed, their double-stranded regions are less susceptible to UV damage compared to single nucleobases, suggesting a protective advantage in RNA stability.

Alternative pre-RNA worlds

The RNA world hypothesis posits that RNA was essential for storing genetic information and catalyzing chemical reactions in early life. However, the challenges that have been discussed here have led to the exploration of alternative pre-RNA worlds, where simpler and more chemically stable molecules might have served as the initial genetic molecules, predating RNA. These hypothetical pre-RNA molecules would not only need to possess the ability to store and replicate genetic information but also exhibit greater resilience in the dynamic environments of early Earth. The pre-RNA world proposals aim to bridge the gap between prebiotic chemistry and the sophisticated RNA-based life forms that eventually arose.

One such alternative molecule is peptide nucleic acid (PNA), a polymer similar to DNA and RNA but with a peptide-like backbone. PNA is proposed as a genetic molecule that may have preceded RNA (Nelson et al., 2000Nelson KE, Levy M and Miller SL (2000) Peptide nucleic acids rather than RNA may have been the first genetic molecule. Proc Natl Acad Sci U S A 97:3868-71. ). Its backbone is composed of repeating aminoethyl glycine units linked by peptide bonds, which makes it more chemically stable than RNA and DNA (Nielsen, 2010Nielsen PE (2010) Peptide nucleic acids (PNA) in chemical biology and drug discovery. Chem Biodivers 7:786-804. ). This stability allows PNA to resist enzymatic degradation under conditions that would typically break down RNA (Nielsen, 1993Nielsen PE (1993) Peptide nucleic acid (PNA): A model structure for the primordial genetic material? Orig Life Evol Biosph 23:323-327.). Additionally, PNA can form stable duplexes with complementary DNA and RNA strands, illustrating its potential role in early genetic systems through PNA/DNA and PNA/RNA hybridization (Egholm et al., 1992Egholm M, Buchardt O, Christensen L, Nielsen PE and Berg RH (1992) Peptide nucleic acids (PNA) - Oligonucleotide analogs with an achiral peptide backbone. J Am Chem Soc 114:1895-1897.).

Similarly, threose nucleic acid (TNA) is a nucleic acid with a threose (a four-carbon sugar) backbone that could have been an early genetic polymer (Yu et al., 2012Yu H, Zhang S and Chaput JC (2012) Darwinian evolution of an alternative genetic system provides support for TNA as an RNA progenitor. Nat Chem 4:183-187.). TNA can form stable double helices and engage in Watson-Crick base pairing with both RNA and DNA, suggesting that TNA could store genetic information and transfer it to RNA and DNA (Chaput et al., 2003Chaput JC, Ichida JK and Szostak JW (2003) DNA polymerase-mediated DNA synthesis on a TNA template. J Am Chem Soc 125:856-857.). In this way, TNA could have served as an intermediate between prebiotic chemistry and the RNA world. These hypotheses highlight the potential diversity of molecules that could have preceded RNA in early evolution, enhancing our understanding of prebiotic chemistry and the transition into more complex biochemical systems.

Transition to the modern DNA-RNA-protein world

Significant challenges and questions persist regarding the transition from the RNA world to the modern DNA-RNA-protein world (Dworkin et al., 2003Dworkin JP, Lazcano A and Miller SL (2003) The roads to and from the RNA world. J Theor Biol 222:127-134. ; Gavette et al., 2016Gavette JV, Stoop M, Hud NV and Krishnamurthy R (2016) RNA-DNA chimeras in the context of an RNA world transition to an RNA/DNA. Angew Chem 55:13204-13209.). In this transition, interactions between DNA, RNA, and proteins led to a specialization and division of cellular activities, with DNA becoming the primary genetic storage molecule, proteins becoming the main catalysts and structural components of the cell, and RNA retaining auxiliary functions between DNA and proteins, with regulatory, structural, and catalytic properties reminiscent of an earlier era (Cojocaru and Unrau, 2017Cojocaru R and Unrau PJ (2017) Transitioning to DNA genomes in an RNA world. Elife 6:e32330.).

One hypothesis addressing these challenges is the RNA/DNA world hypothesis. This hypothesis challenges the traditional view that DNA emerged after an RNA-based system and posits that primordial life forms possessed both RNA and DNA building blocks, with heterogeneous genetic polymers comprising both RNA and DNA sequences (Bhowmik and Krishnamurthy, 2019Bhowmik S and Krishnamurthy R (2019) The role of sugar-backbone heterogeneity and chimeras in the simultaneous emergence of RNA and DNA. Nat Chem 11:1009-1018.; Xu et al., 2020Xu J, Chmela V, Green NJ, Russell DA, Janicki MJ, Góra RW, Szabla R, Bond AD and Sutherland JD (2020) Selective prebiotic formation of RNA pyrimidine and DNA purine nucleosides. Nature 582:60-66.). This scenario simplifies the evolutionary transition by eliminating the need for early organisms to evolve intricate biosynthetic pathways to synthesize DNA from RNA.

Through natural selection, a progressive shift towards more hom*ogeneous polymers would have occurred, characterized by enhanced stability, genome replication fidelity, and catalytic efficiency. This evolutionary trajectory would result in the distinct functional specialization observed in contemporary biology, where RNA primarily facilitates catalysis and information transfer, while DNA functions as the stable repository of genetic information.

Mutual evolution of nucleic acids and proteins

Coevolution models propose that life emerged from a network of interacting molecules that evolved together to form the systems required for cellular function. This approach suggests that complex biochemical systems developed through the mutual evolution of molecules such as nucleic acids and proteins (Saad, 2018Saad NY (2018) A ribonucleopeptide world at the origin of life. J Syst Evol 56:1-13.; Farías-Rico and Mourra-Díaz, 2022Farías-Rico JA and Mourra-Díaz CM (2022) A short tale of the origin of proteins and ribosome evolution. Microorganisms 10:2115.; Tagami and Li, 2023Tagami S and Li P (2023) The origin of life: RNA and protein co-evolution on the ancient Earth. Dev Growth Differ 65:167-174.).

These theories highlight the importance of molecular interactions in the origin of life, suggesting that life arose from cooperative evolution rather than a linear progression of separate systems. This likely created a dynamic environment where evolving molecules influenced each other, fostering the emergence of life from a prebiotic chemical milieu.

One aspect of this theory posits that RNA and proteins coevolved in a symbiotic relationship (Saad, 2018Saad NY (2018) A ribonucleopeptide world at the origin of life. J Syst Evol 56:1-13.; Farías-Rico and Mourra-Díaz, 2022Farías-Rico JA and Mourra-Díaz CM (2022) A short tale of the origin of proteins and ribosome evolution. Microorganisms 10:2115.; Tagami and Li, 2023Tagami S and Li P (2023) The origin of life: RNA and protein co-evolution on the ancient Earth. Dev Growth Differ 65:167-174.). The interdependence between these molecules could have driven the evolution of complex biochemical pathways, with the catalytic abilities of RNA complemented by the structural support provided by proteins. This cooperative relationship may have paved the way for the modern DNA-RNA-protein world.

Although coevolutionary models are attractive, the RNA world hypothesis does not exclude interactions with other types of molecules and, therefore, does not rule out the possibility of coevolution. The RNA world is completely compatible with coevolutionary models.

Non-RNA world proposals

Other proposals that do not contemplate an RNA world include the protein-first hypothesis, which posits that polypeptides were the first self-replicating molecules, with RNA and DNA evolving later as storage media for genetic information (Andras and Andras, 2005Andras P and Andras C (2005) The origins of life - the ‘protein interaction world’ hypothesis: protein interactions were the first form of self-reproducing life and nucleic acids evolved later as memory molecules. Med Hypotheses 64:678-688.; Bernhardt, 2012Bernhardt HS (2012) The RNA world hypothesis: the worst theory of the early evolution of life (except for all the others)(a). Biol Direct, 7:23.). Another hypothesis is the lipid world (Segré et al., 2001Segré D, Ben-Eli D, Deamer DW and Lancet D (2001) The lipid world. Orig Life Evol Biosph 31:119-145.), that suggests that lipid molecules were essential precursors for primitive cell membranes. In this proposal, spontaneously formed vesicles facilitated the development of primitive metabolic networks and catalyzed chemical reactions, playing a key role in the stability and organization of early life, eventually leading to more complex cellular structures (Kahana and Lancet, 2021Kahana A and Lancet D (2021) Self-reproducing catalytic micelles as nanoscopic protocell precursors. Nat Rev Chem 5:870-878.; Subbotin and Fiksel, 2023Subbotin V and Fiksel G (2023) Exploring the lipid world hypothesis: A novel scenario of self-sustained Darwinian evolution of the liposomes. Astrobiology 23:344-357.)

Both the protein-first and lipid world hypotheses, along with coevolution models, offer compelling alternatives to the RNA world. They highlight the diverse pathways through which life might have originated and emphasize the importance of exploring various models to fully understand the complex processes that led to the emergence of life on Earth.

The dynamic nature of RNA viruses and the RNA world intersect in fascinating ways, bringing to light intricate evolutionary processes that have shaped the biological landscape. As previously mentioned, ribocells are hypothetical primordial cellular entities in the RNA world that use RNA, rather than DNA, as their genetic information. Today, RNA viruses and viroids are the only biological entities that use RNA to store genetic material. However RNA viruses are not considered living systems (Colón-Santos et al., 2024Colón-Santos S, Vázquez-Salazar A, Adams A, Campillo-Balderas JA, Hernández-Morales R, Jácome R, Muñoz-Velasco I, Rodriguez LE, Schaible MJ, Schaible GA, Szeinbaum N, Thweatt JL and Trubl G (2024) What Is Life? Astrobiology 24:S-40-S-56. ), they are acellular submicroscopic infectious agents that replicate inside a living host cell. RNA viruses are classified in the realm Riboviria that currently includes all RNA viruses which encode an RNA-directed RNA polymerase (ICTV, 2020ICTV - International Committee on Taxonomy of Viruses Executive Committee (2020) The new scope of virus taxonomy: partitioning the virosphere into 15 hierarchical ranks. Nat Microbiol 5:668-674.). They are also classified based on their genomic structure, genome replication strategies, and evolutionary characteristics (Table 1). The genetic material of RNA viruses is characterized by a compact genome composed of ribonucleic acid which is either single-stranded (ssRNA) or double-stranded (dsRNA). The ssRNA viruses are also classified based on the polarity of their genetic material into positive-sense single-stranded RNA viruses (ssRNA+), whose genomic RNA can be directly translated into proteins by the ribosome of the cellular host (e.g., coronaviruses such as SARS-CoV-2) and negative-sense single-stranded RNA viruses (ssRNA-), which have a genomic RNA that must be transcribed into a ssRNA+ before protein synthesis (e.g., orthomyxoviruses such as influenza viruses). There are also ambisense (negative and positive) RNA viruses that translate genes from both strands (e.g., some bunyavirales) (Holmes, 2009Holmes EC (2009) The evolution and emergence of RNA viruses. Oxford University Press, New York.; Hulo et al., 2011Hulo C, Castro E, Masson P, Bougueleret L, Bairoch A, Xenarios I and Le Mercier P (2011) ViralZone: A knowledge resource to understand virus diversity. Nucleic Acids Res 39:D576-58). The dsRNA viruses use their double-stranded genome as a template by the RNA-dependent RNA polymerase (RdRp) to transcribe a positive-strand RNA (e.g., reoviruses such as Rotavirus) (Hulo et al., 2011). Retroviruses also have an RNA genome that uses a reverse transcriptase (RT) to transcribe RNA into DNA which is then integrated into the host genome (e.g., HIV-1) (Hulo et al., 2011). RdRp and RT are essential enzymes for the replication and transcription of viral genes into mRNA in order to translate viral proteins. The RNA viral genome also has some peculiar characteristics, including secondary structure at both 3’ and 5’ ends to regulate the genome replication process, and segmentation either with a single nucleic acid molecule (monopartite) or multiple genetic fragments (multipartite) (Holmes, 2009). Moreover, some ss- and dsRNA viruses, which infect plants and fungi, have multiple genomic segments contained within different virus particles called multicomponent viruses (Holmes, 2009). Viroids, which are short (250-400 nucleotides), single-stranded, infectious, circular RNA molecules that do not encode any protein, are primarily associated with plant hosts, and replicate in the nucleus or chloroplasts via the cellular RNA polymerase II (Navarro et al., 2021Navarro B, Flores R and Di Serio F (2021) Advances in viroid-host interactions. Annu Rev Virol 8:305-325.) through a rolling circle mechanism (Venkataraman et al., 2021Venkataraman S, Badar U, Shoeb E, Hashim G, AbouHaidar M and Hefferon K (2021) An inside look into biological miniatures: Molecular mechanisms of viroids. Int J Mol Sci 22:2795.). Viroids from the family Avsunviroidae are endowed with hammerhead ribozymes that participate in the cleavage of oligomeric strands (Di Serio et al., 2018Di Serio F, Li SF, Matoušek J, Owens RA, Pallás V, Randles JW, Sano T, Verhoeven JTJ, Vidalakis G, Flores R and ICTV Report Consortium (2018) ICTV Virus Taxonomy Profile: Avsunviroidae. J Gen Virol 99:611-612.; 2020Di Serio F, Owens RA, Li SF, Matoušek J, Pallás V, Randles JW, Sano T, Verhoeven JT, Vidalakis G, Flores R, Zerbini FM, and Sabanadzovic S (eds.) (2020) “Viroids”. ICTV.).

Viral and viroid quasispecies

RNA viruses and viroids are endowed with distinctive evolutionary features, such as short generation times, high rates of genetic recombination and reassortment, and high mutation rates (Duffy, 2018Duffy S (2018) Why are RNA virus mutation rates so damn high? PLoS Biol 6:e3000003.). These characteristics favor their adaptation to changes in their host environment (Dolan et al., 2018Dolan PT, Whitfield ZJ and Andino R (2018) Mechanisms and concepts in RNA virus population dynamics and evolution. Annu Rev Virol 5:69-92.). Such an intrinsically high error rate in RNA viral populations has evolutionary implications including a high prevalence of deleterious mutations and small genomes, ranging from 2,000 to 25,000 nt, although a few may reach up to 40,000 nt (Ferron et al., 2021Ferron F, Sama B, Decroly E and Canard B (2021) The enzymes for genome size increase and maintenance of large (+)RNA viruses. Trends Biochem Sci 46:866-877.). According to Manfred Eigen (1971Eigen M (1971) Self-organization of matter and the evolution of biological macromolecules. Naturwissenschaften 58:465-523.), if the number of errors surpasses the maximum tolerance for mutations of the replicating system, it can result in the generation of numerous defective phenotypes and may drive the system to extinction. RNA viruses are endowed with small genomes that, statistically speaking, are less-prone to acquire a high number of mutations, allowing them to elude Eigen’s limit. It is important to highlight that being endowed with a small genome imposes limitations on the number of proteins that a biological entity may encode. Therefore, RNA viruses maximize the use of their genome space, as the majority of their genome is protein-coding (Holmes, 2009Holmes EC (2009) The evolution and emergence of RNA viruses. Oxford University Press, New York.). These evolutionary characteristics allow the generation of a large number of genetically heterogeneous viroid and RNA viral genomes by a mutation-selection process called quasispecies. This concept was adopted from the study of the origin and early evolution of life to explain the heterogeneity of primitive self-replicative macromolecules with closely related sequences at different levels of organization (hypercycles) based on the mathematical model of Eigen and Schuster (1977Eigen M and Schuster P (1977) The hypercycle. A principle of natural self-organization. Part A: Emergence of the hypercycle. Naturwissenschaften 64:541-565.). According to this model, the different versions of viral genotypes are situated in a part of the genetic fitness landscape that is not particularly advantageous or disadvantageous for their survival (a flat region). In this way, these viral genotypes are more likely to outcompete other variants located in higher but narrower fitness peaks guided by natural selection. Hence, this theory describes the evolutionary dynamics of small RNA replicons with high mutation rates, such as primordial self-replicative RNAs and RNA viral genomes, as potentially-adaptable molecules to their environments. This theoretical model has experimental evidence in mutants of RNA viral populations which infect bacteria, plants, and animals (Domingo et al., 2021Domingo E, García-Crespo C and Perales C (2021) Historical perspective on the discovery of the quasispecies concept. Annu Rev Virol 8:51-72.), as well as in in vitro dynamic systems used for the study of the origin of life, next- generation sequencing data, lethal mutagenesis, antiviral strategies, etc (Domingo and Schuster, 2016Domingo E and Schuster P (2016) Quasispecies: From theory to experimental systems. Springer.).

RNA viruses and viroids: primordial replicons?

Due to their unique and (not so) simple molecular structure and evolutionary features as dynamic heterogeneous quasispecies, RNA viruses have been proposed as relics of the RNA world. Similarly, the error-prone replication, high G+C content, and unique secondary structure of viroids are interpreted as evidence to propose that they could be primordial replicons from the RNA world (Flores et al., 2014Flores R, Gago-Zachert S, Serra P, Sanjuán R and Elena SF (2014) Viroids: survivors from the RNA world?. Annu Rev Microbiol 68:395-414.). Indeed, a modular evolution model for the emergence of protoviroids in the precellular world has been posited, suggesting subsequent increases in genome complexity through the reorganization of modules and mutation (Flores et al., 2022Flores R, Navarro B, Serra P and Di Serio F (2022) A scenario for the emergence of protoviroids in the RNA world and for their further evolution into viroids and viroid-like RNAs by modular recombinations and mutations. Virus Evol 8:veab107. ). However, these same properties do not fully support the primordial origin of viroids. The genome size and “simplicity”, the lack of protein-coding capacity, and their dependence on plant cells exhibit significant differences to the larger, more complex, protein-coding, and self-replicative ribocells.

Therefore, viroids are best understood as specialized RNA infectious agents that may provide insights into the minimal requirements for RNA self-replication. This reinforces the idea that RNA may have played an important role in the origin of life, but viroids should not be considered relics from the RNA world.

Similarly, RNA viruses undergo rapid mutation, reassortment, and recombination, leading to the emergence of genetic diversity, new variants, and adaptability. This ability to evolve quickly highlights the versatility of RNA viral genomes to support the idea of the virus-first hypothesis as precellular genetic elements (Nasir et al., 2012Nasir A, Kim KM and Caetano-Anollés G (2012) Viral evolution: Primordial cellular origins and late adaptation to parasitism. Mob Genet Elements 2:247-252.). Additionally, some authors state that RNA viruses encode hallmark proteins crucial for genome replication, genetic expression, and morphogenesis, which have no cellular hom*ologs. Examples of hallmark viral genes believed to have emerged from the primordial pool of primitive genetic elements in a stage called the ancient virus world are the jelly-roll capsid protein, superfamily 3 helicase, rolling circle replication initiation endonuclease, DNA primase, packaging ATPase, and RNA-dependent RNA polymerase (Koonin et al., 2006Koonin EV, Senkevich TG and Dolja VV (2006) The ancient Virus World and evolution of cells. Biology Direct 1:29.; Koonin, 2016Koonin EV (2016) Viruses and mobile elements as drivers of evolutionary transitions. Phil Trans R Soc B 371:20150442.). However, analyses based on protein structure rather than sequence have demonstrated that some of these hallmark genes, including RdRp and viral capsid proteins, are hom*ologous to cellular components (Krupovic and Koonin, 2017Krupovic M and Koonin EV (2017) Multiple origins of viral capsid proteins from cellular ancestors. Proc Natl Acad Sci U S A 114:E2401-E2410.; Mughal et al., 2020Mughal F, Nasir A and Caetano-Anollés G (2020) The origin and evolution of viruses inferred from fold family structure. Arch Virol 165:2177-2191.; Jácome et al., 2022Jácome R, Campillo-Balderas JA, Becerra A and Lazcano A (2022) Structural analysis of monomeric RNA-dependent polymerases revisited. J Mol Evol 90:293-295.). Different arguments contradict the idea of the primordial origin of RNA viruses in the RNA world. These include their absolute reliance on the enzymatic machinery of host cells, the cellular origin of metabolic and replication genes, and their predominance infecting eukaryotic hosts (Campillo-Balderas et al., 2015Campillo-Balderas JA, Lazcano A and Becerra A (2015) Viral genome size distribution does not correlate with the antiquity of the host lineages. Front Ecol Evol 3:143.). Additionally, the complexity of cellular genomes, the polyphyletic origins of viruses, and their lack of structural components such as membranes further challenge this idea.

Viral and viroid quasispecies dynamics highlight the adaptability and resilience of RNA-based entities in the face of selective pressures. These dynamics echo the evolutionary principles that may have governed early life replicators in the RNA world. The high mutation rates observed in viral quasispecies may mirror the inherent mutability in the RNA world and the importance of genetic diversity as a driving force in evolutionary processes. However, RNA viruses and viroids are RNA-based biological entities that replicate their genetic material through different pathways, neither fully rely on ribozymes, but both are entirely dependent on cellular enzymatic machinery for their replication. Therefore, current evidence indicates that RNA viruses and viroids are not remnants of the RNA world, are not directly related to ribocells, and are unlikely to have been the primordial replicators that preceded cellular life. However, they do have similar population dynamics as quasispecies that may serve as models to help us understand the evolutionary characteristics of the early stages of life (Cruz-González et al., 2021Cruz-González A, Muñoz-Velasco I, Cottom-Salas W, Becerra A, Campillo-Balderas JA, Hernández-Morales R, Vázquez-Salazar A, Jácome R and Lazcano A (2021) Structural analysis of viral ExoN domains reveals polyphyletic hijacking events. PloS One 16:e0246981.; de la Higuera and Lázaro, 2022de la Higuera I and Lázaro E (2022) Viruses in astrobiology. Front Microbiol 13:1032918.).

This review has highlighted the pivotal role of RNA in the early stages of life, emphasizing its extraordinary diversity of functions.

The discoveries in the field of prebiotic chemistry, the many different roles that RNA molecules play in several cellular processes, and the participation of ribonucleotides and their derivatives in regulation, catalysis, and signaling mechanisms have changed our conception of the RNA world.

The synthesis of RNA nucleobases and precursors, leading to the formation of short RNA oligonucleotides, appears to have been a critical step in the prebiotic era. Additionally, the emergence of an RNA-dependent RNA polymerase ribozyme capable of catalyzing its own replication would mark a significant transition from simple chemical systems to an RNA world capable of Darwinian evolution. These insights contribute to a deeper understanding of the origin and early evolution of life, as well as the evolutionary processes that led to the diversity of life forms observed today.

Applying the quasispecies model to contemporary RNA entities, such as RNA viruses and viroids, can enhance our understanding of the evolutionary dynamics of early RNA replicons. Although determining the genomic organization of primordial RNA-based entities remains challenging with existing methodological resources, the diverse genomic architectures of current RNA viruses and viroids may provide clues about the forms adopted by RNA genomes in an RNA world.

Studying RNA and its early evolutionary roles offers valuable insights for current research in molecular biology, genetics, and biochemistry. As we continue to unravel the complexities of RNA and its functions, our understanding of the origin and early evolution of life is set to deepen, presenting exciting possibilities for future scientific exploration.

The authors would like to thank Thalía Garcés-Jurado for creating the figures shown in this review. AC-G is a doctoral student from the Posgrado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM) and received fellowship from CONAHCyT (CVU-1002377). WC-S is a doctoral student from Posgrado en Ciencias Biológicas. We are grateful to DGAPA-INFOCAB PB202223.

Associate Editor:

Carlos F.M. Menck