RNA

In 1986, a class of small, noncoding RNAs was discovered in association with an enormous, enigmatic ribonucleoprotein complex - the "vault" particle - and thus dubbed vault RNAs (vtRNAs). However, its since been recognized that the vast majority ([≥]95%) of these noncoding vtRNA molecules are not associated with the mysterious vaults, raising questions about their potential independent function(s). Moreover, humans express four vtRNAs from two different loci, and debate has arisen about whether the vtRNA paralogs share any functional connection, and whether they should remain classified together. Herein, we report the expression patterns of the four VTRNA paralogs in a variety of human cell lines, including various single- and multi-gene VTRNA-knockout cell lines. Knockout of one or more of the three VTRNA1 genes leads to increased expression of vtRNA2-1, suggesting that their biological functions are related. Additionally, we interrogated the effects of vtRNA knockout on HEK293T cell growth, viability, and viral infection, and were unable to replicate previously reported associations. Collectively, our findings point to a potential functional connection between even the most distantly related human vtRNA paralogs, while reaffirming that their biological roles and mechanisms still require critical study.

RNA molecules form specific 3D structures that facilitate a variety of functions through interactions with other macromolecules. Many RNA viral genomes maintain these structures to interact with and evade host immunity machinery. One such element, the competitive inhibitor RNA (ciRNA), discovered in the protein coding region of the poliovirus serotype 1 (PV1) genome, inhibits a host antiviral protein, ribonuclease L (RNase L). Although some functionally essential structural motifs of the PV1 ciRNA have been studied, the extent of its evolutionary conservation and other structural requirements remained unexplored. Here we combined bioinformatic and biochemical techniques to further define the requirements of a functional ciRNA and assess its phylogenetic distribution. We systematically mutated ciRNA structural features, verifying that ciRNA inhibitory activity requires a conserved loop E motif and a long-range base-pairing interaction, but its peripheral stems are dispensable and in fact a circularly permuted version maintains function. A structure-based homology search identified potential ciRNAs across the Picornaviridae family, but only a subset of those tested were functional - all are in Enterovirus coxsackiepol. When structural features needed for function were transposed from PV1 ciRNA to an RNA unable to inhibit RNase L, the chimeric RNAs did not gain wild-type function, and chemical probing data revealed that these nonfunctional RNAs are unable to form the correct secondary structure. Overall, the dual constraints of encoding a protein and forming a specific functional structure appear to not only limit the sequence diversity, but also the phylogenetic distribution, of ciRNAs.

The immature HIV-1 virion is assembled by the Gag polyprotein using inositol hexakisphosphate (IP6) as an essential assembly co-factor. Gag binds the genomic RNA Psi packaging signal via the nucleocapsid (NC) domain and associates with the plasma membrane via the matrix (MA) domain. Previous studies revealed that Gag exists in both compact (C) and extended (E) conformational states in solution. Only E-Gag formed virus-like particles with the correct size and IP6 shifted the equilibrium of DNA-bound Gag to the E state. The influence of specific RNA elements on this conformational change is unknown. In this work, a dual dye-labeled Gag was prepared for probing the effect of RNA binding on Gag conformation using Forster resonance energy transfer (FRET). In low salt and in the absence of other factors, Gag was primarily in the C state. Psi RNA binding induced a more significant FRET decrease than binding to non-Psi RNAs, consistent with a shift to E-Gag. IP6 alone also promoted the E-Gag state in the absence and presence of RNA. Atomistic molecular dynamics simulations are consistent with and provide detail into the role of NC-Psi RNA binding in the conformational switch of C-Gag to assembly-competent E-Gag. Simulations also showed that this switch is driven by capsid (CA) linker domain orientational flexibility and MA-CA unbinding dynamics. Thus, the highly flexible multi-domain Gag polyprotein leverages both viral and host cell factors to sample and stabilize distinct conformations, thereby orchestrating the viral assembly process.

Premature termination of transcription (PTT), also known as attenuation, is a conserved gene regulatory mechanism that operates across all domains of life and in viruses. Attenuation enables rapid cellular responses to environmental and metabolic changes and fine-tunes expression of biosynthetic genes. In Saccharomyces cerevisiae, attenuation of RNA Polymerase II (Pol II) transcription was first linked to the Nrd1-Nab3-Sen1 (NNS) termination pathway for non-coding RNAs, and the mRNA 3-end processing factor Hrp1 has been implicated more recently. Substitutions in Hrp1 RNA Recognition Motifs (RRMs) cause attenuator readthrough and reduce RNA-binding affinity in vitro, but direct evidence for Hrp1 functioning at attenuators in vivo remains limited. Here, we characterized 5-end RNA terminator elements from several genes, including RAD3, SNG1, MNR2, and CPR8. Readthrough mutations clustered in AU-rich regions resembling polyadenylation site (pA) efficiency elements, consistent with Hrp1 binding targets. Amino acid substitutions of Hrp1 RRM residue F162 revealed a general requirement for aromaticity in RNA recognition that varied to some degree by gene context. To test Hrp1-RNA interactions independent of other yeast factors, we adapted a bacterial 3-hybrid (B3H) assay. Hrp1 interacted with RNA derived from the GAL7 3-end pA site and 5-end terminator regions of RAD3, MNR2, and CPR8. Mutations in AU-rich RNA regions that disrupted Pol II attenuation in yeast generally impaired B3H interactions. However, some Hrp1 mutants (M191T, I270T, D271G, M275V, T280I) retained binding to CPR8 terminator RNA, suggesting their defects require additional yeast components. These results demonstrate that Hrp1 is sufficient to bind multiple UA-rich attenuator RNAs in vivo, expanding Hrp1 function to include early transcription events.

SARS-CoV-2 nonstructural protein 1 (Nsp1) binds to 40S ribosomal subunits and induces host protein synthesis shut off by inhibiting translation initiation and triggering endonucleolytic cleavage of cellular mRNAs. Irrespective of the mode of initiation, Nsp1-mediated cleavage is induced by the cooperative action of the N-terminal domain of Nsp1, the RRM domain of eIF3g and 40S subunits. Using in vitro reconstitution, we determined that cleavage occurs by transesterification following intramolecular nucleophilic attack of the 2’OH of the ribose on the adjacent phosphodiester bond yielding 5’OH and 2’,3‘-cyclic phosphate termini. Cleavage requires a guanosine ∼10-22 nucleotides from the 5’ end of mRNA, occurs within a narrow window upstream of this G, is most efficient between nucleotides at positions -6/-7 and -7/-8 relative to G, and shows a preference for Pu at positions -7 or -8 which provides the 2’OH for the nucleophilic attack. Zero-length UV cross-linking of Nsp1 to nucleotides at positions -1 and -2 suggests that the critical guanosine may be recognized by Nsp1. Resistance to Nsp1-mediated cleavage of SARS-CoV-2 mRNA was ensured both by the relatively long distance between its G 23 G 24 and the 5’end and by the preceding oligoPy stretch lacking purines at positions -7 or -8 upstream of G 23 G 24 .

Deadenylation, the enzymatic shortening of the poly(A) tail, is typically the first committed step of mRNA decay. Deadenylation rates span nearly a 1000-fold range between transcripts and are governed by protein-RNA interactions, including those involving cytoplasmic poly(A)-binding protein (PABPC). Previous work shows that PABPC can straddle the junction between the poly(A) tail and the 3' untranslated region (UTR), but whether this conformation influences deadenylation has not been tested. To investigate how straddling influences deadenylation kinetics, we designed a library of tailed RNA substrates and measured both in vitro deadenylation rates in the presence of PABPC and PABPC binding propensity for each substrate. We found that 3' UTR sequences influence deadenylation through two mechanisms. First, structured UTRs are deadenylated more slowly in the absence of PABPC1, an effect that is alleviated by PABPC1. Second, sequences upstream of the poly(A) tail modulate PABPC1 binding propensity, with tighter binding correlating with slower deadenylation. This relationship is abolished with a PABPC1 mutant lacking UTR-binding capacity. Together, these results show that sequences upstream of the poly(A) tail tune PABPC1 binding and deadenylation rates, likely contributing to the range of deadenylation rates observed for cellular mRNAs.

Small RNAs (sRNAs) regulate the expression of many genes including those involved in antibiotic resistance and bacterial virulence, making them potential therapeutic targets. A molecule that binds an sRNA could interfere with its ability to bind its target mRNA and disrupt the regulation mechanism. Randomization and screening of natural arginine rich motif (ARM) peptides led to peptides capable of interfering with the sRNA MicF's ability to regulate ompF in Escherichia coli. Molecular dynamics simulations suggested that this effect was not a result of a direct disruption of the MicF-ompF interaction. Instead, the peptides interfere with binding of the chaperone Hfq, which is required for MicF-mediated regulation. Subsequent testing demonstrated peptide specificity for MicF over two other Hfq scaffolds and the ability to disrupt regulation of two additional MicF targets. Together, these findings support the use of synthetic ARMs as a potential tool for modulating sRNA function in bacteria.

Alongside their canonical function as adaptors in translation, tRNAs are precursors of tRNA-derived fragments that can regulate diverse aspects of gene expression. Queuosine (Q), present at position 34 of eukaryotic tRNAAsn/Asp/His/Tyr, has been implicated in suppressing tRNA fragmentation. In vertebrates, Q34 of tRNAAsp and tRNATyr is further modified by mannosylation and galactosylation, respectively, catalyzed by QTMAN and QTGAL. However, the interplay between these glycosylations and other anticodon loop modifications, and their impact on tRNA fragmentation, have remained unclear. Here, we define a modification circuit in human tRNAAsp in which Q34 stimulates DNMT2-dependent m5C38 formation, while subsequent Q34 mannosylation does not impact m5C38 installation; reciprocally, m5C38 inhibits Q34 incorporation by the tRNA-guanine transglycosylase TGT. By contrast, anticodon loop modifications of tRNATyr are installed independently, although our data support a hierarchical pathway in which TRMT5-mediated m1G37 formation precedes queuosinylation and galactosylation. Alongside demonstrating that Q34 glycosylation enhances protein synthesis, our data reveal that mannosylation of Q34 protects tRNAAsp from stress-induced cleavage, thus expanding the relevance of Q glycosylation beyond translation.

The subcellular localization patterns of RNAs are controlled by regulatory elements contained within them. However, for most localized RNAs, the identities of these elements remain unknown. We had previously identified several localization elements that are necessary and sufficient for robust, kinesin-dependent RNA targeting to microtubule plus ends in a variety of cell types. Yet the characteristics of these elements that are critical for function remained unclear. To address this, we systematically created tens of thousands of mutant localization elements and quantified their ability to regulate subcellular RNA localization in neuronal cells. We found that the minimally active size of these localization elements is large, approximately 200 nucleotides. These elements contain multiple important subsequences, with some being completely intolerant of any changes and others being tolerant to a shuffling of nucleotide order but not to changes in nucleotide composition. Using single molecule microscopy, we verified these findings in primary rat neurons. Together, these results demonstrate that highly active mammalian RNA localization elements are large, complex, and multipartite and lay a foundation for further mechanistic studies of their function.

Biomolecular condensates formed by liquid-liquid phase separation (LLPS) are commonly studied in vitro using protease-mediated removal of solubilizing tags to induce condensation under controlled conditions. Tobacco Etch Virus (TEV) protease is widely used for this purpose and is generally assumed to remain soluble and inert during condensate reconstitution. Here, we show that in RNA-containing systems, TEV protease variants can interact with RNA, leading to aggregation and changes in the phase behavior of the target protein. Using confocal microscopy, turbidity measurements, and mass photometry, we demonstrate that commonly used TEV protease variants differ in their propensity to undergo RNA-dependent aggregation. The widely used pRK793 TEV protease forms large RNA-associated aggregates. We further show that RNA-TEV aggregation alters the morphology and organization of protein-RNA condensates formed by well-characterized phase-separating proteins, including PGL-3 and FUS. Together, our findings show that TEV protease can directly impact in vitro LLPS assays through RNA binding and aggregation. These results underscore the importance of validating protease-based induction strategies and incorporating appropriate controls when reconstituting biomolecular condensates, particularly in RNA-rich systems.

In eukaryotes, single-stranded DNA (ssDNA) generated during DNA replication, recombination, and repair is rapidly bound and protected by the major single-stranded DNA-binding protein replication protein A (RPA). RPA not only stabilizes ssDNA but also acts as a central platform that coordinates diverse DNA transactions. Exhaustion of RPA due to unregulated ssDNA production leads to replication fork breakage and replication catastrophe, underscoring its critical role in genome stability. However, the direct consequences of RPA limitation remain incompletely understood. Using Xenopus egg extracts, we show that excess ssDNA induces spontaneous priming, a reaction that is otherwise prevented in a physiological nuclear environment. We provide evidence that priming suppression is mediated by stoichiometric binding of RPA to ssDNA. Analysis of the ssDNA-binding proteome reveals that RPA promotes the association of ATR checkpoint factors, Pol-primase, and the RFWD3 ubiquitin ligase with ssDNA. In contrast, RPA depletion induces the recruitment of Rad51, Rad51 paralogs, and Fbh1, a DNA helicase that interacts with both RPA and Rad51 and promotes fork breakage under replication stress. Collectively, our findings suggest that RPA contributes to genome stability by protecting ssDNA from inappropriate DNA synthesis and unscheduled recruitment of recombination and fork-processing factors.

Satellite RNAs (satRNAs) are parasitic subviral agents whose biological activities are mediated largely by specific sequence determinants and structured RNA elements. Establishing biologically supported global RNA structures is therefore essential for understanding how RNA architecture underlies satRNA functions. Cucumber mosaic virus (CMV) satRNA is one of the best-characterized models for investigating satRNA structure-function relationships; however, a biologically supported global RNA architecture of CMV satRNA has yet to be established. Here, we applied AlphaFold3 modeling to predict the global structure of CMV satRNA T1 (sat-T1). Initial full-length structure modeling generated multiple long-distance interactions that lacked biological support. We therefore used fragment-based modeling, combined with chemical probing, evolutionary covariation, and compensatory mutagenesis, to derive a biologically supported global secondary structure. To determine whether structurally distant regions could interact in the context of the full-length RNA, we engineered a structure-guided T1-ZD mutant that preserved the supported secondary structure while reducing alternative base-pairing possibilities. Full-length AlphaFold3 modeling of T1-ZD largely recapitulated the proposed architecture, while one predicted model revealed a long-distance interaction that was subsequently supported by compensatory mutagenesis analysis. These findings suggest that the 3' terminus of sat-T1 may undergo conformational switching between alternative structural states. Together, our work establishes a biologically supported global RNA architecture for CMV sat-T1 and provides a structural framework for investigating the molecular basis of satRNA function.

During translation initiation, the 40S small ribosomal subunit is recruited to the mRNA 5' cap and scans the 5' untranslated region (UTR) to locate the start codon. While the mechanism of 40S translocation remains elusive, the RNA helicase eIF4A has long been suspected as the primary molecular motor driving 40S scanning. In this study, we utilized GFP reporter mRNAs to investigate the impact of 5' UTR length on translational efficiency. We found that an 8-fold variation in the length of unstructured 5' UTRs did not lead to substantial changes in translation efficiency in wheat germ extract (WGE) and human HEK293T cell lysate. By contrast, the presence of a stable stem-loop in the middle of the 5' UTR significantly reduced cap-dependent translation. These results suggest that mRNA scanning is not rate-limiting when the UTR is devoid of secondary structure. Inhibition of eIF4A by hippuristanol in cell-free protein synthesis systems yielded an equivalent decrease in translation for mRNAs with short and long unstructured 5' UTRs, indicating that eIF4A may be dispensable for 40S scanning. Our data suggest that helicase-independent one-dimensional diffusion may be the primary mechanism enabling 40S movement along the 5' UTR during initiation.

Intrinsically disordered regions are widespread in RNA-processing machines. In Escherichia coli, RNase E uses its intrinsically disordered C-terminal domain (CTD) to recruit RNAs to the N-terminal catalytic domain, including mRNAs targeted by regulatory small RNAs (sRNAs), but the basis of substrate recognition and specificity is unclear. We engineered a protease-cleavable RNase E and used split-CRAC to isolate RNAs crosslinked to the AR2 sub-domain of the intrinsically disordered CTD fragment. AR2 preferentially engaged mRNAs and was depleted of sRNAs and sRNA-containing hybrids, supporting recognition of the mRNA. AR2 contacts concentrated on accessible A-rich motifs surrounding ribosome-binding sites and start codons, and purified AR2 recognised this motif in vitro. AR2 also contacted an AUAA motif in the rne translation-initiation region, and AR2 deletion increased RNase E abundance implicating this interaction in autoregulation. These findings define a relatively short AR2-binding motif and are consistent with CTD interactions with the 30S subunit that may provide additional specificity for a subset of mRNA translation initiation regions. SIGNIFICANCE STATEMENTMost RNA turnover in bacterial cells is carried out by the RNA degradosome, yet how this molecular machine checks and selects RNAs for degradation remains incompletely understood. We show that an intrinsically disordered region of the degradosome enzyme RNase E, termed AR2, preferentially binds A-rich sequences near sites of translation initiation. Through recognition of this shared sequence feature in a common functional context, AR2 may help the degradosome recognise messenger RNAs as a functional class. AR2 also contributes to feedback control of RNase E expression by recognising its own messenger RNA.

Background: RNA molecules form thermodynamic ensembles, but interpretation often requires a single representative structure. Existing base-pair centroid estimators assess agreement at the level of individual base pairs and do not directly target nesting depth along the sequence. Methods: We introduce Mountain Centroid, which minimizes expected squared mountain-profile distance, and derive dynamic programming algorithms with and without RNA pairing constraints. We also combine the Mountain Centroid objective with the base-pair centroid gain. Results: Across 21,254 RNAStrAlign sequences, Mountain Centroid had lower median normalized mean squared mountain distance (NMSMD) than minimum-free-energy (MFE) and base-pair centroid ({gamma} = 1) structures, whereas its median base-pair F1 was lower. Imposing RNA pairing constraints improved base-pair F1 for 59.35% of sequences and reduced it for 3.58%. At an illustrative weight, the combined objective had median base-pair F1 similar to MFE while retaining lower median NMSMD than MFE and all tested {gamma}-centroid settings. Conclusions: Mountain Centroid represents an RNA structural ensemble with a single secondary structure that reflects how nesting depth varies across nucleotide positions. Combining mountain-profile and individual-base-pair criteria allows their relative contributions to be varied.

The RNA exosome is an essential, evolutionarily conserved ribonuclease complex that processes and degrades many classes of RNA. The complex is composed of three structural cap subunits (EXOSC1-3/Csl4, Rrp4, Rrp40; H. sapiens/S. cerevisiae), six structural core subunits (EXOSC4-9/Rrp41,Rrp46,Mtr3,Rrp42,Rrp43,Rrp45), and a catalytic ribonuclease (DIS3 or DIS3L/Dis3). Cofactors that associate with the RNA exosome confer specificity to target specific RNAs for processing and/or decay. Missense mutations in genes encoding structural subunits of the RNA exosome have been linked to neurological diseases. Notably, several pathogenic mutations have been identified in EXOSC3 that are associated with pontocerebellar hypoplasia type 1b (PCH1b). These pathogenic alleles cause a broad spectrum of clinical severity, suggesting variant-specific functional consequences. Given the high degree of conservation between the human and budding yeast RNA exosome complexes, we performed a systematic analysis of eight pathogenic EXOSC3 variants modeled in budding yeast Rrp40. We find that two Rrp40 variants cause growth defects, show distinct negative genetic interactions with RNA exosome cofactor mutants, and impair RNA processing in budding yeast. One of these variants, EXOSC3-Y109N/Rrp40-Y64N, had not been previously characterized in any mechanistic studies. Computational stability predictions and immunoblot analyses indicate that most EXOSC3/Rrp40 variants display reduced steady-state protein levels, but decreased protein levels do not strictly correlate with phenotype or disease severity, suggesting that individual variants disrupt RNA exosome function through distinct mechanisms. Collectively, our studies suggest that pathogenic EXOSC3 variants alter RNA exosome function through distinct mechanisms and provide insight into the specific molecular defects that could underlie pathology.

Transposable elements (TEs) not only account for half of the human genome sequence but also generate transcripts that contribute to transcriptomic diversity. Yet, their repetitive nature has hindered accurate quantification of the full TE-derived transcriptome, a challenge that long-read sequencing can overcome. Here, we combined multiplexed arrays isoform sequencing (MAS-ISO-seq) with a dedicated computational framework (TEscape) to perform an in-depth annotation of the human TE transcriptome. To capture the breadth of human transcriptome diversity, we profiled six representative cell types spanning three distinct biological contexts, including metabolism with, primary patient-derived adipogenic cells at two differentiation stages, and iPSC derived hepatic progenitor cells; the nervous system with iPSC-derived neurons, neural progenitor cells (NPCs), and pluripotency using induced pluripotent stem cells (iPSCs). Together, these datasets yielded over 235 million full-length long reads. First, to assess data coverage and transcriptome depth, we quantified protein-coding gene expression, detecting 14,312 genes (73.6% of all annotated protein-coding genes), which is a level consistent with deep and comprehensive transcriptome representation. Second, focusing on TE-derived transcripts, we identified >83,000 previously unannotated isoforms, the vast majority (84%) originating from a complex combination of multi-TEs. We also identified solo TEs, which are predominantly from LINE1 (14%). We confirmed that TE-transcripts are able to be exemplified by signatures detected in Liver Hepatocellular Carcinoma (LICH). Together, MAS-ISO-seq and TEscape establish the first long-read-based, high-resolution atlas of transcribed human TEs, providing a foundational resource for integrative transcriptome analyses and for investigating TE expression and regulation in health and disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/737305v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@18158aeorg.highwire.dtl.DTLVardef@e51fdforg.highwire.dtl.DTLVardef@8f9504org.highwire.dtl.DTLVardef@804113_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

The growing number of approved nucleic acid therapeutics illustrates the potential to treat diseases by targeting their genetic blueprints in vivo. The 10-23 DNAzyme is capable of cleaving a wide range of target RNA with high selectivity. However, its poor performance in vivo restricts its therapeutic application as gene silencing agent. Studies on ribozymes have shown that the crowded environment in cells and associated effects can impact ribozyme folding and thermostability, resulting in a change in activity. This opens up the question whether DNAzymes are also affected by molecular crowding. Here, we investigate the functional and structural influence of molecular crowding conditions on the 10-23 DNAzyme. The stability and activity of a PrP-specific 10-23 DNAzyme were examined in presence of PEG, dextran, and osmolytes. Our results indicate that osmolytes decrease DNAzyme activity in a concentration-dependent manner, while certain PEG and dextran concentrations promote activity. To rationalize our observations, we studied the cosolutes effect on physicochemical solution properties and the structure of the DNAzyme:RNA complex using FCS and SAXS. The data reveal that enhanced activity is observed under conditions where a combination of physiochemical properties matches an optimum that seems to be dependent on the metal ion cofactor. Structural influence under such conditions is indicated less. We propose that a certain degree of molecular crowding is required to favor a state, which allows for higher catalytic turnover. In addition, we show that the requirement for magnesium and manganese as a cofactor remains unchanged under the conditions applied. Our work contributes to a better understanding of how the cellular environment affects DNAzyme structure and function.

Inorganic phosphate (Pi) is a critical building block for key biomolecules including ATP, DNA, RNA, and phospholipids. Consequently, cells monitor phosphate levels and acquire phosphate when intracellular levels become low. Here we demonstrate an unanticipated connection between the enzymatic activity of the S. cerevisiae RNase T2 ortholog, Rny1, and inorganic phosphate availability. Rny1 has been studied for its role in autophagy-linked RNA degradation under starvation conditions. Here we find that in nutrient-rich conditions, cells lacking Rny1 function exhibit phosphate starvation phenotypes and aberrantly activate the PHO signaling pathway despite the presence of high levels of inorganic phosphate in the growth media. This activation is evidenced by increased PHO gene transcript levels and increased nuclear localization of Pho4 in rny1{Delta} strains. Complementation of rny1{Delta} with wild-type RNY1 and human RNase T2 restores PHO transcript levels to those typically observed in exponentially growing cells. This implicates RNase T2-dependent RNA degradation as required for maintaining intracellular phosphate levels, even under phosphate-replete conditions. Furthermore, consistent with its potential role in freeing phosphate from degraded RNA, RNY1 expression is itself further induced under phosphate-limited conditions. These observations suggest that RNase T2-mediated RNA decay is a part of a potentially conserved metabolic recycling pathway that provides Pi to growing cells. These findings reframe RNA as a key metabolic resource and positions RNase T2 enzymes, and their function in RNA degradation, as an unexpected player in phosphate homeostasis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/739639v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@17a63b3org.highwire.dtl.DTLVardef@278addorg.highwire.dtl.DTLVardef@db33forg.highwire.dtl.DTLVardef@172ff96_HPS_FORMAT_FIGEXP M_FIG C_FIG

Previous studies revealed a crosstalk between intronic polyadenylation (IPA) and the DNA damage response (DDR). Indeed, genotoxic agents, including radiations and anticancer drugs (e.g., cisplatin that crosslinks DNA), regulate the ratio of IPA to last-exon transcripts in many genes. Conversely, multiple genes involved in the DDR, especially homologous recombination, are regulated at the IPA level. The U1 small nuclear RNA (snRNA) widely represses IPA, thereby enhancing full-length gene transcription. However, besides its implication in IPA regulation by ultraviolet-C radiation, little is known about U1 snRNA effects on the DDR and on cell sensitivity to genotoxic agents. Here, we show that U1 snRNA blockade using an antisense oligonucleotide (U1-AMO) in lung cancer cell lines enhances cell growth inhibition by cisplatin, through an increase in cisplatin-induced DNA damage. 3-seq analysis indicates that U1 snRNA blockade represses full-length mRNA expression of multiple genes of the nucleotide-excision repair and Fanconi anemia pathways, which are involved in the repair of cisplatin-DNA crosslinks. Our 3-seq analyses also reveal that moderate doses of U1-AMO and cisplatin upregulate the IPA:LE isoform ratio in overlapping but distinct sets of genes, and that U1-AMO prevents cisplatin effects on the IPA:LE ratio in a large subset of genes. Altogether, these data extend the crosstalk between IPA and the DDR and suggest that U1 snRNA targeting may be used to sensitize cancer cells to genotoxic agents.