RNA Biology

Pseudouridine ({Psi}) is an important post-transcriptional modification of many noncoding RNAs that is under-characterized in microRNA (miRNA) due to historical limitations in pseudouridine mapping methods. {Psi} modification stabilizes RNA duplex structures and could therefore play an important role in miRNA target binding and repression. To investigate the extent to which mammalian miRNAs are modified with {Psi}, we profiled the modification landscape of short (<30 nt) RNA in human cells and mouse tissues using bisulfite sequencing. Our approach was powered to detect small RNA pseudouridylation based on robust detection of known {Psi} positions in tRNA fragments (tRFs), some of which show tissue-specific patterns of modification. In contrast with tRFs, we find that miRNA pseudouridylation is exceedingly rare, with a single modified miRNA (miR-3068-5p) identified in mouse tissues. Pseudouridylated miR-3068-5p diSerentially repressed predicted miRNA targets with less stable miRNA:mRNA pairing modes. This study fills a long-standing gap in transcriptome-wide {Psi} profiling and reveals a new potential function for {Psi} as a modulator of activity of small regulatory RNAs.

Eukaryotes use several distinct quality control pathways to resolve aberrant ribosomes and mRNAs. For example, the no-go decay mRNA pathway is stimulated after ribosome collisions caused by stalled ribosomes translating damaged or truncated mRNAs. Separate decay pathways for non-functional 40S and 60S subunits containing rRNA mutations affecting decoding and peptidyl transferase activity, respectively, have also been elucidated. To our knowledge, whether eukaryotes have evolved a quality control pathway to sense and process globally stalled ribosomes is unclear; however, such a pathway would be advantageous to eukaryotes during exposure to natural elongation inhibitors such as ricin and diphtheria toxin. Here, we test how prolonged robust inhibition of elongation using a high dose of cycloheximide (CHX) affects ribosome turnover. Despite no decrease in cell viability and that mammalian ribosomes have been classically characterized of having a half-life of 3-5 days, a single 24 hr high dose of CHX resulted in drastically shortened half-lives (<24 hr) of 28S and 18S rRNA in A549 cells. A [~]2-fold reduction in nearly all ribosome species was observed by polysome analysis in HeLa and A549 cells after prolonged CHX treatment. Depletion of ribosomes was also evident when assessing ribosomal proteins from both the 40S and 60S subunits by Western blot. Literature supports that ribosomes can be degraded by autophagy and the ubiquitin (Ub)-proteasome system. Upon testing inhibitors of both pathways, only proteasome inhibitors (i.e., MG132 and bortezomib) rescued both rRNA and ribosomal protein levels. Proteasome inhibitors also rescued ribosome levels in polysome profiling experiments. Remarkably, rRNA levels were not rescued during CHX treatment when co-treated with the Ub activating enzyme E1 inhibitor, TAK243. Polysome analysis also showed that the high prolonged dose of CHX did not cause robust accumulation of collided ribosomes compared to control treatments. Proteasome-dependent turnover of rRNA was also observed with high doses of other elongation inhibitors, namely anisomycin, homoharringtonine, and lactimidomycin. The recognition capabilities of the pathway were further expanded as we observed that 80S ribosomes not trapped on the mRNA were also targeted for degradation by the proteasome. Together, our findings define the framework of a regulatory pathway in mammalian cells that degrades both ribosomal subunits in response to prolonged periods of robust elongation inhibition.

The prevalent transcriptional repressor NrdR binds to highly conserved prokaryotic sequences in the promoter regions of operons encoding the essential enzyme ribonucleotide reductase. The NrdR binding sites consist of two partially palindromic 16 bp sequences (NrdR boxes) separated by a 15-16 bp linker sequence. We have assessed the requirement of both boxes for binding, the propensity of different NrdRs to bind to heterologous binding sites, and that the linker sequence is only limited to length and not sequence conservation. As we have observed several deviations from the conserved sequences of the NrdR boxes, we here test the conservation requirements of individual basepairs in the NrdR boxes using a synthetic DNA fragment (Synt DNA) to which the NrdR proteins from the actinomycete Streptomyces coelicolor and the gammaproteobacterium Escherichia coli bind equally well as to their homologous binding sites. By introducing isolated mutations to Synt DNA and testing the binding capacity of NrdR from S. coelicolor and E. coli we expand our understanding of what criteria are needed to build a functional binding site for the NrdR repressor.

BackgroundMetazoan adenosine-to-inosine (A-to-I) mRNA editing temporospatially diversifies the neuronal transcriptome and proteome. The limited read length from next-generation sequencing (NGS) constrains the quantification of the potentially differential editing levels across different splicing isoforms, restricting our understanding of the extent to which RNA editing contributes to molecular diversity and its interplay with splicing. MethodsWe employed reverse transcription nested PCR (RT-nPCR) and developed a novel interfering-Primer PCR (iPrimer PCR) technique to distinguish different transcripts of any gene. We selected multiple essential genes exhibiting RNA editing in coding sequences (CDSs) or untranslated regions (UTRs) for isoform-specific amplification and Sanger sequencing. ResultsNine different Adar isoforms together with pre-mRNA had distinct editing levels at the S>G auto-recoding site, which was predicted to have isoform-specific effects on catalytic activities. Although pre-mRNA editing might exert isoform-dependent promotion/suppression of splicing, closely located editing sites, such as those in neuronal genes qvr and stj, still exhibited high correlation in editing levels due to co-editing. iPrimer strategy further discovered differential recoding levels between the long/short 3UTR isoforms of gene jef. ConclusionsWe provide the first comprehensive solution for isoform-specific PCR amplification of any gene, enabling quantification of RNA editing level of different isoforms. Our results offer insights into how RNA editing interplays with splicing, and highlight its complicated role in expanding molecular diversity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/725286v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1ebc82org.highwire.dtl.DTLVardef@1ea365dorg.highwire.dtl.DTLVardef@1971aceorg.highwire.dtl.DTLVardef@160d053_HPS_FORMAT_FIGEXP M_FIG C_FIG We developed isoform-specific PCR followed by Sanger sequencing, and achieved the quantification of differential RNA editing levels in different transcripts of a gene.

MicroRNAs are short noncoding RNAs that regulate gene expression and are commonly profiled by small RNA sequencing (miRNA-seq). Despite the widespread use of miRNA-seq, datasets are often analyzed with RNA-seq method such as DESeq2 or edgeR, which do not take into account the specific characteristics of miRNA-seq data. Here, we present a benchmark study of normalization and differential expression approaches using a realistic ground-truth dataset. By mixing mouse RNA of two organs, we generated expression trends while capturing biological and technical variability. Using monotonicity across the dataset and expected fold changes from the mixture design, we assessed normalization and differential expression methods. Normalization benchmarking showed that within-sample scaling, particularly Read Per Million (RPM), best preserved the expected monotonic trends, outperforming cross-sample methods such as TMM, rlog, and VST. These approaches sometimes recovered apparent monotonicity among abundant miRNAs, but inspection of individual profiles suggested likely over-correction. Regarding differential expression, edgeR consistently ranked among the best-performing methods across several metrics, including log2 fold-change estimation, with performance comparable to miRNA-seq-specific tools such as miRglmm and NBSR. DESeq2, edgeR-v4, and limma-based approaches tended to systematically underestimate log2 fold changes. Applying a common RPM-based normalization substantially improved the performance of cross-sample methods, highlighting the strong influence of normalization on differential expression analysis. Overall, our findings support within-sample scaling methods such as RPM for normalization, and edgeR, miRglmm, or NBSR for differential expression. The dataset has been made publicly available, providing a valuable resource for objective method comparison and future miRNA-seq software development.

Variants affecting RNA splicing are a major contributor to human disease, yet the consequences of variants outside of the canonical splice motifs are often difficult to determine. Here, we present a protocol for minigene-based evaluation of candidate splice-altering variants. The methodology described includes locus-specific insert design, commercial gene fragment synthesis, and long-read sequencing. The combined approach enables rapid assay development and nucleotide level resolution of the effect on splice isoforms in vitro, providing a scalable framework for functional validation of predicted cryptic splice variants. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/723105v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1a88cb5org.highwire.dtl.DTLVardef@adda98org.highwire.dtl.DTLVardef@1ea587corg.highwire.dtl.DTLVardef@574a63_HPS_FORMAT_FIGEXP M_FIG C_FIG

Upstream open reading frames (uORFs) in the 5' leader of bacterial mRNAs can modulate gene expression, yet genome-wide identification remains limited. We combined bioinformatic prediction of ribosome-binding sites (RBSs) - a Shine-Dalgarno sequence and a start codon - with experimental validation to uncover new uORFs in Sinorhizobium meliloti 2011. From totally 1106 predicted upstream RBSs (uRBSs), we first examined 15 candidates using eGFP reporters and integrating existing RNA-seq and Ribo-seq data. Translation was detected at 13 sites, with fluorescence intensity broadly correlating with predicted initiation rates. Two uRBSs correspond to gene start sites, thereby refining gene annotations. In nine cases, uRBS mutations affected downstream gene expression in reporter fusions. Among others, the data suggests that a Type I secretion system operon, the RNA chaperone gene hfq, and metabolic genes are regulated by uORFs. Four uORFs acted through translational coupling. We also identified uRBSs that were ribosome-occupied yet (nearly) silent in eGFP assays, and closely spaced to the downstream main RBS (mRBS). These uRBSs probably mediate ribosomal occlusion downregulating lacR and SM2011_RS36230. A re-screen of the prediction set revealed 335 close uRBS/mRBS pairs. Three of them were analyzed, supporting the proposed ribosomal occlusion mechanism for SM2011_RS03630 and SM2011_RS22110, while for glnK translational coupling to an uORF was suggested. These results indicate that uORFs are more widespread in bacteria than previously recognized and suggest that direct ribosomal occlusion of the mRBS is a novel mechanism for down-regulating protein synthesis.

N6-methyladenosine (m6A) is the most abundant modification in eukaryotic mRNA and can influence gene expression, but the molecular mechanisms by which it does this at an individual gene level are poorly understood. The eukaryote with the highest known percentage of adenosine in its RNA is P. falciparum, causative agent of the deadliest form of malaria. P. falciparum has a high genomic AT content (>80% A+T) and a particular A-bias in mRNA - coding strands have an average adenosine content of 44%. Plasmodium parasites employ extensive transcriptional and post-transcriptional control over gene expression to differentiate within and between sexual and asexual stages in response to changing environments and hosts. We used direct Nanopore RNA sequencing and GLORI-seq to interrogate m6A methylation of the asexual Plasmodium falciparum transcriptome, revealing m6A depletion in protein coding regions relative to the 3UTR, where m6A is deposited in precise patterns approximately 50 nt upstream of the polyadenylation site. We used an inducible protein mislocalisation system to disrupt the methyltransferase, which writes m6A to mRNA, and observed m6A depletion associated with transcriptional readthrough and chimeric transcripts. We show that m6A in transcript 3UTRs is required for faithful positioning of polyadenylation and transcription termination. This work highlights the importance of m6A to mRNA processing in P. falciparum, and potentially a wider role in post-transcriptional regulation.

Microproteins are proteins of <100 amino acids. They represent a major, and until recently, overlooked fraction of the human proteome. However, it has now been demonstrated that many of these proteins play key roles in cellular physiology. Our group reported the expression of a microprotein expressed from an ioORF within the 53BP1 CDS arising as a result of delayed translational reinitiation mediated by a small uORF within the 5 TL. We named this microprotein SEP53BP1. We have sought to expand these studies with the ultimate aim of establishing a function for this microprotein. Although this remains elusive, we report findings providing new insights into the elements regulating its translation and demonstrate that the SEP53BP1 sequence serves as a Golgi targeting tag. Lastly, despite the fact that subunits of the proteasome feature prominently on interactome studies we were unable to demonstrate an impact of microprotein over-expression on the activities of both the proteasome and immunoproteasome.

Some long non-coding RNAs (lncRNAs) are known to regulate gene expression. However, the underlying temporal dynamics of lncRNAs influencing gene and epigenetic regulation and mechanisms of lncRNA regulation in trans are less understood. To investigate this, we genetically engineered 17 doxycycline-inducible lncRNA transgenes for ectopic expression at the H11 safe harbor locus in human pluripotent stem cells (hiPSCs), and we generated high-density temporal RNA-seq and ATAC-seq profiles. Most lncRNA transgenes were induced at 2 hours and maintained expression through the 96-hour time course. Surprisingly, when we sought to identify gene expression changes due to the lncRNAs, we found that the global transcriptional landscape was dominated by a strong systemic response triggered by doxycycline exposure. We rigorously defined this cohort of genes as a Doxycycline-Responsive Gene Signature (DRGS). The DRGS was also present in at least 28 public datasets from dox-inducible transgene studies involving diverse cell types. Next, we determined which lncRNAs exhibited trans-regulatory events. We identified DANCR, FENDRR, LINC00667, LINC00847, LNCPRESS1, and PNKY as lncRNAs that regulate specific transcript expression in trans. The downstream target genes encoded 53 mRNAs and 10 lncRNAs. None of the target lncRNAs altered gene expression proximal to their own loci (i.e., triggering secondary cis-effects). Surprisingly, the target genes of LINC00847 (transcribed from chromosome 22) were substantially enriched on chromosome 19, with a preponderance of target genes encoding RNA metabolism and RNA splicing factors. Collectively, our study provides a resource to discern artifacts in the doxycycline-inducible system and identifies temporally regulated targets of 6 lncRNAs for future mechanistic studies.

O_LISymbiosis between legumes and rhizobia is beneficial on nutrient-poor soils, as it enables the fixation of atmospheric N2. To establish this symbiosis, gene expression in both the host plant and the symbiont has to be regulated. To understand the underlying RNA-mediated regulation of host gene expression, we designed experiments to identify competing endogenous networks involving circular RNA, microRNA, and linear transcripts during symbiosis, using wt and symbiosis-deficient Lotus japonicus mutants with the rhizobium Mesorhizobium loti (M. loti). C_LIO_LICircRNA, miRNA, and linear transcripts were identified from Lotus japonicus wildtype and CCamK mutant (ccamk-13; snf-1) seedlings without inoculation or with M. loti inoculation using deep short-read sequencing with rRNA-depletion and random primers. C_LIO_LIDifferentially expressed miRNAs showed negative correlations to predicted target genes and may regulate symbiotic processes. The symbiosis essential iron-sensor LjnsRING/BRUTUS expresses a circRNA which was upregulated in symbiotic treatments. This circRNA may act as a target mimic and contribute to nodule longevity. CircRNAs are predicted to act predominantly as trans-regulatory molecules with similar frequencies in Arabidopsis thaliania, Oryza sativa, and Lotus japonicus. C_LIO_LIWe identified novel miRNAs, long noncoding RNAs, and circRNAs, and nominated several as potential new regulatory non-coding RNAs that may act as target mimics to stabilize genes and support symbiosis. C_LI SummarySymbiosis between Lotus japonicus and Mesorhizobium loti involves treatment-specific regulation of competing endogenous RNA networks involving circular RNA, miRNA, and linear transcripts.

Splicing is governed by interactions between the spliceosome and precursor RNA sequence and structural elements. However, the relative contributions of RNA sequence and structural elements remain unclear. Here, we systematically dissect these determinants using a high-throughput mutagenesis approach with the MAP3K7 intron reporter. The MAP3K7 gene encodes a serine/threonine kinase involved in response to environmental stress. MAP3K7 precursor RNA contains a cryptic 3 splice site that increases in use when the core spliceosomal protein SF3B1 is mutated. SF3B1 mutations are known to promote aberrant splicing and are associated with cancer, particularly the lysine 700 to glutamate mutation (K700E). We designed a pooled library of 249 MAP3K7 mutants targeting branch points, RNA-binding protein motifs, nucleotide composition and predicted structural elements. The impact of these mutants on splicing was measured in the context of normal and SF3B1 K700E expression. RNA structure was assessed in parallel using in vitro high-throughput SHAPE-MAP chemical probing. We found that branchpoint mutations drive the strongest increases in cryptic splice-site use. There is no overall correlation between cryptic splice-site use and structural similarity to the wild-type MAP3K7 RNA. However, mutants within an RNA binding protein hotspot (containing U2AF2, U2AF1, KHSRP and SRSF2 sites) are associated with cryptic splice-site use and structural similarity to wild-type MAP3K7 RNA. These structural changes are associated with increased ensemble diversity. Our results demonstrate that although there are key structured regions within an RNA, there is also extensive variability where divergent RNA structures allow for accurate splicing.

Nearest neighbor parameters are widely used in software for estimating the conformational stability of an RNA sequence folding into a specific structure. Folding stability for RNA with canonical nucleotides A, C, G, and U has been widely studied, but the same is not true for most modified nucleotides. In this work, we present a comprehensive set of nearest neighbor parameters for estimating the folding stability of RNA including pseudouridine in helical or loop contexts. These parameters are derived from 210 optical melting experiments involving helices with pseudouridine-A and pseudouridine-G pairs and with pseudouridine in loop motifs. The experiments include sequences with pseudouridine and U in the same strand, including U-A and U-G pairs, allowing us to consider the folding stability of sequences with both U and pseudouridine. On average, pseudouridine stabilizes RNA folding compared to U in an analogous motif, although this effect is sequence-context dependent. These parameters improve the modeling of folding stability for RNA secondary structures containing pseudouridine. We demonstrate that these parameters successfully model the secondary structure change for Saccharomyces cerevisiae U2 snRNA when two additional inducible pseudouridines are present. These parameters are freely available and incorporated into the RNAstructure software package. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/725682v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@e1167aorg.highwire.dtl.DTLVardef@18ac7f0org.highwire.dtl.DTLVardef@4c909eorg.highwire.dtl.DTLVardef@aa8bca_HPS_FORMAT_FIGEXP M_FIG C_FIG

Acropora spp. are the dominant reef-builders of the Indo-Pacific but are also amongst the most stress-sensitive corals. For these reasons, Acropora spp. have become the most studied of corals, two species (A. digitifera and A. millepora) often essentially serving as the basis for understanding molecular responses and processes across the sub-order Refertina and corals in general. The early development of these species has been well-characterised in terms of morphology and gene expression but as yet we have a limited understanding of how transcription is regulated during development. In "higher" animals (bilaterians) microRNAs (miRNAs) are critical regulators of gene expression but until now their involvement in coral development has not been investigated. Building on the existing developmental data for Acropora spp., we catalogued microRNAs (miRNAs) expressed during the early development of Acropora digitifera and profiled their expression in 21 stages from unfertilised eggs to 24h after treatment with a natural settlement cue (CCA chips). 157 miRNAs were recognised, many of which ([~]60%) were novel. These fell into three distinct groups, corresponding to three distinct developmental phases: (1) those present in eggs through to gastrulation (2) a larvally expressed group and (3) those expressed following settlement induction. Exposure of competent larvae to a natural settlement inducer resulted in major changes in the miRNA profile within 10 minutes, indicating that miRNAs may be particularly important in mediating the larva/polyp transition but are also likely to play important regulatory roles throughout early coral development in addition to possible roles in disease resistance.

Conformational plasticity of RNAs plays important roles in recognizing RNA-binding proteins, and is often modulated by their binding partners. Here, we investigate RNA conformational preferences in a non-redundant dataset of 263 protein-RNA complexes to characterize the structural landscape associated with protein recognition. RNA dinucleotide segments are analyzed using seven backbone torsion angles ({delta}1, {varepsilon}1, {zeta}1, 2, {beta}2, {gamma}2, and {delta}2), two glycosidic torsion angles ({chi}1 and {chi}2) and the pseudo-torsion angle . Focusing on dinucleotide steps present in both interface and non-interface regions, we performed density-based clustering using selected backbone torsion angles to identify recurrent conformational states. We identify 28 distinct RNA dinucleotide conformers containing at least ten members each. Among these, eight conformers represent previously unreported nucleotide conformers (NtCs), including the transitional and the non-canonical states AB06, AB07, BB21, BB22, OP32, OP33, IC08 and IC09. Several of these conformers are preferentially enriched at protein-binding interfaces, suggesting their involvement in local conformational adaptation during protein-RNA recognition. The newly identified conformers span transitional A-B geometries, distorted B-like states, open conformations and compact intercalated structures, highlighting the remarkable structural plasticity of RNA in ribonucleoprotein complexes. Overall, this study expands the current understanding of RNA conformational space and provides a refined RNA dinucleotide conformer library for protein-RNA complexes. These findings will facilitate the identification of novel RNA structural motifs and improved RNA structural modeling, docking protein-RNA complexes and deep learning-based prediction frameworks for describing RNA tertiary structures.

Hepatocyte transplantation is a promising alternative to liver transplantation; however, it currently serves only as a temporary treatment until a donor organ becomes available. In contrast, animal studies have demonstrated "liver repopulation", a phenomenon in which transplanted hepatocytes progressively replace host hepatocytes. Despite extensive documentation, the mechanisms driving this process remain poorly understood. More fundamentally, it remains unclear whether liver repopulation is driven by active cell-cell interactions between host and transplanted hepatocytes that induce host cell death, or whether it can be explained solely by intrinsic differences in proliferation and survival between these populations. To address this, we performed computational simulations using an agent-based model constrained by experimental data from repopulation in uninjured rat livers. Our analysis reveals that host hepatocyte death rate is the dominant determinant of repopulation kinetics, whereas variations in proliferation rate have only a limited impact. Notably, experimentally observed repopulation dynamics were only reproduced when cell- cell interactions were incorporated, or alternatively when host hepatocyte lifespan was set to unrealistically short values (approximately 25 days). These findings support a model in which cell- cell interactions play a critical role in efficient liver repopulation. More broadly, this study establishes a conceptual and computational framework for evaluating the requirement for cell-cell interactions in tissue replacement, even in the absence of a defined molecular mechanism.

Double-stranded RNA (dsRNA) is recognized by cellular receptors as a sign of viral infection, triggering the innate immune response. Increasing evidence shows that cellular dysregulation, for example in immune disorders and neurodegenerative diseases, can also lead to accumulation of endogenously produced dsRNA that stimulates a viral-like immune response. Additionally, dsRNA contamination in RNA therapeutics can lead to harmful side effects via a similar pathway. Despite the clinical relevance of dsRNA, reliable tools for its detection remain limited. At present, dsRNA detection relies almost exclusively on the monoclonal antibodies J2 and K1, which suffer from sequence bias and low sensitivity, limiting their reliability. To address this challenge, we aimed to repurpose naturally occurring dsRNA-binding domains (dsRBDs) to produce reliable, pan-specific affinity reagents for dsRNA. We first systematically screened the dsRBDs of the three human adenosine deaminases acting on RNA (ADARs). This analysis identified ADAR3 dsRBDs as promising candidates due to their reduced sequence dependence compared to the dsRBDs of ADAR1 and ADAR2. We then engineered ADAR3-derived dsRBD constructs having varying linker lengths and domain combinations, allowing us to specifically vary the length cutoff of dsRNA detected, thus creating dsRNA accumulation detected by ADAR3 RBDs (dsRADAR) affinity reagents. Finally, we demonstrate the superior performance of dsRADAR over currently available dsRNA antibodies in a cell model of viral infection and a tissue model of gastric inflammation. Together, dsRADAR provides a sensitive and reliable approach for imaging and quantifying diverse dsRNA structures in a variety of biological contexts. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/724404v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1d89c30org.highwire.dtl.DTLVardef@1f64fc1org.highwire.dtl.DTLVardef@1ee391forg.highwire.dtl.DTLVardef@e834a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Nucleobindin 1 (NUCB1) is a multifunctional conserved protein located in Golgi luminal, nucleus, extracellular and cytosolic pools. NUCB1 is multidomain protein comprised of a signal peptide, a DNA-binding domain, a leucine zipper and Ca2+ -binding domain. The multiple domains and localization of NUCB1 potentiates its interactions with various partners, such as DNA, Gi3 protein, cyclooxygenase 2, LRP10 and RNA suggests its importance in the regulation of many cellular events. We revealed that NUCB1 contains three RNA-binding regions and able to interact with two RNA fragments. It was suggested possible variants of the participation of NUCB1 in the interaction of the two partially complementary RNAs. The RNA-binding properties of the NUCB1 were also confirmed in vivo experiments.

Polycomb Repressive Complex 2 (PRC2) maintains repression of genes specific for other cell differentiation states. PRC2 binds RNA in vitro with a preference for G-rich sequences. UV-based crosslinking coupled with immunoprecipitation (CLIP) experiments have shown that PRC2 also binds RNA in cells. Recently, Guo et al reported that a stringent denaturing variant of CLIP called CLAP did not detect PRC2 RNA binding in cells. We present a reanalysis of CLAP data that supports direct interaction of PRC2 with RNA in cells. CLAP for Halo-tagged PRC2 subunits from mixed populations of human and mouse cells specifically enriched for RNA from the species in which the proteins were tagged. The lack of apparent PRC2 RNA binding in Guo and colleagues analysis stems from a scaling step that deflates enrichment scores for low-complexity CLAP samples. Our findings pave the way for studies seeking to determine the physiological roles of PRC2 RNA binding activity.

We previously found that high genome replication fitness of the hepatitis C virus (HCV) was associated with severe disease in immunocompromised patients. Elevated replication fitness was mediated by accumulation of mutations in the replication enhancing domain (ReED) within domain (D) 2 of non-structural protein (NS) 5A. NS5A is a partially unstructured phosphoprotein lacking enzymatic activity but fulfilling a key role in HCV replication due to interacting with various cellular and viral proteins. It can exist in a variety of dimeric and oligomeric conformations mediated by NS5A D1 with clinically approved NS5A inhibitors proposed to exert their antiviral function by fixing these dimers in distinct conformations. In this study, we aimed at elucidating the ReEDs mode of action. AlphaFold modelling indicated a so far unrecognized NS5A dimerization site in the ReED. Indeed, split nano luciferase assays revealed a significantly stronger NS5A dimerization of high replicator ReED variants, suggesting that high replication fitness is mediated by enforcement of NS5A self-interaction. This hypothesis was supported by the effect of low dose (1 pM) NS5A inhibitor treatment, increasing replication fitness and phenocopying the effects of ReED mutations. Furthermore, we found that HCV isolate JFH1, replicating with very high efficiency, is completely resistant to the regulatory function of the ReED. Chimeric replicons composed of ReED resistant JFH1 and the ReED sensitive isolate J6 identified NS3 helicase and NS5B polymerase as critical genetic elements mediating ReED sensitivity/resistance. Our data overall suggest that NS5A is a negative regulator of HCV replication fitness with dimerization releasing the inhibitory interaction with helicase and/or polymerase, thereby likely facilitating initiation of RNA synthesis.