RNA Biology

Escherichia coli YicC enzyme is the founding member of a family of endoribonucleases that is encoded in virtually all bacterial species. Previous structural studies revealed that this ribonuclease binds RNA by a novel mechanism in which the hexameric apoprotein presents an open channel that undergoes a large rotation upon RNA binding and clamps down on the RNA. The current study follows up on these findings by examining the cleavage of various oligonucleotide substrates designed to probe recognition elements required for YicC binding and cleavage. A 26-nucleotide RNA oligomer (oligo), with a KD in the low micromolar range, was the standard to which numerous oligos with altered sequence were compared. In vitro RNase assays and fluorescence anisotropy binding measurements indicated that the preferred substrates for YicC were relatively small RNAs that contain some secondary structure. Larger RNAs or highly structured RNAs were less-than-optimal substrates. Similarly, RyhB RNA, a [~]90-nucleotide, iron-responsive RNA of E. coli, which has been described as a target of YicC binding and/or cleavage, was a poor YicC substrate in our assays. These results suggest that the native substrates for YicC-family members are very small RNAs or RNA fragments derived from larger RNAs.

Regulation of RNA pools allows for adaptation to changing environments and stress, which is especially important in pathogenic bacteria such as Mycobacterium tuberculosis. RNA degradation is a significant contributor to RNA abundance, and Ribonuclease (RNase) E has a rate-limiting role in degradation of a majority of mycobacterial transcripts. However, many open questions remain about the RNA substrate requirements and specificities for efficient cleavage by mycobacterial RNase E. Here, using both Mycolicibacterium smegmatis and M. tuberculosis RNase E, we demonstrate that this enzyme is only active on substrates with a minimum length of approximately 27 nt. Furthermore, we show that mycobacterial RNase E prefers substrates with 5 monophosphates rather than 5 triphosphates, and that the positions of cleavage events within substrates are dictated by both sequence and distance from the RNA ends. Our results also suggest that RNase E may be affected by product inhibition. Finally, we show that M. smegmatis RNase E behaves similarly to M. tuberculosis RNase E, validating the use of this model organism for RNA degradation studies.

Even though teleost fish and mammals share the same mitochondrial gene content and organization, the teleost mitochondrial transcriptome is still poorly understood. We characterized the mitochondrial transcriptome during zebrafish (Danio rerio) early development by long-read direct RNA sequencing. All heavy-strand specific mRNAs were found to carry 3 poly-A tails of approximately 50-60 residues, and the transcriptome profile was distinctive but practically invariant between stages. Three unusual transcripts were however noted. These included two mRNAs (COI and ND5 mRNAs), with significant 3 untranslated regions corresponding to antisense gene sequences, and a previously not described noncoding RNA named here lncOriL. The ND5 mRNA was found to carry one third of all detected m6A methylation sites in the zebrafish mitochondrial transcriptome. The 313 nt-long lncOriL transcript had an abundance comparable to that of ND5 mRNA and it mapped to mitochondrial genome region covering the origin of light strand replication and four flanking antisense tRNAs. A mitochondrial tRNA-derived fragment (tiRNA5-Asn), with a 35 nt perfect pairing-potential to lncOriL, was present at all stages. Additional analyses including adult zebrafish, scissortail (Rasbora rasbora), and monkfish (Lophius piscatorius) strongly corroborate the results of COI mRNA, ND5 mRNA, and lncOriL transcript prevalence among teleost fish. Surprisingly, our findings in zebrafish were further supported by mitochondrial transcriptome analyses in domestic pig (Sus scrofa) and human (Homo sapiens), including tiRNA5-Asn commonly present in human tissues, suggesting that lncOriL is ubiquitously expressed and regulated in vertebrates. Author SummaryMitochondria contain their own genome and produce essential RNAs needed for energy production. Although fish and mammals share the same mitochondrial gene organization, less is known about how mitochondrial RNAs are processed and regulated in teleost. Using Nanopore direct RNA sequencing, we examined mitochondrial RNAs during early zebrafish development and discovered three unusual transcripts that include extended non-coding regions. Two of these molecules, COI and ND5 mRNAs, carry long 3' untranslated regions formed by antisense gene sequences, suggesting previously unrecognized regulatory potential. We also identified lncOriL, a highly structured long noncoding RNA that spans the origin of light-strand replication and is abundant during development. Strikingly, the same RNA feature, including lncOriL and a matching tRNA-derived small RNA (tiRNA5-Asn), was found not only in zebrafish but also in human mitochondrial transcriptomes. These findings support conservation of regulatory mitochondrial RNAs across main groups of vertebrate species. Our work reveals a new layer of mitochondrial RNA regulation and expands the current understanding of how mitochondrial gene expression is controlled.

Accurate detection of RNA splice variants is often hindered when transcripts lack large distinguishable exonic regions, making conventional PCR strategies challenging. We developed a simple melting temperature (Tm)-guided exon-exon junction (EEJ) RT-PCR method to enable variant-specific detection under these conditions. Uni-directional primers spanning exon-exon junctions were designed so that approximately each half anneals to adjacent exons. The Tm of each half-site was set >7{degrees}C below the annealing temperature, preventing stable binding to individual exons and enforcing junction-dependent amplification. The method was evaluated using HTRA1-AS1 long noncoding RNA variants that share overlapping exon sequences but differ in splice connectivity. HTRA1-AS1 comprises five variants, only one with a large distinguishable exon. Tm-guided EEJ primers robustly discriminated the remaining four variants. After optimization, amplification yielded sharp, single bands with minimal cross-reactivity. Compared with conventional designs, this approach reduced heteroduplex and heteroquadruplex formation, improving band clarity. Sanger sequencing confirmed junction specificity, and the method performed well in multiplex settings. Overall, Tm-guided EEJ RT-PCR is a cost-effective, high-resolution approach for detecting RNA variants lacking easily distinguishable exonic regions, readily compatible with standard RT-PCR and qPCR workflows.

Since the discovery of viral Internal Ribosome Entry Sites (IRESes), researchers have sought to find similar elements in mammalian host genes, termed "cellular IRESes". However, the plasmid systems used to measure cellular IRES activity are vulnerable to false positives due to promoter activity in candidate IRESes. Orthogonal methods are needed to validate putative IRESes while carefully avoiding artifacts known to cause false positives. Recently, Koch et al. proposed approaches for studying IRESes, primarily circular RNA-generating plasmids, and for validating mRNA transcripts using smFISH and qRT-PCR. Here, we demonstrate confounding variables and artifacts in each of these approaches that can lead to inappropriate conclusions about potential cellular IRES activity. We show the back-splicing circRNA plasmid creates linear mRNA artifacts associated with false-positive IRES signals. Using orthogonal, gold-standard assays validated with viral IRESes, we find putative cellular IRESes reported using the back-splicing plasmid have no IRES activity. Furthermore, we demonstrate that smFISH and qRT-PCR can misidentify nuclear non-coding RNAs as mRNAs and we validate a single molecule sequencing assay for identifying genuine mRNA 5 ends. Our work establishes reliable methods for robust transcript annotation and IRES studies that avoid documented artifacts arising from bicistronic and back-splicing circRNA plasmid reporters.

Human mitochondrial genome (mtDNA) encodes multiple proteins in the oxidative phosphorylation complexes as well as the ribosomal and transfer RNAs (tRNAs) needed for in situ translation. These genes are transcribed from only three promoters, producing polycistronic transcripts that are co-transcriptionally cleaved by mitochondrial RNase enzymes to release majority of individual gene products. tRNAs separate many of these genes and are thought to serve as "punctuation" marks that enable RNase recognition, binding, and hydrolysis of the 5' "leader" and 3' "trailer" sequences flanking the tRNA. Mutations in the tRNA genes dominate the mtDNA-linked mitochondrial pathologies; yet a systematic study of the impact of tRNA sequence variation on the RNase-catalyzed processing is lacking. Here, we employed human mitochondrial tRNATyr as a model system to dissect the effect of tRNA variants on the in vitro 5' leader and 3' trailer hydrolysis. We found that nucleotide variations located near the catalytic interfaces - particularly within or near the tRNA acceptor stem - showed the strongest defects in 5' processing and prevented release of the downstream tRNA in a tRNA cluster where multiple tRNAs are transcribed in tandem. This work provides mechanistic insight into how mutations disrupt coordinated mitochondrial tRNA processing and establish a framework for predicting variant effects based on their structural position relative to the processing enzymes.

Muscleblind-like (MBNL) RNA-binding proteins (RBPs) possess modular domains that mediate regulation of alternative splicing and RNA localization. Myotonic Dystrophy Type 1 is a CTG repeat expansion disorder where MBNL is sequestered into intranuclear RNA foci, impairing its function. Previous studies found that MBNL self-associates through its exon 7, but the nature of this interaction is not well understood. We identified a cysteine in MBNL1 exon 7 that enables dimerization through formation of an intermolecular disulfide bond. We likewise demonstrate that MBNL2 dimerizes by forming disulfide bonds between multiple cysteines in its carboxy-terminus. Nucleocytoplasmic fractionation revealed a greater proportion of MBNL1 dimer in the nucleus, suggesting a nuclear function for the MBNL1 dimer. We investigated a connection between MBNL1 dimerization and MBNL1-mediated regulation of alternative splicing. To accomplish this, we mutated the MBNL1 cysteine in question to alanine (C325A) and performed RNAseq. We uncovered novel splicing events sensitive to MBNL1 dimerization. We also found that MBNL1 C325A, when co-expressed with expanded CTG repeats, produces smaller, more numerous foci, suggesting a role for the MBNL1 dimer in maintaining foci integrity. These results provide insight into biological and pathological mechanisms of MBNL1 dimerization and suggest other RBPs might similarly dimerize to regulate function. GRAPHICAL ABSTRACT

A morphospace is an abstract space of theoretically possible biological traits, shapes, or property values. It is interesting to explore which parts of a morphospace life occupies, as compared to those parts which could be occupied, but are not. Comparing random and natural non-coding (nc) RNA secondary structures is an established approach to studying morphospace occupation for RNA structures. Most earlier studies have focused on the minimum free energy (MFE) structure, while relatively few have looked at the Boltzmann distribution, describing the ensemble of energetically suboptimal RNA folds. These suboptimal structures may have important roles and functions, and hence should be examined carefully. Here we compare random and natural ncRNA in terms of their Boltzmann distributions, finding that natural RNA tend to have very similar profiles to random RNA, with the main difference being that natural RNA are slightly more energetically stable, except for very short sequences (20 to 30 nucleotides) which tend to be slightly less stable. We infer that natural ncRNA occupy similar parts of the morphospace that random RNA do, indicating that the biophysics of the genotype-phenotype map largely determines the ensemble properties of ncRNA.

Pre-mRNA splicing is an essential step in eukaryotic gene expression during which spliceosomes remove introns from nascent RNAs while ligating the adjacent exons. Spliceosomes are cellular nanomachines composed of five small nuclear (snRNA) components and dozens of proteins, most of which are highly conserved. Despite the high conservation of many splicing factors between S. cerevisiae and H. sapiens, several protein components of the S. cerevisiae spliceosome are not essential for growth under normal laboratory conditions. This is particularly surprising for nonessential factors whose conserved domains contact the spliceosomes catalytic core. Uncovering a function for these splicing factors can be challenging since they are not required for viability, may engage in functionally redundant interactions, and may display only weak phenotypes in the absence of secondary mutations in other spliceosome components. One such nonessential factor is the Cwc15 protein. Cwc15s highly conserved N-terminus directly contacts the U2/U6 di-snRNA within the spliceosome catalytic core; yet its precise role in splicing has not been defined in any organism. In this work, we use molecular genetics in S. cerevisiae combined with splicing reporter assays to study Cwc15p function. We propose that Cwc15p not only promotes active site stability during 5 splice site cleavage but also impacts structural transitions into and out of this spliceosome conformation. This function may be critical for splicing in S. cerevisiae under nonoptimal conditions, facilitating use of weak or alternate splice sites, and could have implications for proofreading of spliceosome active site formation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/713263v1_ufig1.gif" ALT="Figure 1"> View larger version (74K): org.highwire.dtl.DTLVardef@b296c5org.highwire.dtl.DTLVardef@c87b91org.highwire.dtl.DTLVardef@287011org.highwire.dtl.DTLVardef@d59741_HPS_FORMAT_FIGEXP M_FIG C_FIG Article SummaryPre-mRNA splicing is carried out by large macromolecular machines called spliceosomes which are composed of several snRNAs and dozens of proteins. Despite decades of study, the functions of many splicing factors such as S. cerevisiae Cwc15p remain unknown. Cwc15p is highly conserved among eukaryotes and directly contacts the spliceosome catalytic core. Here, we have used genetic and splicing reporter assays to study the function of Cwc15p during splicing in vivo. We propose that Cwc15p both stabilizes the spliceosome active site during 5 splice site cleavage and impacts remodeling of that site.

Background: Vascular smooth muscle cells (VSMCs) phenotypic plasticity can modulate atherosclerosis progression. Although several gene regulatory steps towards pro-inflammatory phenotypes have been well-studied, epitranscriptomic changes during this transition and their regulatory roles remain unexplored. Methods and Results: Primary human VSMCs were stimulated with TGF-{beta}1 to induce an atheroprotective, contractile, and matrix-producing state and with IL1-{beta} plus PDGF-BB to induce a highly energetic, pro-inflammatory state, confirmed by Illumina bulk RNA sequencing and proteomics. Untargeted screening of mRNA base modifications using Oxford Nanopore Technologies direct RNA sequencing and xPore analysis revealed enhanced uridine modification within a GUUUU motif in pro-inflammatory VSMCs. Modified uridines were enriched in 3'-UTR and accessible RNA structures, with implications on Poly(A) tail dynamics and miRNA binding. Conclusions: Atheroprotective and pro-atherogenic treatments induce distinct epitranscriptomic landscapes composed of different modification types, often co-localized in the same transcript. Modified uridines in mRNAs are abundant in a high-energy, pro-inflammatory VSMC state and associated with post-transcriptional regulation. In summary, epitranscriptomics adds a novel regulatory layer to VSMC phenotypic transitions critical for atherosclerosis development and progression.

The ability to access, search, and analyse large collections of RNA molecules together with their secondary structure and evolutionary context is essential for comparative and phylogeny-driven studies. Although RNA secondary structure is known to be more conserved than primary sequence, no existing resource systematically associates individual RNA molecules with curated phylogenetic classifications. Here, we introduce PhyloRNA, a curated meta-database that provides large-scale access to RNA secondary structures collected from public resources or derived from experimentally resolved 3D structures. PhyloRNA allows users to search, select, and download extensive sets of RNA molecules in multiple textual formats, each entry being explicitly linked to phylogenetic annotations derived from five curated taxonomy systems. In addition to taxonomic information, each RNA molecule is accompanied by a rich set of descriptors, including pseudoknot order, genus, and three levels of structural abstraction--Core, Core Plus, and Shape--which facilitate comparative analyses across sets of molecules. PhyloRNA is publicly available at https://bdslab.unicam.it/phylorna/ and is regularly updated to incorporate newly available data and revised taxonomic annotations.

Efficient protein synthesis in eukaryotic cells typically requires a 5' cap structure on messenger RNAs (mRNAs). However, under stress conditions or in viral infection, translation can also occur independently of the cap via internal ribosomal entry sites (IRES). IRES elements are therefore key regulators of protein expression in both viral and cellular contexts. Here we describe a cell-free protocol to quantitatively assess IRES-mediated translation using wheat germ extract (WGE) and a firefly luciferase (FLuc) reporter. The protocol includes template preparation, RNA synthesis and luminescence measurement following in vitro translation in WGE. This method enables rapid and robust comparison of IRES activity under controlled conditions and can additionally be applied to evaluate mRNA modifications designed to enhance translation efficiency. Key featuresO_LIStringent in vitro workflow from DNA template preparation through RNA synthesis and protein synthesis to reporter readout, including quality controls. C_LIO_LIEvaluation of IRES-driven translation suitable for testing combinations of IRES and CDS. C_LIO_LItranslation analysis without radioactive labeling. C_LI Graphical overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/716985v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@417649org.highwire.dtl.DTLVardef@1bcd186org.highwire.dtl.DTLVardef@15fecb3org.highwire.dtl.DTLVardef@acdf8d_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical AbstractPipeline for the production and evaluation of IRES-firefly luciferase constructs using wheat germ extract. (1-4) Preparation: IRES-firefly luciferase constructs are amplified in E. coli and isolated from bacterial cells. Plasmids are linearized to prepare for in vitro transcription. (5-6) Transcript synthesis and verification: In vitro transcription is followed by electrophoretic validation to confirm integrity and correct molecular weight. (7-8) Translation and detection: Translation is executed in wheat germ extract and quantified by measuring reporter activity in a luminometer.

Background: Ribavirin is a guanosine analogue with clinical antiviral activity against a range of RNA viruses including hepatitis C virus (HCV), respiratory syncytial virus and Lassa virus. Several potential mechanisms of action have been proposed, but there is limited data supporting them clinically. Methods: We studied 196 HCV-infected participants from a trial of short-course directly antiviral therapy (STOPHCV-1) which included a factorial randomisation to ribavirin versus no ribavirin. Deep sequencing of the HCV genome was performed on samples with detectable viremia from three time-points: baseline (n = 191), day 3 of treatment (n = 25) and post-treatment failure (n = 47). Results: Ribavirin exposure significantly increased total mutational load at treatment failure (P = 0.0065) and enriched classical ribavirin-associated transitions, including G->A (P = 0.026) and C[->]U (P = 0.004), along with other key changes including A->G (P = 0.005), U->C (P = 0.023), C->G (P = 0.010), and U->A (P = 0.026). The resulting mutational signature was broad, not dominated by G-related changes. Region-specific analyses demonstrated this increase was broadly distributed across the viral genome, without strong evidence for protection of specific regions. Non-synonymous to synonymous mutation ratios (dN/dS) rose at day 3 (P = 5.5e-5) before declining at failure (P = 8.5e-7), with trends toward higher dN/dS in the ribavirin group at day 3 (P = 0.06). Conclusions: Ribavirin acts as a potent in vivo mutagen, driving viral populations toward genome-wide diversity rather than selecting a few highly fit drug-resistant clones. These findings support an error-catastrophe model.

Rotavirus is a major cause of severe diarrheal disease in children under the age of five, with reduced vaccine effectiveness in low-resource settings causing substantial morbidity and mortality. In the absence of approved antiviral therapeutics, treatment is largely supportive, urging the need for targeted and precision-based interventions. VP4 protein plays an essential role in viral attachment, entry, and infectivity, making it a suitable target for targeted therapy. In this context, RNA interference is a specific method for inhibiting viral gene expression with its efficacy depending on sequence conservation, target accessibility, and compatibility with the RISC-loading machinery. In the present study, an integrative in silico approach was employed to design and evaluate siRNAs targeting conserved regions of the VP4 gene across six geographically diverse countries. Candidate siRNAs were screened using established design rules and regression-based scoring with off-target filtering. Three optimized siRNAs were further assessed through structural modeling, molecular docking, and molecular dynamics simulations to examine interactions with human Dicer, TRBP, and Argonaute-2. Comparative dynamic analyses identified one siRNA with enhanced structural compatibility, reduced conformational fluctuations, and stable interactions with RISC-loading proteins. These findings provide a rational computational basis for VP4-targeted siRNA development, facilitating experimental validation.

Anti-hormonal therapies such as selective estrogen receptor modulators like tamoxifen or aromatase inhibitors like letrozole represent a cornerstone for breast cancer prevention and therapy of estrogen receptor-positive breast cancer. Therapeutic monitoring can include blood tests and imaging; however, genetically-based approaches are not yet in practice. Ideally, a test would be able to detect a positive molecular response across different estrogen pathway-suppressive approaches. Circular RNAs are a species of non-coding RNAs detectable in plasma that have been proposed as non-invasive therapeutic biomarkers. To determine whether a set of specific circular RNAs is altered across estrogen-suppressive pathway approaches, we analyzed mammary gland-specific total RNA sequencing data from two individual genetically engineered mouse models (GEMMs) of estrogen pathway-induced breast cancer, with or without exposure to tamoxifen or letrozole. The nf-core/circrna pipeline was used to identify circRNAs that were differentially expressed in response to either tamoxifen or letrozole. We then screened for circRNAs that were differentially regulated by both anti-hormonals. Four up-regulated and 31 down-regulated circRNAs with host genes known to be expressed in human breast epithelial cells were identified as showing reproducible differential regulation in response to anti-hormonal treatment.

Alternative splicing (AS) is traditionally understood to increase transcript and protein diversity by generating multiple coding isoforms from a single gene. In addition, many alternative isoforms are degraded through non-sense mediated decay (NMD). Furthermore, we show here that AS also produces nuclear long non-coding RNAs (lncRNAs) from protein-coding genes, which can serve regulatory functions rather than expanding proteomic complexity. Focusing on the Arabidopsis thaliana SR protein gene At-RS31, we found that a non-coding isoform (mRNA3) accumulates in the nucleus under dark conditions. This transcript binds the protein product of its own transcriptional unit, modulating splicing decisions and balancing gene activity in response to environmental cues. Overexpression of mRNA3 down-regulates the action of At-RS31 on its target genes in trans and restores the phenotype induced by intron-less At-RS31 accumulation. Further sub-cellular fractionation and RT-PCR analyses show that many SR genes generate nuclear-retained non-coding isoforms, especially under dark conditions, suggesting a widespread mechanism. These findings redefine the role of AS in plants, highlighting its capacity to generate regulatory lncRNAs to fine-tune gene expression beyond protein diversification. Highlights- Alternative splicing generates regulatory lncRNAs from protein-coding genes - A nuclear-retained isoform forms an autoregulatory RNA-protein feedback loop - Non-coding isoforms of SR genes accumulate in the nucleus under dark conditions - Co-expression of the non-coding isoform rescues splicing factor overexpression phenotypes

Small regulatory RNAs (sRNAs) are major post-transcriptional regulators in bacteria and, together with transcriptional regulators such as the two-component systems (TCSs), participate in the rapid adaptation of these microorganisms to changing environments. Several examples of paralogous sRNAs with overlapping functions have been reported, that could in theory integrate different environmental cues. Consistent with this idea, we have identified the acid-responsive RstB-RstA two-component system, important for virulence of multiple bacterial species, as a specific multicopy activator of the Escherichia coli OmrB sRNA, but not of the paralogous sRNA OmrA. Further characterization of this regulation unexpectedly revealed the asr-ydgU operon, itself a target of RstB-RstA, as a dual modulator of this TCS via two opposite effects. First, the 27 aminoacids YdgU small protein exerts a negative feedback by directly interacting with RstB and, second, Asr in contrast mediates a positive feedback on RstB-RstA activity via a not completely elucidated mechanism. These results provide a new example of retro-control of a TCS, here RstB-RstA, by one of its direct targets. They further highlight the major role of small proteins in controlling TCS activity and ydgU was thus renamed samT, for Small Acid-responsive Modulator of the RstB-RstA TCS.

RNA thermometers (RNATs) are temperature-responsive structures in 5' untranslated regions (UTRs) of bacterial messenger RNA (mRNA) that control translation by modulating ribosome access. The Listeria monocytogenes prfA RNAT represses translation of PrfA (positive regulatory factor A), the master virulence regulator, at ambient temperature and activates it near the human host temperature ([~]37 {degrees}C) by modulating ribosome binding site (RBS) accessibility. However, the prfA RNAT shares no homology with known RNAT classes, and its unfolding mechanism remains unclear. Here, we used analytical ultracentrifugation and single-molecule kinetic analysis of RNA transient structure (SiM-KARTS) to map prfA RNAT unfolding. SiM-KARTS analysis demonstrates that thermal opening occurs predominantly at the RBS, while the upper helix of the RNAT hairpin remains largely folded at 37 {degrees}C. The RBS binding kinetics increases with temperature in parallel with translation output, establishing a quantitative link between structural unfolding and function. Mutations in the upper helix impair thermal regulation, indicating that this region tunes switching even as it stays structured at host temperature. Together, these data reveal a hierarchical unfolding pathway in which initial RBS opening triggers activation, whereas the upper helix remotely tunes temperature sensitivity. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/717274v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@a95daborg.highwire.dtl.DTLVardef@144bc42org.highwire.dtl.DTLVardef@1a39bf6org.highwire.dtl.DTLVardef@545e60_HPS_FORMAT_FIGEXP M_FIG C_FIG

In Archaea, a prevalent class of circular RNAs corresponds to 16S and 23S ribosomal RNA intermediates (circ-pre-rRNAs). A conserved bulge-helix-bulge (BHB) motif within the 16S and 23S rRNAs processing stems and adjacent to the circularization site in Euryarchaeota and TACK superphylum suggests that pre-rRNAs circularization is widely conserved across Archaea. Using genome-wide transcriptomic data obtained on total RNAs of wild-type Thermococcus barophilus cells, we recovered the canonical circularization junctions of the 16S and 23S circ-pre-rRNAs at the predicted BHB motifs. We also identified three alternatives 23S circular junctions introducing variability at the 3 end of the mature rRNA. We investigated the different forms of 16S and 23S by performing primer extension and RACE experiments. We showed that while the 16S rRNA has standard 5 and 3 extremities, the main form of 23S rRNA is circularly permuted, with helix H99 now at its 5 end. This permutation most probably emerged from the deletion of helix H98 from the 23S circ-pre-rRNA. Interestingly, we showed that the permuted 23S rRNA is incorporated into ribosome subunits and 70S monosomes. The significance of this event in generating functional 50S particles remains to be determined.

Synonymous codons encode the same amino acid but can differ in their usage and translational properties. In previous work we reported statistical differences in backbone dihedral angle distributions associated with synonymous codons in the Escherichia coli proteome. This finding has been questioned due to concerns regarding the statistical methodology used. Here we revisit the dataset using corrected statistical procedures and alternative statistical tests. Across multiple frameworks, the real dataset consistently shows an excess of small p-values relative to randomized controls, indicating detectable codon-associated differences in backbone conformation.