RNA-catalyzed RNA replication is widely believed to have supported a primordial biology. However, RNA catalysis is dependent upon RNA folding, and this yields structures that can block replication of such RNAs. To address this apparent paradox, we have re-examined the building blocks used for RNA replication. We report RNA-catalysed RNA synthesis on structured templates when using trinucleotide triphosphates (triplets) as substrates, catalysed by a general and accurate triplet polymerase ribozyme that emerged from in vitro evolution as a mutualistic RNA heterodimer. The triplets cooperatively invaded and unraveled even highly stable RNA secondary structures, and support non-canonical primer-free and bidirectional modes of RNA synthesis and replication. Triplet substrates thus resolve a central incongruity of RNA replication, and here allow the ribozyme to synthesise its own catalytic subunit ‘+’ and ‘–’ strands in segments and assemble them into a new active ribozyme. Life as we know it relies on three types of molecules: DNA, which stores genetic information; proteins that carry out the chemical reactions necessary for life; and RNA, which relays information between the two. However, some scientists think that before life adopted DNA and proteins, it relied primarily on RNA.Like DNA, strands of RNA contain genetic data. Yet, some RNA strands can also fold to form ribozymes, 3D structures that could have guided life’s chemical processes the way proteins do now. For early life to be built on RNA, though, this molecule must have had the ability to make copies of itself.This duplication is a chemical reaction that could be driven by an ‘RNA replicase’ ribozyme. RNA strands are made of four different letters attached to each other in a specific order. When RNA is copied, one strand acts as a template, and a replicase ribozyme would accurately guide which letters are added to the strand under construction. However, no replicase ribozyme has been observed in existing life forms; this has led scientists to try to artificially create RNA replicase ribozymes that could copy themselves.Until now, the best approaches have assumed that a replicase would add building blocks formed of a single letter one by one to grow a new strand. Yet, although ribozymes can be made to copy straight RNA templates this way, folded RNA templates – including the replicase ribozyme itself – impede copying. In this apparent paradox, a ribozyme needs to fold to copy RNA, but when folded, is itself copied poorly. Here, Attwater et al. wondered if choosing different building blocks might overcome this contradiction.Biochemical techniques were used to engineer a ribozyme that copies RNA strands by adding letters not one-by-one, but three-by-three. Using three-letter ‘triplet’ building blocks, this new ribozyme can copy various folded RNA strands, including the active part of its own sequence.This is because triplet building blocks have different, and sometimes unexpected, chemical properties compared to single-letter blocks. For example, these triplets work together to bind tightly to RNA strands and unravel structures that block RNA copying.All life on Earth today uses a triplet RNA code to make proteins from DNA, and these experiments showed how RNA triplets might have helped RNA sustain early life forms. Further work is now needed to improve the ribozyme designed by Attwater et al. for efficient self-copying. The premise that some RNA sequences can catalyse and template their own replication - reciprocally synthesizing their own ‘+’ and ‘–’ strands - underpins current thinking about early genetic systems . Any ancIn a contrasting approach, RNA polymerase ribozymes (RPRs) have been developed that can use general monomer building blocks (ribonucleoside 5’ triphosphates (NTPs)) in RNA-templated RNA synthesis , akin toWe wondered whether this paradox might be avoided through a re-consideration of plausible building blocks for early RNA replication. Models of non-enzymatic polymerisation of all four activated ribonucleotides – the presumed source of the first RNA sequences – yield pools of di-, tri- and tetranucleotide etc. length oligonucleotides (in decreasing abundance) dominating the population alongside longer products . Here, wWe set out to explore the potential of short RNA oligonucleotides as substrates for RNA-catalyzed RNA replication. To do this, we required a ribozyme capable of general, iterative RNA-templated oligonucleotide ligation. Previously-described RNA polymerase ribozymes such as the ‘Z’ RPR  can use To be able to properly examine such RNA trinucleotide triphosphates (triplets) as replication substrates, we first sought to convert Zcore into an effective triplet polymerase ribozyme using in vitro evolution. We devised a selection strategy that required iterative templated triplet ligation by ribozymes to achieve their covalent linkage to a tagged primer . This en15 Zcore variants with a new random 3’ N30 region under eutectic phase conditions that increase RNA half-life and enhance ribozyme activity  receptive to activity enhancement by type 1 RNA . The catIn complex with type 5 , type 1 boosts polymerization of triplets (or longer oligonucleotides) to enable synthesis of long RNAs . Here, i+1’ , diversifying the previously-fixed 3’ domain reverse transcription primer binding sequence. This reselection yielded a shorter final heterodimeric triplet polymerase holoenzyme, hereafter termed ‘t5+1’ . This ro+1 activity, we revisited triplet-based RNA synthesis on structured templates. To provide a stringent test of template structure inhibition, we now examined hairpin-containing templates  with increasing RNA hairpin stability and estimated TMs of up to 93˚C (8S). The latter had previously strongly arrested even the most advanced mononucleotide RPRs at higher temperatures . Encouragingly, t5+1 could synthesise both a functional fluorescent ‘+’ strand of the 52 nt Broccoli RNA aptamer  could reduce tertiary structures  for synthesis of full-length α+ and ε+ segments  was similar to that observed when t5+1 used an unstructured model template . We found that this synthesis product could regenerate fresh δ- segment using t5+1 ribozyme-synthesised δ+ (left over from ribozyme assembly) as a template , efficiency of both segment synthesis and assembly will need to be increased significantly to realise a full self-replication cycle .At this point, we tested whether the broad oligonucleotide ligation capacity of t5+1  might al strands . Through strands , the ribtemplate , recapitTemplated ‘+’ strand self-synthesis is a central element of ribozyme self-replication. However, a limitation of our above strategy in the context of triplet-based self-replication is the continued requirement for some pre-synthesized longer oligonucleotides to act as primers and occasional substrates . In particular, some specific oligonucleotide substrates were required for efficient synthesis of α+ and ε+ segments to compete out inhibitory mutual hybridisation between ‘−’ strand template and corresponding ‘+’ strand unstructured elements in the t5 ribozyme . In vitrThe majority of specific oligonucleotides, however, were provided as primers to initiate syntheses, as required by all RPRs akin to the activity of replicative polymerases in biology. As a consequence of this, the priming sequence would effectively be excluded from evolution during replication. Furthermore, RNA oligonucleotides able to act as specific primers are unlikely to be prevalent in prebiotic substrate pools, and their depletion during successive replication cycles could lead to loss of sequence at genome ends. This ‘primer problem’ has previously been noted in the context of nonenzymatic replication  as one o+1 can extend primers bidirectionally, in both the canonical 5’−3’ as well as the reverse 3’−5’ directions , as exemplified here for the β+ segment  could be used as substrates by t5+1 in segment syntheses in place of defined triplet sets  and collated, which allowed an estimation of ribozyme misincorporation tendencies. On average, the starting Zcore ribozyme exhibited ~91% fidelity per position  synthesised by t5+1 using pppNNN indicated similar aggregate fidelity could be achieved during longer product synthesis excluding the final triplet  reverted the fidelity profile towards that of Zcore (Having established that accurate triplet-based copying is possible (in at least some sequence contexts), we sought to understand how the triplet polymerase ribozyme achieves it. Investigating the fidelity contributions of different t5of Zcore . Presencof Zcore . Overallof Zcore , though of Zcore . The ε dof Zcore .Tetrahymena group I intron folding  rendered the ε fidelity domain unable to discriminate mismatches . Previou folding  and the  folding .pppGCC:pppGGC (than pppACC:pppGGU) dimer formation would selectively reduce the effective concentration of free pppGCC vs. pppACC upon inclusion of their complementary pppGGC and pppGGU.An important contribution to triplet fidelity also appears to arise from unexpected behaviours of the triplet substrates themselves. We observed that in some direct pair-mispair triplet contests, inclusion of their complementary triplets caused a striking (~3 fold) drop in misincorporation errors . A potenpppNNN substrate pools  binding strength.These pairwise reductions were recapitulated in the presence of random te pools . Indeed,ror rate . Althougror rate . Dimer fpppNNN pool formulated with a reduced G content  with a robust ability to polymerize RNA trinucleotide triphosphate (triplet) substrates. Unusually, this triplet polymerase ribozyme comprises a heterodimer of a catalytic triplet polymerase subunit (t5) and a non-catalytic RNA cofactor (type 1), which enhances triplet polymerase activity and abrogates the need for template tethering. Such a quaternary structure - involving a heterodimer of a full-length and a truncated subunit - is reminiscent of the processivity factors of some proteinaceous polymerases such as the heterodimeric p66/p51 HIV reverse transcriptase holoenzyme , with particularly helpful stability contributions from intra-triplet base stacking . FurtherTogether these favourable molecular traits serve to pre-organize the template towards a double-stranded RNA duplex with triplet junctions poised for ligation. A triplet/template duplex presents a more ordered, regular target for sequence-general ribozyme docking (by e.g. the ε domain) than a single stranded template (variably prone to secondary structure formation or sequence-specific interactions with the ribozyme ). Such g+1 ribozyme fidelity is due to both a readout of cognate minor groove interactions by the ribozyme ε domain , suggestive of convergent adaptive solutions to the challenges of replication and decoding. It has long been speculated that the decoding centre of the small ribosomal subunit might have had its origins in an ancestral RNA replicase, but the implied triplet-based character of such a replicase was conspicuously discordant with modern mononucleotide-based replication . The utiIn conclusion, the unexpected emergent properties of triplets – including cooperative binding and unfolding of structured RNA templates, enhanced incorporation of AU-rich substrates, and error attenuation (resulting from triplet pool interaction networks) – argue that short RNA oligonucleotides may represent predisposed substrates for RNA-catalyzed RNA replication. Some of these benefits might also extend to codon/anticodon dynamics in early translation, and to the non-enzymatic replication of RNA , where d2 and 0.5 μl of 1 M tris•HCl pH 8.3  then added on ice, and left for >5 min to ensure folding. This was added to 5 pmol each of primer and template and 50 pmol of each triplet pre-annealed in 5 μl water, then frozen on dry ice (10 min) and incubated at −7˚C in a R4 series TC120 refrigerated cooling bath ) to allow eutectic phase formation and reaction.Standard ribozyme activity assays (modified where specified) comprise 5 pmol of each ribozyme annealed in 2.5 μl water , with 2 μl of 1 M MgCl2, 50 mM tris•HCl pH 8.3). Supercooled reactions  led to a higher final reaction volume, but eutectic phase equilibration restored standard operational concentrations, also applicable to the four-fold-diluted extensions with the fragmented ribozyme , with pre-annealed primer/template/substrates (0.5/0.5/5 pmol) added in 0.75 μl water. These reactions, and preparative syntheses , with urea to a 6 M final concentration and a 10–20 fold molar excess over template of complementary competing oligonucleotide see  to preve+/γδε+, +, δ+, ε+, + synthesised in + synthesis was denatured directly from the ligation reaction in 60% formamide with excess EDTA.To avoid using potentially confounding competing oligonucleotide when purifying extension products, reactions with a biotinylated primer or template (stopped as above) could be purified by bead capture using MyOne C1 (Invitrogen) streptavidin-coated paramagnetic microbeads (using 5 μg pre-washed beads per pmol biotinylated RNA) in 0.5 × − 0.8 × bead buffer , 1 mM EDTA, 0.1% Tween-20). After washing twice in BB to remove unbound components, beads were incubated (1 min) in 25 mM NaOH, 1 mM EDTA, 0.05% Tween-20 to denature the duplexes . To reco2) for separation by native PAGE  2) kept at 4˚C in a circulator bath for 6–8 hr at 10 W), then SYBR Gold stained as below.For gel mobility shift assays , ribozymFluorescent primer extension products were detected using the appropriate laser wavelength on a Typhoon Trio scanner  ); gel densitometry allowed quantification of RNA synthesis efficiency. Gel contrasts in figures were linearly adjusted to optimize display of bands of differing intensities.The gel in +) alongside known amounts of the positive controls indicated, followed by SYBR-Gold staining. To purify, bead-eluted products were run similarly, and excised using UV shadowing. Products were then eluted from the gel fragments in 10 mM tris•HCl pH 7.4, and Spin-X column filtrate ) precipitated in 75% ethanol with 1 μl 1% glycogen carrier (omitted for β+). Recovered full-length product yields were calculated similarly to reaction yields for αβ+/δε+/γδε+, or using A260s for β+, δ+, ε+.Full-length product yields in the +/γδε+ fragments were sequenced by first ligating a 3’ adaptor (10 U/μl T4 RNA Ligase 2 truncated KQ in 1 × RNA ligase buffer (New England Biolabs (NEB), ) with 15% PEG-8000 and 2 μM AdeHDVLig at 10˚C overnight). These reactions were bound to MyOne C1 microbeads (ThermoFisher (Invitrogen)), washed with BB to remove unligated adaptor, and reverse transcribed (50˚C 30 min) with 1 μM HDVrec primer using Superscript III (Invitrogen). Beads were washed again then PCR amplified  using GoTaq HotStart master mix ) and 0.8 μM each of primers P3HDV, and P5Xα8 or P5Xγ7, for high-throughput sequencing  MiSeq or HiSeq) after PCR product agarose gel purification. β+ syntheses’ cDNAs were amplified with P3HDV and P5Xβ6.Purified ribozyme- and TGK-synthesized αβpppNNN on templates encoding CCC-XXX-CCC, where XXX were 12 different triplet sequences evenly exploring base composition and distribution . The 12 pairs were combined in 0.27M MgCl2/67 mM tris•HCl pH 8.3 on ice to discourage primer-template assortment . 36 pmol of each triplet in pppNNN  were added to a reaction vessel in 10.8 μl water, to which 5.4 μl of the primer/template/buffer mix was added followed by 7.2 pmol of ribozyme pre-annealed  in 1.8 μl water . Reactions were frozen and incubated (7 days at −7˚C) as described above.Each primer/template pair (0.45/0.525 pmol per reaction) was annealed in 4 mM MgClReactions were stopped with 3.6 μl 0.44 M EDTA and 10.5 pmol of each template's competing oligonucleotide , denatured with 6 M urea, and urea-PAGE separated. After alignment with a fluorescence scan of the gel, a region of the sample lane corresponding to primers extended by +4 to +14 nt was excised (encompassing 2–4 triplet additions), and extension products were eluted, precipitated in 77% ethanol with 1 μl 1% glycogen carrier, washed in 85% ethanol and resuspended in water.th reverse transcription mix, 0.8 μM each of primers P3HDV and P5GGGX) for sequencing as above (yielding 2 × 105 – 4 × 106 sequences per ribozyme assay).These extension products were 3’ adaptor ligated as for fragment sequencing. Products were reverse transcribed  and then PCR-amplified  were combined for purification and sequencing as above, to allow isolation of the ribozyme-mediated errors resulting from inclusion of the other 62 (61 for ACC) triplets in the reaction . The counts of cognate triplet (C) and each error triplet (E) in the positive control (p) reduced error counts in the experimental samples (x) to yield ribozyme-mediated error counts (Er) thusly: Er = Ex - Ep*(Cx/Cp)  remained uncorrected).After processing and 3’ adaptor trimming, sequences corresponding to primer extended by CCC +1–3 additional triplets were collated for analysis. Variations in upstream primer sequences see  allowed For each template, counts were then collated at the first/second/third positions to yield base-specific mutation rates for each position . Across pppGU, encoding UAC yielded just pppAC) or use oligonucleotides generated during transcription to re-initiate .Triplets (and some other short oligonucleotides) were prepared from NTPs by T7 RNA polymerase run-off transcription of a 5’ single-stranded DNA overhang downstream of a DNA duplex T7 promoter sequence. In most cases, the 5’ overhang encoded (was the reverse complement of) the desired oligonucleotide. These oligonucleotides were short enough to synthesise during the abortive initiation stage of transcription, attenuating sequence constraints on the first bases of the transcript. However, T7 RNA polymerase exhibited tendencies to skip the first (or even second) base , and reduced recruitment as initiators of products with bases not complementary to the introduced first position template base .These tendencies could be subverted by encoding additional first bases . This initiated the oligonucleotide at the second position where skipping tendencies were lower  in 1 × MegaShortScript kit buffer with 1.5 μl MegaShortScript T7 enzyme (ThermoFisher). Also present were 15 pmol of each DNA oligonucleotide forming the transcription duplex target ).Each 30 μl transcription reaction contained 72 nmol of each desired product base as an NTP (Roche) ); incorporation and migration were similar between the two, and their concentrations were calculated from A260nm by comparison to the A260nm of mixtures of the component ribonucleotides with UTP vs. U2STP. Triplets with 2’-fluoro, 2’-deoxy positions could also be transcribed, with lower efficiency, by substituting the corresponding triphosphate (Trilink Biotechnologies). The biotinylated pppGAU–Bio triplet used in γ segment synthesis ), quantified via by comparison to the A290nm of mixtures of the component ribonucleotides.3’-deoxy triphosphorylated ‘terminator’ triplets were transcribed as above but using a 3’ deoxynucleoside 5’ triphosphate (Trilink biotechnologies) for the last position, migrating faster during PAGE than the equivalent all-RNA triplet. Triplets with 2-thiouridine residues were transcribed as for their corresponding U, replacing UTP with Uynthesis  was tranLonger triphosphorylated oligonucleotides used in ribozyme self-synthesis were generated similarly, but using ~200 ng of fully double stranded DNA as a template. Candidate product bands were purified and the desired oligonucleotide identified by ribozyme-catalysed in-frame incorporation and, for some, fragment sequencing.Transcriptions were performed on ~15 ng/μl dsDNA using MegaShortScript enzyme and buffer (ThermoFisher) with 7.8 mM of each NTP, or, to yield a 5’ monophosphate on the product to avoid aberrant ligation, 10 mM GMP (guanosine monophosphate) and 2 mM of each NTP (‘GMP transcription’).dsDNA templates for some of these in  were gen2) to extend 5’ biotinylated RNA primers (0.75 μM) on DNA templates (1 μM) using 2.5 mM of each NTP . Biotinylated products were bead-purified as above.Some 5’ biotinylated RNAs were synthesized using the TGK polymerase   cycloaddition (3’ biotinylation of RNAs was achieved in two stages: 3’ azidylation (at 2 μM with 25 U/μl yeast poly-A polymerase (ThermoFisher) and 0.5 mM 2’-azido-2’-deoxycytidine triphosphate (Trilink Biotechnologies) for 1 hr at 37˚C) with subsequent acidic phenol/chloroform extraction and 75% ethanol precipitation, then copper-catalysed biotin-(PEG)addition  with subRound one libraries were synthesised by mutual extension of 4 nmol of oligonucleotides 1baN30 and 1GMPfo or 1GTPfo at 1 μM each in 1 × isothermal amplification buffer (NEB) with 250 μM each dNTP, annealed  before addition of 0.4 U/μl Bst 2.0 (NEB) and 30 min 65˚C incubation.15 molecules) were transcribed in 5 ml transcription reactions (36 mM tris•HCl pH 7.9 (at 25˚C), 1.8 mM spermidine, 9 mM DTT, 10.8 mM MgCl2, 2 mM each NTP, 1% 10 × MegaShortScript buffer, 2% 1:9 MegaShortScript:NEB T7 RNA polymerase, 37˚C overnight). These were treated with DNase, acid phenol/chloroform extracted and 73% ethanol precipitated prior to urea-PAGE purification, elution, filtering (Spin-X) and re-precipitation, yielding the 1GTP Zcore selection construct  derived from amplification of recovered PCR products (see later).After purification, 375 μg of each DNA . DNA encoding type 5s amplified with AACAt5s was used to generate reselection libraries by PCR amplification using primers TriGAA7GAAM and T5ba13N/T5ba20N/T5ba28N; 5 pmol of the three dsDNA products were transcribed to generate reselection constructs.For round 8, 700 pmol DNA was formed, with Tri3CUUQ amplifying round seven merged output (50 pmol), round seven merged output recombined by StEP   or 2 μM HOCUG (Rounds 8–18) with 30 U/ml T4 RNA Ligase 2 (NEB)). After incubation at 4˚C for 1 hr, these were stopped with 2.2 mM EDTA and acid phenol/chloroform treated.At the end of incubation the reaction was thawed on ice. To link the primer 3’ hydroxyl to the 5’ monophosphate of GMP constructs, selection constructs were buffer-exchanged directly after thawing using a PD-10 column (GE) in a cold room, into 3 ml ligation mix (optimised to prevent ligation over gaps) (2 mM MgClConstructs were then precipitated with glycogen carrier and 0.3 M sodium acetate in isopropanol (55%) before resuspension and denaturation . For the reselection rounds, constructs were then treated with polynucleotide kinase (NEB) before denaturation to resolve the HDV-derived 2’, 3’-cyclic phosphates and allow later adaptor ligation.Constructs were urea-PAGE separated alongside FITC-labelled RNA markers equivalent to successfully ligated constructs. The marker-adjacent gel region in the construct lane was excised, excluding the bulk unreacted construct (judged by UV shadowing). Biotinylated (primer-linked) constructs were eluted overnight into BB with 100 μg MyOne C1 beads. After 30 μm filtering )) of the supernatant to remove gel fragments, the beads were washed in BB then denaturing buffer  to confirm covalent linkage of construct to primer, before further BB washing and transfer (to a fresh microcentrifuge tube to minimize downstream contamination). At this stage in the reselection, 3’ adaptors were then ligated to bead-bound constructs as above for 2 hr (with buffer/enzyme added after bead resuspension in other reaction components including 0.04% Tween-20), and beads BB washed and transferred again.Bead-bound constructs were now reverse-transcribed using 1 μM RTri (or HDVRec for the reselection) by resuspension in a Superscript III reaction with added 0.02% Tween-20 (50˚C 30 min). Beads were BB washed and the RNA-bound cDNA 3’ end blocked by incubation with terminal deoxynucleotidyl transferase (ThermoFisher) and 0.2 mM dideoxy-ATP (TriLink) with 0.02% added Tween-20 (37˚C 30 min), and beads were BB washed and transferred again.cDNAs were eluted (10 μl 0.1 M NaOH 0.1% Tween-20 20 min), neutralized and plus strands regenerated with 0.2 μM rescue oligonucleotide in an IsoAmp II universal tHDA kit (NEB) reaction (65˚C 60 min) to read through the structured product region. Whilst at this temperature, reactions were stopped with 5 mM EDTA and one volume of BB with 50–100 μg beads to bind the nascent biotinylated plus strands at room temperature. These beads were then BB washed, NaOH washed again to discard cDNAs (and recover only correctly-primed plus strands), and washed and transferred again.260nm quantified and added to construct synthesis PCR in 3 × molar amount of the anticipated recovered RNA (judged by test extensions) that yielded it.Each 50 μg of beads were then subjected to plus strand recovery PCR in a 100 μl GoTaq HotStart reaction with 0.5 μM each RTri (or HDVrec for reselection) and RecInt (rounds 1–5)/RecIntQ (rounds 6–9)/RecIntL /RecIntQL (rounds 15–18). The product was agarose size-purified, AThis final PCR for construct transcription in the subsequent selection round used 1 μM of the indicated construct synthesis primer, plus 1 μM RTri (or HDVrt for the reselection), in GoTaq HotStart reactions or (where indicated in source data) the GeneMorph II kit for mutagenesis ).Conditions for selections are included as source data 1 for  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and James Manley as the Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: Nebojsa Janjic (Reviewer #3).Thank you for submitting your article "Ribozyme-catalysed RNA synthesis using triplet building blocks" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. The most important thing to improve is the written clarity of the manuscript. There was a lot of agreement between the reviewers' comments so we attach their full reviews below for your attention. All their concerns should be attended to.Reviewer #1:Attwater et al. report an advance in efforts to develop a robust self-replicating RNA by presenting an example of an RNA polymerase ribozyme (RPR) capable of transcribing RNA from structured RNA templates of substantial length with fairly good fidelity. This RPR uses a trinucleotide building block (in contrast to NTPs used by standard polymerases), that the authors speculate could have implications in the evolution of mRNA decoding by the ribosome.The major findings from this work are as follows:+1 which was used for downstream studies. This RPR could use structured hairpins as templates, even those with Tm >90oC that were intractable to previous RPRs that use NTPs as substrates. The authors demonstrate the efficiency of this RPR by transcribing both + and – strands of the Broccoli aptamer, a structured and fluorescent RNA.1) The authors exploit the ligase activity in the catalytic core of their previously reported RPR Z and evolve a suite of catalytic sequences capable of extending an RNA primer using RNA triplets as substrates. Through further iterations of in-vitro selection for triplet dependent RPRs, an RNA sequence emerged without any catalytic activity itself, but it enhanced triplet extension by these newly evolved RPR sequences (named t2 through t6) through a bimolecular interaction between the catalytically proficient sequence. Further rounds of selection using the cofactor t1 resulted in the most efficient triplet RPR, t5+1 RPR to replicate fragments of itself (both + and – strands), using triplet building blocks (and in some cases hexamers). The complete active RPR could be reconstituted from the five individual components of the RPR transcribed by the same RPR. Although this replicase ribozyme is still not able to replicate itself in its entirely, it does give rise to a functioning replicase by using building blocks as small as trimers, Previous efforts had primarily used the ligase activity of the RPRs to assemble oligonucleotides representing their sequence to assemble a 'replica' of itself. Considering the challenges of self-replication, even incremental advances in the right direction are significant.2) The authors used t5Overall this work introduces a new RPR system that brings the field closer to a self-replicating system, which is one of the cornerstones of Origin of life research. The authors explore properties of this replicase including fidelity and catalytic efficiency and explain the molecular basis for these properties. This work also introduces trinucleotides as plausible substrates on which ancient RNA-based replicases could have acted. Considering the relative ease of non-enzymatic polymerization of monomers into triplets, the presence of these triplets in a prebiotic setting seems plausible. The current work is detailed and the authors have explored multiple facets of the new system they have setup. This new RPR presents a major advance in terms of its ability to create a fully functional copy of itself . As such, this would be a great addition to the current understanding of RNA-based self-replication systems.1) Overall, the manuscript was very difficult to follow. It is up to the authors to make their presentation as simple and direct as possible. Given the availability of supporting information, there is no reason why the authors could not add schemes where appropriate to clarify their presentation, logic, and workflow.2) Regarding the dimerization, it is not clear how the authors implicated the 5'-hairpin to begin with, leading to the experiment that showing that a single mutation in that loop inhibits dimerization.3) The assertion that the two RNAs form a 1:1 complex is not quantitatively substantiated from Figure 3B.4) Considering their success in replicating structured hairpins and achieving primer free synthesis of an 18 nt strand (β+), it would be interesting (though not necessary for publication) to see how much of the Broccoli RNA could be transcribed without the addition of a primer sequence. If this is not possible, the authors could demonstrate the complete replication of a functional RNA of tractable length, like the Hammerhead ribozyme, to illustrate the ability of their trinucleotide RPR to replicate entire sequences of functional RNAs by exclusively using triplets as substrates.5) The fidelity analysis is based on testing 12 of the possible 64 trinucleotides, so one cannot be convinced of the generality of their observations and inferences. The authors need to underscore this in the discussion of fidelity. The authors show that increased concentrations of triplets help fidelity, however in the absence of the 'correct' triplet, it is likely that RPR will incorporate 'mismatch' triplets. The authors should comment on this aspect.Reviewer #2:1) In this paper, the authors demonstrate interesting and novel results that provide further evidence for RNA's capacity to act as prebiotic genetic material. The key feature associated with this study involved using trinucleotide triphosphates (triplets) rather than NTPs as substrates for a novel RNA polymerase ribozyme. The use of triplets allowed structured templates to be unfolded and in some cases avoided the need for primers, circumventing an ongoing obstacle to RNA self-replication. This study thus makes some significant steps towards identifying a truly self-replicating prebiotic biopolymer. Literature precedents are well-discussed, and the results are interpreted in a detailed and convincing manner. I would nonetheless encourage the authors to try to make these important results as accessible as possible to readers who are unfamiliar with in vitro evolution.Specific comments:2) Subsection “in vitro evolution of triplet polymerase activity”, last paragraph. The use of "in-ice evolution" (a technique developed by the authors) is reported. A sentence could be added describing why this technique is being used / why it is necessary.3) For certain experiments (e.g. subsection “Fidelity of triplet-based RNA synthesis”), random triplet pools were used. What kind of triplet pools were used in other sequence-copying experiments? Were they non-random? This needs to be more clearly articulated.4) "… we found that using modified substrates with a disrupted minor groove hydrogen bond acceptor at the 3rd position…" – What is the modification? This should be stated in the body-text.+1 ribozyme? Triplets reportedly incur a lower entropic cost than NTPs , but what would happen when a mixture of the two were used? Would both the NTPs and triplets be incorporated?5) What is the scope of substrates for the t5If these points are satisfactorily addressed, then I am in favour of acceptance.Reviewer #3:Attwater et al. used an in vitro evolution method to enhance the trace amount of ligase activity with short (3-nucleotide) substrates inherent in the truncated version of the Z RPR ribozyme . This was achieved by adding a random region of 30 nucleotides at the 3' end of Zcore and a template at the 5' end, followed by selection for variants able to extend a primer hybridized to the 5' template with two 5'-triphosphate triplets and ultimately ligate the extended primer to the 5' terminal triphosphate. After 7 rounds of activity enrichment, the authors identified a sequence that represented 25% of the activity-enriched pool called "type 0" that, upon truncation to its minimal active sequence ("0core"), was able to catalyze multiple cycles of templated ligation with triplet substrates, including the ability to extend thought double-stranded regions of the template by cooperative invasion through the secondary structure of the template .+1. t5+1 was able to catalyze the extension through templates with Tm values of 93 ⁰C, and was capable of generating both the functionally active Broccoli RNA aptamer and its complementary template. Finally, t5+1 was able to replicate its own t5 sequence split into five segments, mostly from triplets, with some help from longer substrates (hexamers) needed for two of the segments, thus falling short of having the capacity for a full replication cycle, but nonetheless, exhibiting impressive catalytic efficiency.After additional 14 rounds selection, the composition of the pool showed considerable changes: type 0 sequence had disappeared and was replaced by six new types of sequences, types 1-7. Remarkably, the most prevalent type 1 sequence (50% of the pool) was completely inactive and types 2-6 were less active than the activity enriched pool. This mystery was solved by the observation that a truncated version of the type 1 sequence, while being inactive, can form augment the activity of type 2-6 ribozymes by forming heterodimeric complexes. In the presence of type 1 sequence, the best stand-alone ribozyme, type 5, no longer required the template to be tethered to the 5' end of the type 5 ribosome, in a remarkable example of an intermolecular complex between the dimeric type 1:type5 ribozyme, the untethered primer template, and the triplet substrates. Further optimization of the type 5 ribozyme by re-randomization of the previously fixed 3' end sequences and reselection in the presence of minimal type 1 sequence  resulted in the final heterodimeric holoenzyme called "t5+1 can extend primers in both the canonical 5'-3' as well as the reverse 3'-5' direction raises the potential for primer extension from short sequences in a primer-free manner, therefore potentially solving the "primer problem" (depletion of longer but presumably sparse primers needed for replication in prebiotic conditions). Impressive overall positional fidelity of more than 97% was achieved by counter-selection for mispairing triplets with a clever inclusion of excess 3'-deoxy terminator triplets during late rounds of selection. Attwater et al. mapped the region responsible for fidelity to the 3' domain of t5. Further reduction in mis-incorporation comes from cognate triplet-triplet interactions have a self-correcting feature of countering the effect of triplets with strong base-pairing tendency.The surprising observation that t5eLife. [Editors’ note: this reviewer’s minor comments have not been included.]The authors make a strong case that triplets represent relatively short and yet powerful substrates able to invade highly stable structural features in RNA templates. The evolution of a heterodimeric ribozyme capable of generating functional RNAs with triplet substrates represents a major accomplishment that advances the plausibility of RNA-catalyzed RNA replication in prebiotic environment. Despite its considerable length, as well as its remarkable breadth and scope, the manuscript reads like a riveting novel. It is a tour de force scientific accomplishment, and as such, it will be of considerable interested to a wide audience of readers of  Reviewer #1:[…] 1) Overall, the manuscript was very difficult to follow. It is up to the authors to make their presentation as simple and direct as possible. Given the availability of supporting information, there is no reason why the authors could not add schemes where appropriate to clarify their presentation, logic, and workflow.We apologize for the poor presentation and have made several changes to enhance clarity. These include textual changes to reduce ambiguity and lay out our reasoning more thoroughly as well as explanatory schemes of ribozyme activity in Figure 1 and Figure 1—figure supplement 1. Furthermore, we have redesigned Figures 3 and 7, and now provide an additional dataset presenting substrate concentration-dependent error rates in a more straightforward manner in Figure 9—figure supplement 1.The manuscript structure is complicated by the multiple stages of RNA evolution and characterization involved in the project, and to help navigate the progress of our ribozyme development, we now provide the new Figure 4—figure supplement 1 detailing all the stages of evolution and engineering of the different triplet polymerase ribozymes to provide a “roadmap”.2) Regarding the dimerization, it is not clear how the authors implicated the 5'-hairpin to begin with, leading to the experiment that showing that a single mutation in that loop inhibits dimerization.We welcome the opportunity to clarify this. We had originally noticed the ‘cap-’ mutation in the most abundant isolated type 1 clones, and when testing this in different constructs observed that it quenched type 1 enhancement pointing towards the 5’ hairpin region as the critical interaction site. We now refer to this in the manuscript text.Our knowledge about this dimerization interface is currently limited and we have therefore generally refrained from speculation. However, it seems possible that the ‘cap-’ mutation was enriched in these type 1 sequences to diminish homodimerisation of type 1 that may interfere with heterodimerisation with active triplet polymerase in the selection pool.3) The assertion that the two RNAs form a 1:1 complex is not quantitatively substantiated from Figure 3B.We now include quantitative data on the stoichiometry of the complex as observed by gel shift (EMSA) titration in Figure 3B, and activity tests in Figure 3—figure supplement 1. These support a 1:1 stoichiometric complex in the active ribozyme as expected from the final selection pool composition.4) Considering their success in replicating structured hairpins and achieving primer free synthesis of an 18 nt strand (β+), it would be interesting (though not necessary for publication) to see how much of the Broccoli RNA could be transcribed without the addition of a primer sequence. If this is not possible, the authors could demonstrate the complete replication of a functional RNA of tractable length, like the Hammerhead ribozyme, to illustrate the ability of their trinucleotide RPR to replicate entire sequences of functional RNAs by exclusively using triplets as substrates.The reviewer highlights an important challenge that such ribozymes must ultimately face – copying without primers both strands of functional RNAs to achieve their complete replication using only short oligonucleotide substrates. With the current triplet polymerase ribozyme, while primer-initiated synthesis is almost general (as we show by synthesizing both + and – strands of the Broccoli RNA aptamer), we cannot currently achieve primer-free synthesis of all sequences. The latter includes Broccoli where yields of full-length RNA are poor using triplets alone. This may reflect inefficient initiation of synthesis or poor upstream inclusion of some 5’ terminal triplets.Nevertheless, the current triplet polymerase ribozyme can achieve this for some functional sequences, most relevantly from t5 itself. To demonstrate this, we have now included data on the primer-free synthesis and replication of the Υ segment. Specifically, we describe primer-free synthesis using triplets alone of the γ+ strand segment with sufficient yield to use this ribozyme-synthesized RNA as the template for primer-free γ- strand synthesis, demonstrating complete primer-free sequence replication using only triplets. These new data are now included as Figure 7C, replacing the (now in our view redundant) primer-free γ+ syntheses.5) The fidelity analysis is based on testing 12 of the possible 64 trinucleotides, so one cannot be convinced of the generality of their observations and inferences. The authors need to underscore this in the discussion of fidelity. The authors show that increased concentrations of triplets help fidelity, however in the absence of the 'correct' triplet, it is likely that RPR will incorporate 'mismatch' triplets. The authors should comment on this aspect.We measured fidelity of triplet incorporation with all64 triplet substrates present but in the context of 12 defined template triplet sequences flanked by conserved CCC incorporation sites. These were chosen to be compositionally balanced, including both GC-rich and AU-rich template triplets, to provide fair estimates of ribozyme fidelity, as well as an effective tool to compare the fidelities of different ribozymes.This assay was designed to allow us to separate incorporation fidelity (i.e. discrimination among 64 triplets for decoding a defined anti-triplet) from each ribozyme’s extension capabilities by providing a uniform, tractable sequence context.The important qualification regarding this assay, we feel, is therefore that while it provides an accurate representation of fidelity in a defined sequence context (triplet-triplet junction), it may not provide a comprehensive picture of triplet polymerase fidelity performance in all sequence contexts. We now provide this caveat in the text.Reviewer #2:1) In this paper, the authors demonstrate interesting and novel results that provide further evidence for RNA's capacity to act as prebiotic genetic material. The key feature associated with this study involved using trinucleotide triphosphates (triplets) rather than NTPs as substrates for a novel RNA polymerase ribozyme. The use of triplets allowed structured templates to be unfolded and in some cases avoided the need for primers, circumventing an ongoing obstacle to RNA self-replication. This study thus makes some significant steps towards identifying a truly self-replicating prebiotic biopolymer. Literature precedents are well-discussed, and the results are interpreted in a detailed and convincing manner. I would nonetheless encourage the authors to try to make these important results as accessible as possible to readers who are unfamiliar with in vitro evolution.As discussed above, we have made a significant number of changes to the manuscript in order to present our results more clearly.These include textual changes to improve clarity and remove ambiguities. We have also made an effort to lay out our experimental progress and reasoning more thoroughly, using explanatory schemes of ribozyme activity in Figure 1 and Figure 1—figure supplement 1, redesigning Figures 3 and 7, and providing an additional more straightforward dataset in Figure 9—figure supplement 1. We hope this will make the manuscript easier to read and more accessible to the general reader.Specific comments:2) Subsection “in vitro evolution of triplet polymerase activity”, last paragraph. The use of "in-ice evolution" (a technique developed by the authors) is reported. A sentence could be added describing why this technique is being used / why it is necessary.We have expanded our description of the mechanisms of in vitro evolution and discuss in more detail the reasoning behind our use of eutectic ice phases and in-ice evolution. As the properties of ice as a medium for RNA-catalyzed RNA synthesis, replication and evolution are discussed at length in the cited references, a fairly brief discussion must suffice. We hope nevertheless that this will now clearly illustrate our rationale in using this technique in our work.3) For certain experiments (e.g. subsection “Fidelity of triplet-based RNA synthesis”), random triplet pools were used. What kind of triplet pools were used in other sequence-copying experiments? Were they non-random? This needs to be more clearly articulated.We generally list the triplet substrates used for different experiments but apologise for any ambiguity.In general for preparative syntheses we have avoided using pools of random triplets, providing instead a set of defined triplets as specified by the template sequence to minimise substrate consumption. We have however verified that random triplet pools can be used for key triplet based RNA synthesis experiments including hairpin invasion  and ribozyme segment synthesis , as well as in experiments on ribozyme fidelity . We now explicitly state the substrate composition in the text and figure legends for all experiments described.4) "… we found that using modified substrates with a disrupted minor groove hydrogen bond acceptor at the 3rd position…" – What is the modification? This should be stated in the body-text.We apologize for this omission and have amended this. We now state in the text that uracil was replaced by 2-thio uracil in the triplets.+1 ribozyme? Triplets reportedly incur a lower entropic cost than NTPs , but what would happen when a mixture of the two were used? Would both the NTPs and triplets be incorporated?5) What is the scope of substrates for the t5This is an interesting suggestion considering that abiotically-generated substrate pools would likely include a mixture of different length substrates including mono- and dinucleotides. We have performed additional experiments to explore this and now include data on their incorporation during segment synthesis in Figure 6—figure supplement 5.+1 does incorporate both dinucleotide triphosphates and NTPs, but with reduced efficiency. Furthermore, they appear to serve as poor substrates for segment synthesis with triplets incorporated much more efficiently. Increasing amounts of NTP and dinucleotide substrates vs. triplets  do lead to diversification of extension products outside the initial triplet register, but do not notably affect the total amount of ligation performed.In summary, t5As seen in Figure 6—figure supplement 2, more length-heterogenous substrate pools (including longer oligomers) may offer both advantages and disadvantages, but we feel that a more systematic investigation of diverse substrate pools and their effect on yield and fidelity of RNA-catalyzed RNA replication goes beyond the scope of the current manuscript.


The hepatitis C virus RNA genome possesses a variety of conserved structural elements, in both coding and non-coding regions, that are important for viral replication. These elements are known or predicted to modulate key life cycle events, such as translation and genome replication, some involving conformational changes induced by long-range RNA–RNA interactions. One such element is SLVI, a stem-loop (SL) structure located towards the 5′ end of the core protein-coding region. This element forms an alternative RNA–RNA interaction with complementary sequences in the 5′ untranslated regions that are independently involved in the binding of the cellular microRNA 122 (miR122). The switch between ‘open’ and ‘closed’ structures involving SLVI has previously been proposed to modulate translation, with lower translation efficiency associated with the ‘closed’ conformation. In the current study, we have used selective 2′-hydroxyl acylation analysed by primer extension to validate this RNA–RNA interaction in the absence and presence of miR122. We show that the long-range association (LRA) only forms in the absence of miR122, or otherwise requires the blocking of miR122 binding combined with substantial disruption of SLVI. Using site-directed mutations introduced to promote open or closed conformations of the LRA we demonstrate no correlation between the conformation and the translation phenotype. In addition, we observed no influence on virus replication compared to unmodified genomes. The presence of SLVI is well-documented to suppress translation, but these studies demonstrate that this is not due to its contribution to the LRA. We conclude that, although there are roles for SLVI in translation, the LRA is not a riboswitch regulating the translation and replication phenotypes of the virus. Hepacivirus in the family Flaviviridae and, despite the recent development of novel and effective therapies (Hepatitis C virus (HCV) belongs to the genus herapies , infectsherapies . Like otherapies . The virThe limited coding capacity of small RNA viruses necessitates the genome being multi-functional, with control of key events in the replication cycle being influenced by the presence of RNA secondary structures involved in either RNA–RNA interactions and/or the recruitment of viral or host factors . Domains SLII-IV of the 5′UTR, along with the first 12–30 nucleotides of the core protein coding sequence, form the internal ribosome entry site (IRES) involved in cap-independent initiation of viral translation . Two-addIntriguingly, a sequence spanning the miR122 seed sites has also been demonstrated to anneal to complementary sequences that form the basal stem of SLVI. Mutation analysis of sequences in these regions had previously suggested a role for such an interaction in controlling translation . SubsequIn this study, we have utilised a panel of defined 5′UTR and SLVI mutations to investigate a translation-modulating role for the switch from the ‘closed’ to the ‘open’ conformation of this potential molecular switch. Selective 2′-hydroxyl acylation analysed by primer extension SHAPE;  analysis2, in Dulbecco’s modified minimal essential medium (DMEM)  supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS), 1% non-essential amino acids and 2 mM l-glutamine . HeLa cells were maintained at 37 °C, 5% CO2, in DMEM supplemented with 10% (v/v) FBS. Cells were seeded in 24-well plates at 1 × 105 cells/well for translation assays, or 0.8 × 105 cells/well for replicon assays, in DMEM-FBS 18 h prior to transfection.Huh 7.5 human hepatocellular carcinoma cells were maintained at 37 °C, 5% COTransfections were carried out with 500 ng RNA and two μl of Lipofectamine 2000  as per manufacturers’ instructions. In addition, for translation assays five ng of a capped and polyadenylated Renilla luciferase RNA was added as a transfection control. Transfection media was replaced at 4 h with fresh DMEM-FBS. Cell lysates were harvested for analysis at 4 h (translation assay) or 4, 21, 28 and 45 h (replicon assay). Briefly, cells were washed twice with PBS and lysed with 0.1 ml/well Glo Lysis buffer  for 15 min with shaking. Luciferase readings were determined with Dual-Luciferase (translation assays), or Luciferase (replicon assays), Assay System Kits  as per manufacturers’ instruction.A variety of numbering or naming schemes have been used to specify RNA stem-loop structures in the HCV genome. Although the most logically extendable scheme involves numbering structures according to the location of the 5′ nucleotide within the relevant genome region in an H77 reference sequence , for conAgeI/XbaI fragment was ligated into similarly digested pJFH1-luc-trans:ΔNS5B to generate pJFH1-CEtrans.A JFH-1 based translation-only reporter construct containing a core-extended (CE) sequence, to include SLV and VI—designated pJFH1-CEtrans—was generated via modification of pJFH1-luc-trans:ΔNS5B described previously . Overlap25A and G28C were introduced into the miR122 S1 site to generate pJFH1-CEtrans-S1 (F:5′-GGCGACACACCCCCATGAATCACTC-3′ and R:5′-GAGTGATTCATGGGGGTGTGTCGCC-3′). To disrupt the SLVI structure point mutations C436G and A439U (F:5′-CCAGATCGTTGGGGGTATACTTGTTGC-3′ and R:5′-GCAACAAGTATACACCCCCAACGATCTGG-3′), and/or U496A and G499C (F:5′-CGACAAGGAAAACATCCGAGCGGTCCCAGC-3′ and R:5′-GCTGGGACCGCTCGGATGTTTTCCTTGTCG-3′), were introduced into the 5′ and/or 3′ stem of SLVI to generate pJFH1-CEtrans-L, pJFH1-CEtrans-R and pJFH1-CEtrans-L/R, respectively. Mutants pJFH1-CEtrans-S1/L, pJFH1-CEtrans-S1/R and pJFH1-CEtrans-S1/L/R, containing both the miR122 S1 and SLVI mutations were generated by site-directed mutagenesis of pJFH1-CEtrans-L, pJFH1-CEtrans-R and pJFH1-CEtrans-L/R with the pJFH1-CEtrans-S1 primers described above. All mutations were confirmed by sequence analysis.Mutations at miR122 seed site 1 (S1) or/and SLVI were introduced into pJFH1-CEtrans using the QuikChange II site-directed mutagenesis kit as per manufacturers’ instructions . To disrupt miR122 binding, point mutations USbfI/NotI fragment from pJFH1-5′UTR-Con1b-rep containing the JFH-1 5′UTR, poliovirus IRES and Firefly luciferase, was inserted into similarly digested pJFH1-EMCV to generate pJFH1-rep. An SbfI/PmeI fragment of pJFH-1-CEtrans was inserted into similarly digested pJFH1-rep to generate the core-extended JFH-1 replicon, pJFH1-CErep. Mutations at miR122 S1 and/or SLVI were introduced through SbfI/PmeI digestion of the appropriate pJFH1-CEtrans plasmid, and ligation into pJFH1-CErep. This resulted in replicon constructs pJFH1-CErep-S1, pJFH1-CErep-L, pJFH1-CErep-R, pJFH1-CErep-L/R, pJFH1-CErep-S1/L, pJFH1-CErep-S1/R and pJFH1-CErep-S1/L/R.A JFH-1 replicon, containing the extended core sequence as above, and designated pJFH1-CErep, was designed based on Con1b-luc-rep . OverlapGGGUGUGUGACAAUGGUGUUUGU-3′) were synthesized by Integrated DNA Technologies along with an RNA oligonucleotide corresponding to nucleotides 1–50 of the JFH-1 strain of HCV (JFH11–50).Native (miR122), complementary (miR122-Comp) and S1 mutant (S1-miR122) miR122 RNAs  was duplexed with miR122-Comp (10 mM) in a final concentration of 100 mM HEPES, five mM MgCl1–50 RNA (10 pmol) was heated for 5 min at 65 °C followed by cooling to 35 °C for 1 min in a 5 μl volume containing 100 mM HEPES (pH 7.6), 100 mM KCl and five mM MgCl2. miR122 RNAs were added at molar ratios of 0, 0.5, 1, 1.5, 2, 3 and 5 and incubated for a further 30 min at 37 °C. An equal volume of loading dye  was added and RNA complexes separated by non-denaturing gel electrophoresis  at 150 V, 4 °C, 3 h using a BioRad MiniProtean III gel system. Gels were stained with SYBR Gold  and RNA visualized using a Typhoon FLA 9500 .For electrophoretic mobility shift assays (EMSA) JFH1BspHI (for translation templates) or terminated with a 3′ cis-acting ribozyme from an MluI linearized template (for replicon templates). Newly transcribed RNA was column-purified using a GeneJET RNA Purification Kit .RNA transcripts were synthesized using a HiScribe™ T7 High Yield RNA Synthesis Kit (NEB), as per manufacturers’ instructions, with one μg of DNA template linearized with 2 folding buffer . Samples were allowed to refold at 37 °C for 20 min before being divided in half and incubated with either one μl of 100 mM N-methylisatoic anhydride (NMIA) dissolved in DMSO, or one μl of DMSO, at 37 °C for 45 min. For reactions in the presence of miR122 a three molar excess of miR122 was added prior to addition of folding buffer. Modified RNAs were column-purified using a GeneJET RNA Purification Kit  to remove miR122 prior to reverse transcription (RT).A total of 10 pmol of translation construct-derived RNA transcripts were prepared in 10 μl of 0.5× Tris-EDTA (pH 8.0) (TE), denatured at 95 °C for 3 min and incubated on ice for 3 min prior to addition of six μl of either a five mM or 10 mM MgCl32P]-ATP  at 37 °C for 30 min followed by heat inactivation at 65 °C for 20 min. Radiolabelled primers were purified by separation on Sephadex G-25 Quick Spin Oligo Columns .A total of 60 μM of primer was incubated with 10 units of T4 polynucleotide kinase (NEB), two μl of supplied 10× buffer and 10 μl γ-[N-methylisatoic anhydride- or DMSO-treated RNA in 0.5× TE (10 μl) was mixed with three μl of radiolabelled primer, denatured at 95 °C for 5 min, annealed at 35 °C for 5 min and chilled on ice for 2 min. RT mix (six μl) was added  and samples incubated at 55 °C for 1 min prior to addition of one μl of SuperScript®III  and further incubation at 55 °C for 30 min. The RNA template was degraded by addition of one μl of 4M NaOH and incubation at 95 °C for 5 min before addition of 29 μl of acid stop mix  and further incubation at 95 °C for 5 min. Dideoxynucleotide (ddNTP) sequencing markers were generated by the extension of unmodified RNA with addition of two μl of 20 mM ddNTP  prior to addition of RT mix. The cDNA extension products were separated by denaturing electrophoresis (7% (19:1) acrylamide:bisacrylamide, 1× TBE, 7M urea) at 70 W for 3–5 h depending on product sizes to be analysed. Gels were visualised with a phosphorimager (Typhoon FLA 9500) and densitometry analysis carried out with ImageQuant TL 8.1 software . Normalised reactivities indicating exposure of nucleotides in predicted RNA structures were calculated as described previously  and U496A and G499C in the 3′ stem of SLVI (designated ‘R’ mutants) independently prevented base pairing of the basal stem of SLVI. Both C436 and A439 are implicated in the formation of the ‘closed’ structure and consequently, ‘L’ mutants were predicted to additionally prevent the LRA due to disruption of the complementarity with the miR122 seed site 1 (S1). Conversely, ‘R’ mutants would free the 5′ sequences forming the basal stem of SLVI to contribute solely to formation of the ‘closed’ structure. However, since formation of the ‘closed’ structure would also be dependent on the S1 site being unoccupied by miR122, we also introduced substitutions into the latter (at positions U25A and G28C) that were predicted to prevent miR122 binding and at the same time would restore complementarity with the ‘L’ mutations in SLVI . With the addition of miR122 to unmodified JFH11–50 we observed the expected two complexes with reduced mobility, representative of binding of miR122 to both S1 and S2 seed sites. Saturation of both seed sites was achieved upon addition of a 3:1 molar ratio of miR122:JFH11–50  and it is not possible to deduce the precise structure from the NMIA-reactivity plots.We first analysed those templates predicted to preferentially form the ‘open’ conformation . Modific of SLVI . NMIA-re35 motif  and 4D. 35 motif  and 4D. 434G435 motif, as well as the overall lack of reactivity in the 3′ basal stem of SLVI that would be expected . In contrast, JFH1-CEtrans-S1/L, shown to preferentially form the ‘closed’ structure, did not show the predicted reduction in translation and was unchanged from parental JFH1-CEtrans. Similarly, despite small increases and decreases in translation levels of JFH1-CEtrans-L/R and JFH1-CEtrans-S1/L/R, respectively, these were not significantly different when compared to the parental template. This suggests that, even though the wild type SLVI structure is not fully restored, with reduced base pairing remaining in the basal 5′ stem, this did not impact translation.We therefore compared translation from JFH1-CEtrans with selected modified templates that had shown a distinct conformation in SHAPE analysis. We selected JFH1-CEtrans-S1 as a representative of the ‘open’ conformation due to the SHAPE analysis most closely matching the SLVI structure observed for parental JFH1-CEtrans. For the ‘closed’ conformation we were limited to the study of JFH1-CEtrans-S1/L, as the only modified template in which the LRA was demonstrated. In addition, we investigated JFH1-CEtrans-L/R and JFH1-CEtrans-S1/L/R, for which the SHAPE reactivities suggested that the 5′ and 3′ stem mutations did not recapitulate wild type SLVI as expected due to increased reactivity in the 5′ basal stem, but neither were they representative of the ‘closed’ conformation  and 5B. The limited changes in phenotype observed strongly suggested that the difference observed between JFH1-luc-trans:ΔNS5B and JFH1-CEtrans is due to an as yet unidentified role for SLV and VI, and is not miR122 or LRA dependent. In the presence of miR122 both these templates exhibit the ‘open’ conformation as determined by SHAPE analysis  and 4A. p < 0.01)  . This is < 0.01) . The add < 0.01) .These results demonstrate that, while miR122 binding has a profound effect on the replication of the HCV genome, neither replication nor translation phenotypes are significantly influenced by modifications to SLVI that preferentially form the ‘open’ or ‘closed’ conformations of the LRA.How are the competing events of single-stranded, positive-sense, RNA virus translation and replication separated? At least early in the replication cycle, before compartmentalization into membrane-bound replication vesicles, these must involve interaction of the translating ribosome or the viral polymerase with the same template. One strategy, typified by poliovirus, requires the accumulation of one or more viral translation products to initiate genome replication . In thisThe HCV genome is extensively structured  and a nuTo investigate the structure and function of the ‘open’ and ‘closed’ conformations in greater detail we have mapped the native structure using SHAPE in the presence and absence of miR122. We have additionally extensively mutagenised sequences implicated in formation of both the ‘open’ and ‘closed’ conformations, mapped their structure by SHAPE and investigated the resulting influence on translation and genome replication.Although the strong stem-loop (SLI) in the 5′UTR confounded SHAPE interrogation of sequences forming the S1 site of miR122 binding, those contributing to the basal stem of SLVI were readily mapped. Having determined the influence on miR122 binding of mutations in the S1 site  we inferHowever, incorporation of 5′ (L) and 3′ (R) mutations to SLVI did lead to structural changes within the stem-loop that were not predicted by mfold. With the exception of JFH1-CEtrans-S1/L, which clearly adopts the ‘closed’ conformation, all the tested substitutions to the basal stem of SLVI increased the NMIA-reactivity of the structure  and 5, iNotwithstanding the potential for the LRA forming during HCV infection in vivo, we went on to analyse HCV translation and replication from templates in which we engineered the ‘open’ or ‘closed’ conformations by mutagenesis of the complementary pairing critical for LRA formation. We reasoned that, if dramatic differences were observable, this might indicate a role for the LRA and our ability to detect the ‘closed’ structure in vitro might not be representative of conditions in vivo. For example, cellular proteins could influence the formation of the LRA or miR122 may be limiting or absent at certain stages of the replication cycle.We investigated translation from a core-extended template transfected into Huh 7.5 cells. As expected from previous studies , extensi10 at 28 h post-transfection  Predicted structure of JFH1-CEtrans in the absence of miR122 binding generated using mfold prediction software. Structure shows the formation of the LRA with the 5’ basal of stem of SLVI base paired with complementary nucleotides of the miR122 binding site in the 5’UTR. Predictions were computed from sequence in which the nucleotides comprising SLII-V were represented by a series of n’s to simplify the folding. dG = −52.80 (B) Predicted structure of JFH1-CEtrans bound to one molecule of miR122 generated using bifold prediction software. Structure shows miR122 bound to seed site 1, blocking LRA formation, and the resulting SLVI structure. Predictions were computed from sequence in which the nucleotides comprising SLII-V were represented by a series of x’s to simplify the folding. dG = −66.7. Blue and yellow highlighted sequences show the S1 and 5’ SLVI sequences respectively, which overlap in the miR122:S1 and S1:SLVI binding motifs.Click here for additional data file.10.7717/peerj.5870/supp-2Supplemental Information 22 folding buffer, and is included for comparison of reactivities to a demonstrated ‘open’ conformation. A maximum negative reactivity was set at -0.1. Data was derived from one replicate folding reaction.SHAPE reactivities are shown for JFH1-CEtrans plus miR122 when folded in a reaction buffer containing 10 mM MgCl2, with predicted (P) and observed (O) conformations given top right below template name. Black bars show normalised SHAPE reactivities of nucleotides 427–447 and 487–507, encompassing the 5’ and 3’ basal stems of SLVI respectively. Nucleotides with a reactivity of <0.4 are considered unreactive and therefore base-paired. Shaded regions highlight nucleotides of importance in determining ‘open’ or ‘closed’ conformations: specifically the 5’ G434G435 motif and 3’ nucleotides 494-507. The superimposed red line indicates the exposure of JFH1-CEtrans plus miR122 in 5 mM MgClClick here for additional data file.10.7717/peerj.5870/supp-3Supplemental Information 3Click here for additional data file.


Trypanosoma brucei, the causative agent of sleeping sickness, cycles between an insect and a mammalian host. Here, we investigated the presence of pseudouridines (Ψs) on the spliceosomal small nuclear RNAs (snRNAs), which may enable growth at the very different temperatures characterizing the two hosts. To this end, we performed the first high-throughput mapping of spliceosomal snRNA Ψs by small RNA Ψ-seq. The analysis revealed 42 Ψs on T. brucei snRNAs, which is the highest number reported so far. We show that a trypanosome protein analogous to human protein WDR79, is essential for guiding Ψ on snRNAs but not on rRNAs. snoRNA species implicated in snRNA pseudouridylation were identified by a genome-wide approach based on ligation of RNAs following in vivo UV cross-linking. snRNA Ψs are guided by single hairpin snoRNAs, also implicated in rRNA modification. Depletion of such guiding snoRNA by RNAi compromised the guided modification on snRNA and reduced parasite growth at elevated temperatures. We further demonstrate that Ψ strengthens U4/U6 RNA–RNA and U2B"/U2A’ proteins-U2 snRNA interaction at elevated temperatures. The existence of single hairpin RNAs that modify both the spliceosome and ribosome RNAs is unique for these parasites, and may be related to their ability to cycle between their two hosts that differ in temperature.The parasite  O-methylation (Nm) and pseudouridinylation (Ψ). These modifications are guided by the C/D or H/ACA small nucleolar RNAs (snoRNAs), which guide Nm and Ψ, respectively  are 2′-ectively . Ψ increectively . Recent ectively .Whereas the modifications on rRNAs are performed in the nucleolus, guiding of modifications on spliceosomal U1, U2, U4 and U5 snRNAs as well as snRNP assembly takes place in the Cajal bodies (CB). These modifications are guided by small Cajal-body RNA species (scaRNA) that are bound by WDR79 protein . snRNA mTrypanosoma brucei is an important parasite that cycles between two hosts; such cycling requires major adaptation to changes in temperature and nutrient levels  donates a 5′ exon to all mRNAs , the homologue of human WDR79  of T. brucei 427 (cell line 131–514) was aerobically cultivated at 37°C under 5% CO2 in HMI-9 medium supplemented with 10% fetal calf serum, 2 μg/ml G418 and 2.4 μg/ml phleomycin (Procyclic form (PCF) Stem-loop constructs were generated to silence MTAP and snoRNA, using primers listed in N-cyclohexyl-N-β-(4-methylmorpholinium) (CMC) in bicine buffer  at 37°C for 20 min. Excess CMC was removed by ethanol precipitation. To remove CMC groups attached to G and U, the CMC-treated RNA was subjected to alkali hydrolysis with Na2CO3  at 37°C for 4 h, as previously described   Figure . RNA ext) Figure .To perform small RNA Ψ-seq, an adaptor was ligated to the 3′ end of the PRS RNA (without fragmentation) or total RNA (upon fragmentation) before and after CMC treatment, and cDNA was prepared using AffinityScript reverse transcriptase (Agilent). The cDNA was then ligated to an adaptor, PCR amplified, and after size selection on E-Gel EX (Invitrogen), the samples were sequenced in an Illumina NextSeq machine in paired end mode (40 million reads for each sample).http://hannonlab.cshl.edu/fastx_toolkit), and reads of 15 bases or less were discarded from subsequent analysis. The remaining reads were mapped to the T. brucei genome (TriTrypDB-2.5 http://tritrypdb.org/common/downloads/release/) using SMALT v0.7.5  with the default parameters. Only properly paired partners were retained. Each read pair was ‘virtually’ extended to cover the area from the beginning of the first read to the end of its partner. For each base, the number of reads initializing at that location as well as the number of reads covering the position were calculated. A combination of Bedtools (http://bedtools.readthedocs.io/en/latest/) and in-house Perl scripts was used to calculate the Ψ-ratio and Ψ-fc (fold change), as previously described.The 42 bp sequence reads obtained from the Illumina Genome Analyzer were first trimmed of Illumina adapters using the FASTX toolkit (2) was computed as the log2-fold change of the Ψ-ratios in (–CMC) versus the (+CMC) samples (2) of 1.15 or greater , a Ψ-ratio of CMC treated sample >0.01, and with a minimum of 800 reads initiating at the (n + 1) nucleotide. We applied this threshold to each PCF and BSF sample. Our small RNA Ψ-seq protocol calls a uridine as Ψ only if it was detected in at least five independent biological replicates, and was also reduced under CBF5 silencing experiments. We then merged the positions to generate a list of all positions meeting the criteria (2) (BSF/PCF) >1.3  in at least two independent biological replicates . This was performed for both (–CMC) and (+CMC) samples. The Ψ-fc. The mixture was incubated at 37°C for another 2 h. The reaction was analyzed using LC–MS/MS. The nucleosides were separated by ultra-performance liquid chromatography (Vanquish UHPLC Thermo Fisher scientific) on a Hyprsil GOLD aQ column (Thermo Scientific 25303-152130), and then detected by Q Exactive™ HF hybrid quadrupole-Orbitrap mass spectrometer (Thermo Scientific) in Parallel Reaction Monitoring Mode (PRM). The mass transitions of m/z 243.06 to 153.03, 183.04 (pseudouridine), m/z 243.06 to 110.02, 82.02 (U) in the negative mode, and mass transitions of m/z 268.10 to 136.06, (A), m/z 284.1 to 152.06 (G) and m/z 244.1 to 112.05 (C) in the positive mode were monitored and recorded. Concentrations of nucleosides in RNA samples were deduced by fitting the signal intensities using standard curves.RNA (300 ng) was digested using 4U of P1 nuclease (Sigma N8630) in 25 mM NaCl 2.5 mM ZnCl9 cells were prepared and loaded on a Superdex 200 gel filtration column (Amersham BioSciences), equilibrated with 20 mM HEPES (pH 7.9), 10 mM MgCl2, 150 mM KCl, at a flow rate of 0.5 ml/min. Fractions of 0.5 ml were collected. RNA and protein from every second fraction were analyzed by western and northern blotting. The elutions of bovine serum albumin (66 kDa) and β-amylase (200 kDa) were used as markers to follow the fractionation  was cloned into the pET28 vector. Primers are listed . RecombiT. brucei cells were harvested and resuspended at 5 × 107 cells/ml, and washed twice with PBS. Cells (∼109) were concentrated and incubated on ice. 4′-Aminomethyl-trioxsalen hydrochloride (AMT) (Sigma) was added to the cells at a concentration of 0.2 mg/ml. Cells treated with AMT were kept on ice and irradiated using a UV lamp at 365 nm at a light intensity of 10 mW/cm2 for 30 min. Next, the cells were washed once with PBS and deproteinized by digestion with proteinase K (Roche) (200 μg/ml in 1% SDS for 60 min). RNA was prepared using TRIzol (Sigma) reagent. Approximately 250 μg of RNA was used for affinity selection, essentially as described  were incubated with AMT-psoralen, and extracts and PRS were prepared as described above. RNA was prepared from the PRS and was subjected to mild fragmentation in Tris buffer (10 mM Tris–HCl (pH 8.0 at 37°C), 5 mM MgCl2, 0.1 M KCl, 0.02% Triton X-100 and 0.1 mg/ml BSA) by boiling at 94°C for 3 min. The RNA was dephosphorylated using alkaline phosphatase and purified on SPRI-beads (Agencourt® AMPure® XP). The RNA was then ligated using RNA ligase (Thermo Scientific) at 25°C overnight, and again purified; cross-linking was reversed by irradiation at 254 nm. The recovered RNA was then used for library preparation, as previously described  from ilencing  was suspMicrofluidic devices were fabricated as previously described from polydimethylsiloxane (PDMS)  . BrieflyThe template for N' terminal cMYC, and C' terminal 6 x HIS U2B" (Tb927.3.3480) or U2A’ (Tb927.10.14360) synthetic gene was created by assembly PCR  using prhttp://statpages.org/nonlin.html): x, [B]y and [AxBy] are the equilibrium concentration of A, B and AB respectively.Binding affinity was determined by normalizing the Cy5 tagged RNA signal to the protein signal. The dose response relative binding was then fitted to the following equation using non-linear least squares minimization  enriched in small RNAs  (CMC) that selectively binds to Ψ (CBF5 depletion (see below), and the scatterplots show a high correlation between the experiments (r = 0.9 for modified positions)  Figure Bii. Our q Figure . Howeverq Figure Ci; Ψ47, q Figure Cii and Ψq Figure  which weOur small RNA Ψ-seq also detected Ψs on other small RNA species such U3, C/D and H/ACA snoRNA, 7SL RNA, and also verified the presence of Ψ on SL RNA , and theT. brucei possess the highest number of Ψs (61 Ψ) reported thus far on these RNA types. T. brucei U1 snRNA has 4 Ψs compared to 2 Ψs in human and 2 Ψs in yeast. Similarly, T. brucei U2, U4, U5 and U6 snRNA has 34, 14, 2 and 7 Ψs respectively compared to 14, 3, 4 and 3 Ψs in human, whereas yeast has only 5 Ψs in U2, and 1 Ψs in U5 snRNA.The quality of PCF and BSF small RNA Ψ-seq was validated by the detection of a single Ψ28 on SL RNA and Ψ74 on 5.8S rRNA that remained invariant between the two life stages . It was T. brucei spliceosomal snRNAs is depicted in Figure T. brucei BSRR is compared to homologous domains in other organisms, showing conservation in this functional domain   was highly significant (P < 0.001)  . All dets Figure , indicats Figure , the H/A< 0.001) . This re< 0.001) . Interes< 0.001) . The red< 0.001) , and on < 0.001)  upon CBF6 snRNAs . The sma6 snRNAs .T. brucei MTAP was disovered as the protein that assoicates with the complex that governs the methylation of SL RNA  and major reductions in spliceosomal snRNA modifications, but no reduction in rRNA modification  of the snoRNA cross-linked to U2 shows that only a subset of snoRNAs were efficiently selected with U2 snRNA Figure . A line trans-splicing was also observed based on the accumulation of the SL RNA, as a result of its not being utilized in trans-splicing . To assess the stability of the U4/U6 dimeric complex, total RNA was denatured and annealed with oligonucleotides A and B , compared to U2B"-U2 snRNA with U instead (Kd = 317.5 pM) but only at 37°C, suggesting that Ψ strengthens the RNA-protein interaction mainly at an elevated temperature. Similarly, U2A’ had stronger affinity towards pseudouridylated U2 snRNA (Kd = 1030 pM) compared to RNA with Us (Kd = 1618 pM) at 37°C . Th. ThT. brt Figure  is surprt Figure . In factt Figure . Future T. brucei spliceosomal snRNAs deviate from the consensus Sm site and are not rich in uridines but carry Ψs, in contrast to the homologous human sites . Of. OfT. brrmed SSm ,43. Howermed SSm , and henDrosophila, several scaRNAs were shown to modify both rRNA and snRNA using unexpected degree of plasticity (T. brucei single hairpin H/ACA snoRNAs have at least a dual function. The dual functionality observed here may be more general. Many ‘orphan’ snoRNAs exist in mammals, and these may guide novel substrates including mRNA, possibly using highly flexible pockets (In asticity . However pockets . Interes pockets . The con pockets . This flAn important mystery that is highlighted in this study is how U2 and U4 snRNA modifications are introduced at neighboring positions by snoRNAs with non-canonical guiding pockets. The key to this enigma may lie in the known ability of Ψ to base-pair with any of the four major bases . Thus, tT. brucei spliceosomal snRNAs, which are found not only on RNA interacting domains but also on protein binding sites, underscoring the effect of this modification on protein binding. The strengthening of protein binding to pseudouridylated RNA is likely to be beneficial especially when the parasite propagates in the mammalian host. However, obtaining further support for this notion awaits in vivo experiments demonstrating that depletion of individual Ψs on the protein binding site affects the association specifically with U2 snRNA.This study describes the unprecedented degree of Ψ modifications on trans-splicing. However, we can not rule out the possibility that the growth arrest also stems from depleting the modification on rRNA or any other yet uncharacetrized RNA target guided by the same snoRNA. Nevertheless, the finding that MTAP silencing exclusively affects growth and trans-splicing demonstrates the importance of this modification for the splicing reaction. The high level of Ψs found on both rRNAs (This is the first system wherein a change in a single Ψ guided by a single hairpin H/ACA snoRNA is shown to have an effect on growth and th rRNAs  and snRNth rRNAs .The small RNA Ψ-seq and small RNA interactome sequencing data have been deposited in the NCBI SRA database under the accession number PRJNA476671.gkz477_Supplemental_FilesClick here for additional data file.