Transfer RNA and Origins of RNA Interference.

Almost 30 years ago the first microRNA (miRNA) was detected (Lee et al., 1993), later put in mechanistical context by the discovery of the RNA interference (RNAi) and RNA-induced silencing complex (RISC). miRNA is the variable RISC element (guide) that recognizes messenger RNA (mRNA) targets in the RNAi process facilitated by Argonaute (Ago) proteins, the fixed key players in RISC. Alas, poetically sounding Argonautes were named not after the adventurers from Greek mythology but octopus-shaped leaves of an Arabidopsis thaliana mutant (Bohmert et al., 1998). Production of miRNAs is performed by a sophisticated orchestra of players with even groovier names, like Drosha, Pasha or Dicer. While roles of miRNA in post-transcriptional regulation have been established, its evolution has been typically described as expansion of new miRNA genes from the existing ones, not their first appearance (Berezikov, 2011). The emergence of RNAi has been attributed to its defense against pathogens, which involves other participants, e.g., double-stranded RNA binding proteins (dsRBPs). But details of such emergence are lacking. This is a short opinion piece, not a full review, so I apologetically skip some of these players and hundreds of relevant miRNA/RNAi citations (and those for subsections below). The complexity of the RISC is bewildering. Intricate steps of miRNA production in the nucleus are followed by unequal strand loading to Ago, and finally resulting in cytoplasmic events, sometimes involving “slashing” of mRNA. All that (for simplicity, ignoring nuclear events upon Ago transport from cytoplasm) is based on weak and imperfect binding of short “seeds” (5′ 6-8 nucleotides in length in animals) to targets and on weak outcomes of slight tuning of target translation. How can such weak binding be a driving force for emergence and evolution of this complicated system and its parts, acting in concert in different cellular compartments? Plant RNAi requires full-length small RNA hybridization, significantly limiting the number of target genes, yet the system appears even more complex (four Dicers, seven dsRBPs versus a single Dicer/couple of dsRBPs in humans). Further, the miRNA generation machineries are not entirely homologous, mature miRNAs are produced and modified in the plant nucleus, but these processes are divided between the nucleus and cytoplasm in animals. How would these different processes evolve in parallel with creating the elaborate and divergent Ago functionalities (four different Agos in humans, ten in A. thaliana, 26 in Caenorhabditis elegans), able to utilize these precisely cut miRNAs for mRNA regulation? This scenario would require a small RNA already acting together with some Ago prototypic protein. Compatible with this view, the last common ancestor of eukaryotes likely appeared after the RNAi functional principles (and some components, except for miRNA) had been invented (Cerutti and Casas-Mollano, 2006). And many prokaryotes do not have miRNAs but possess Ago protein homologs. Transfer RNAs and Their Fragments Enter transfer RNAs (tRNAs), fundamental elements in mRNA translation. Each tRNA is a link of informational (anticodon) and corresponding chemical (aminoacid) units, which jointly provide the basis of the central dogma. These are ancient molecules, potentially capable of performing primitive replication (Kuhnlein et al., 2021) or aiding in it (Maizels and Weiner, 1994). While the genetic code requires <64 anticodons for translation, tRNA genes are very numerous, with several hundred copies in human genome. From a regulation standpoint, these numbers far exceed potential codon adaptation mechanisms. Perhaps fitting to this view, many tRNA genes have been described as inactive, raising questions about their actual role (Torres, 2019). The early sequence/structure determination and clearly defined function of tRNAs established their place in molecular biology textbooks, hardly revisited until the arrival of tRNA fragments (tRFs). Hypotheses of further roles were occasionally entertained, based on additional function observed, e.g., in viral replication, etc. (see also a review (Avcilar-Kucukgoze and Kashina, 2020) in this article collection for more details on these roles). However, an avalanche of data from small RNA sequencing experiments have challenged such perceptions, revealing numerous and ubiquitous tRFs in a multitude of sequenced samples. These fragments, detected in datasets produced to study miRNA, have been mostly dismissed as noise. It took >10 years to get acceptance even after detailed studies (Cole et al., 2009; Lee et al., 2009; Haussecker et al., 2010; Ivanov et al., 2011; Gebetsberger et al., 2012), even though tRNA breakage products were detected in urine of cancer patients much earlier (Speer et al., 1979). The finding of Ago proteins loaded with tRFs, in addition to their cargo of miRNAs, have prompted speculations about similar functionality of tRFs and search for their targets (Table 1). TABLE 1 Representative studies on detection and analysis of Argonaute-loaded tRFs in different organisms.