RNA

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.

The bacterial three-hybrid (B3H) assay is a powerful genetic tool for detecting interactions between RNA and RNA-binding proteins (RBPs) and assessing the consequences of RBP mutations. This transcription-based system connects the strength of an RNA-protein interaction to the expression of a lacZ reporter gene in Escherichia coli cells. This in vivo approach allows researchers to dissect RNA-protein interactions within a cellular environment, bypassing the need for biochemical purification of RNAs or proteins. This chapter details a three-day protocol for generating quantitative B3H data. Since a significant challenge in B3H assays is RNA misfolding, we describe a recently optimized set of B3H constructs that mitigates this issue by isolating bait RNAs as discrete folding units.

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.

Transposable element-derived long intergenic noncoding RNAs are increasingly recognized as context-dependent regulators of gene expression, but the functional consequences of their ectopic activation in somatic cells remain poorly understood. We previously showed that U7 snRNA represses a subset of LTR12-associated lincRNAs, including lnc-ARRDC4-1 and lnc-ADCYAP1-2, two testis-enriched lincRNAs with minimal expression in somatic cells. Here, we examined the consequences of their increased expression in somatic cells. We showed that overexpression of either lincRNA led to overlapping transcriptomic and proteomic changes, impaired migration, altered adhesion and proliferation, and a [~]50% reduction in protein synthesis. Furthermore, we identified lnc-ARRDC4-1 as an upstream regulator of lnc-ADCYAP1-2 transcription. Downstream of this event, lnc-ADCYAP1-2 interacts with the RNA helicase DHX36, a regulator of G-quadruplex-containing mRNAs. lnc-ADCYAP1-2 activation reduces DHX36 protein levels which is accompanied by decreased protein output from a subset of DHX36 mRNA targets. At the cellular level, these effects correlate with altered cell proliferation, migration, adhesion, and global translation. Our results suggest a lnc-ARRDC4-1: lnc-ADCYAP1-2 : DHX36 regulatory cascade linking de-repression of LTR12-containing lincRNAs to reduced protein synthesis and altered cellular processes in somatic cells.

Processing of the precursor ribosomal RNAs (pre-rRNAs) is a key aspect of ribosomal subunit assembly that is closely coordinated with other maturation events. The ribonucleases that mediate pre-rRNA cleavages require regulation to ensure that their activities are exerted in a timely manner. Post-translational modifications can influence protein functions, and although many human ribosome assembly factors are reported to be post-translationally modified, most of these sites remain unconfirmed and functional insights are lacking. Here, we show that NOB1, the PIN domain endoribonuclease responsible for cleavage of the 3' end of the 18S rRNA, is phosphorylated within an evolutionarily conserved acidic tract that can be modified by casein kinase II in vitro. Association of NOB1 with pre-ribosomes is independent of these phosphorylations, and lack of NOB1 phosphorylation only mildly perturbs the efficiency of SSU maturation events upstream of 3' end cleavage of the 18S rRNA. Interestingly, our analyses of pre-rRNA levels in cells depleted of NOB1 or lacking its catalytic activity revealed not only accumulation of the 18SE precursor of the 18S rRNA, but also altered levels of pre-rRNAs containing 5' external transcribed spacer (ETS) sequences (43S, 26S and 30S). This suggests that lack of NOB1-mediated pre-rRNA cleavage impairs recycling of assembly factors required during early biogenesis steps, leading to altered kinetics of 5' ETS processing. Taken together these data provide new insights into the role of NOB1 during SSU biogenesis and the post-translational regulation of this ribonuclease.

The RNA-binding proteins Musashi1 and Musashi2 (MSI1 and MSI2) regulate stem cell function and tissue plasticity by modulating mRNA translation. While typically known as translational repressors, the MSI1 and MSI2 proteins can also act as context-dependent activators of mRNA translation, although the mechanism of MSI-mediated translational activation are unknown. Here, we identify Embryonic Lethal Abnormal Vision-like (ELAVL) proteins as essential co-regulators of MSI1-dependent translational activation. In Xenopus laevis oocytes, antisense oligonucleotide knockdown of Elavl4 inhibited progesterone-stimulated maturation and blocked polyadenylation and translation of key MSI target mRNAs, including the Mos and Cyclin B5 mRNAs. Exogenous expression of ELAVL4 rescued these defects, confirming its necessity for maturation and cell cycle progression. Mechanistically, we determined that the ELAVL4 C-terminal domain interacts with the N-terminal RNA recognition motifs of MSI1 in an RNA-independent manner. Mass spectrometry and functional assays revealed this interaction is evolutionarily conserved: mouse ELAVL1 interacts with MSI1 in the pituitary, and human ELAVL1 rescues Elavl4-depleted Xenopus oocytes. Furthermore, knockdown of Elavl1 in a mammalian cell line abrogated MSI-dependent translational activation of a pituitary Prop1 3-UTR mRNA reporter. Our results establish a conserved mechanism where ELAVL family members interact with MSI to promote MSI-dependent mRNA translational activation.

Non-coding RNAs play diverse roles in a wide range of cellular processes, with their spatial structure being pivotal to their function. RNA secondary structure is a key determinant of its overall fold. Given the scarcity of experimentally determined RNA 3D structures, understanding secondary structure is vital for discerning RNA function. Currently, there is no universally effective solution for de novo RNA secondary structure prediction. Existing methods are becoming increasingly complex without marked improvements in accuracy and often overlook critical features such as pseudoknots and alternative folds. Here, we introduce SQUARNA, a new approach to de novo RNA secondary structure prediction that is suitable for both individual RNA analysis and large-scale structural searches. SQUARNA revisits the concept of base pair maximization and develops it into a stem maximization idea coupled with the widely used free energy minimization (MFE) framework. SQUARNA can predict alternative structures and handle pseudoknots of arbitrary complexity. Benchmarking shows that SQUARNA outperforms existing methods, including deep learning models, in both single-sequence and alignment-based RNA secondary structure prediction. SQUARNA seamlessly integrates sequence and alignment information with experimental data, such as residue reactivities obtained by chemical probing, as well as other structural restraints, including automated searches for Rfam database templates, G-quadruplex patterns, and protein-binding motifs. SQUARNA is available as a standalone tool at https://github.com/febos/SQUARNA and as a web server at https://larnal.imol.institute.

Analysis of RNA-binding proteome data from different bacterial species revealed many cell wall metabolic enzymes cross-linking to RNA in vivo, hinting that these proteins directly bind RNA. Surprisingly, penicillin-binding proteins (PBPs) were also abundantly identified as putative RNA-binding proteins. The cell surface localisation properties of many of these proteins therefore beg the question at what stage of their cellular life cycle these proteins interact with RNA and what the functional significance is. Here, we characterised the RNA-binding activity of PBP2a, the alternative transpeptidase that confers {beta}-lactam resistance in MRSA. Using in vivo RNA-binding assays, we show that PBP2a interacts with hundreds of transcripts without apparent sequence specificity. Computational analyses identified a possible RNA-binding cleft in PBP2a proximal to its active site. Mutation of only two predicted positively charged residues located in this cleft substantially reduced cross-linking in vivo, implying that RNA recognition is largely dictated by RNA backbone interactions. While PBP2a does not regulate RNA steady-state levels, RNA-binding appears important for proper protein function: an RNA-binding deficient mutant exhibits reduced oxacillin resistance. These findings establish PBP2a as an RNA-binding protein in vivo and provide a framework to investigate how this non-canonical interaction may relate to cell wall biogenesis and {beta}-lactam resistance.

Small RNAs (sRNAs) rarely cause strong growth phenotypes upon overexpression, complicating efforts to link regulatory interactions to physiological outcomes. Here, we report that high levels of the Escherichia coli sRNA OmrA, but not its sibling OmrB, severely inhibit growth in glucose minimal medium. Genetic, biochemical, and physiological analyses indicate that OmrA-dependent toxicity results from reduced flux through the tricarboxylic acid (TCA) cycle. A UV-based suppressor screen identified mutations in the gene encoding Hfq, the RNA-chaperone that aids sRNA-mRNA interactions. Secondly, three independent mutations clustered in the ribosome-binding site of ppc, encoding phosphoenolpyruvate carboxylase, a key anaplerotic enzyme. OmrA directly inhibits Ppc translation via Hfq-dependent base-pairing in the ppc 5' UTR, including the mutated nucleotides obtained in the genetic screen. OmrA is significantly more effective than OmrB in ppc repression in vivo and in vitro, consistent with sequence divergence in their central regions. Supplementation with glutamate, glutamine, or downstream TCA cycle metabolites fully restores growth, linking reduced Ppc levels to metabolic limitation. These results identify ppc as a physiologically relevant OmrA target and suggest how RNA toxicity can uncover central metabolic nodes used by sRNAs to modulate bacterial physiology.

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.

Small regulatory RNAs (sRNAs) are key players in bacterial adaptation to stress. They often occupy central positions in regulatory networks and control the expression of multiple targets. In a striking example of this, the enterobacterial OmrA and OmrB paralogous sRNAs are known to regulate about ten different targets, with extensive data suggesting the regulon is in fact much larger. Here we performed transcriptome and proteome analyses and identified more than fifteen new targets of Escherichia coli OmrA and OmrB. We validated several, including genes involved in central carbon metabolism and fatty acid synthesis, among which ppc, actP and fabA. Consistent with a role in carbon metabolism, overproducing OmrA or OmrB inhibited growth on glucose minimal medium. The analysis of suppressor mutants shows that this is due to a decreased carbon flux through the TCA cycle. Incorporating other datasets such as RIL-seq, we generated a multi-omics-based prediction of target candidates. Together, our results show that OmrA/B base-pair to various regions of their mRNA targets, and therefore likely act through diverse regulatory mechanisms. Hence, this work extends the OmrA and OmrB regulons, establishes an unsuspected connection with carbon usage, and shows the benefits of combining global analyses to investigate sRNA regulons.

Interleukin 27 (IL-27) is an anti-HIV cytokine that induces 14 novel microRNAs (miRNAs) in T cells. We previously reported that transfection of two of these miRNA mimics, miRTC10 and miRTC14, differentially induced interferons (IFN)A2, A8, A13, and L1 expression in human primary macrophages. However, the mechanism underlying this activation remains unclear. Here, we show that miRTC14 does not directly target IFN-regulatory genes but instead engages cytosolic RNA-sensing proteins. Using miRNA pull-down coupled with mass spectrometry and immunoblotting, we identified a metabolic enzyme, pyruvate carboxylase (PC) and laboratory of genetics and physiology 2 (LGP2/DHX58) as direct binding partners of miRTC14. Functional analyses revealed that miRTC14 induces IFN expression by more than100-fold (p < 0.001), whereas PC and LGP2 depletion markedly attenuated this response (50-100 fold reduction, p < 0.01). Reconstitution of PC and LGP2 in deficient HEK293 cells restored miRTC14-driven IFN induction. We found that miRTC14-induced IFN activation depends on sequence features at the duplex termini and is unlikely to arise from canonical miRNA-mediated gene silencing. These findings establish PC as a novel miRNA-binding protein and define a previously unrecognized RNA-sensing mechanism by which miRTC14 drives IFN production, linking metabolic enzymes to RNA sequence-dependent innate immunity.

Bacterial ribosomal RNA (rRNA) methylations are important for accurate translation. Four distinct methylations incorporated by RsmE, RsmF, and RsmH/ RsmI form a cluster of three modified 16S rRNA nucleotides (m3U1498, m5C1407, and m4Cm1402) surrounding the decoding center of the 30S subunit. Given their common substrate requirement of a late-stage intermediate 30S subunit, these enzymes likely act contemporaneously during subunit biogenesis, but whether there exists a required modification order is unknown. Here, using hypomethylated 30S subunits obtained from a collection of rsmH/I/E/F-deleted Escherichia coli strains, we identify RsmF activity to be highly dependent on prior modification of h44 both in vitro and in E. coli. RsmF activity on hypomethylated 30S subunits could be partially rescued by prior in vitro methylation using RsmE and RsmH, indicating that incorporation of these methyl groups directly shapes h44 for recognition by RsmF. RNA structure probing using SHAPE-MaP and molecular dynamics simulations reveal specific alterations in 16S rRNA structure and dynamics in the absence of the m4C1402 (RsmH) and m3U1498 (RsmE) modifications that likely restrict RsmF action. These studies thus uncover a previously unappreciated "order of operations" for 16S rRNA modification during ribosome biogenesis with important implications for studies on the collective functions of these modifications.

The Illumina TruSeq Stranded and Illumina Stranded mRNA protocols are commonly used for strand-specific bulk RNA-seq and they typically yield >99% antisense reads. We show that these protocols can generate sense-oriented reads for transcripts with extremely low U content (<3%). Indeed, such regions can bypass the dUTP-based blockade of cDNA second strand amplification. A small number of genes are affected by this issue (three in Drosophila melanogaster, including the glue gene Sgs3, and 46 in Mus musculus). To prevent overestimation of expression levels, we recommend excluding sense reads for all genes.

The unique solvent milieu found in biomolecular condensates can control cellular enzymatic reactions and shift reaction kinetics by modulating reactant concentrations, structural dynamics, and enzyme activities. Here we explore the interplay of multiple regulatory factors within a condensate to control poly(A) RNA deadenylation, the first and rate-limiting step in mRNA turnover. The deadenylase CNOT7, a subunit of the CCR4-NOT deadenylation complex, localizes to cytoplasmic RNA granules and shows increased degradation activity in vitro in condensates formed by the C-terminal low complexity disordered region of CAPRIN1, a component of RNA granules. We use a combination of enzymatic assays, kinetic modeling, microscopy, Nuclear Magnetic Resonance (NMR) spectroscopy, and molecular dynamics simulations to deconvolute and define the components that underlie this enhancement. We found that enzyme and RNA are concentrated in condensates relative to buffer, which increases CNOT7 activity, while the equilibrium between CNOT7's active and inactive states remains unchanged. The concentration-dependent increase in enzymatic rates is counterbalanced by a substantial decrease in the enzyme's catalytic efficiency, likely due to slower diffusion of CNOT7 and RNA within the condensates, which lessens the probability of enzyme-substrate complex formation. Molecular dynamics simulations reveal CNOT7-CAPRIN1 interactions that rely on conserved CAPRIN1 sequence features, hinting at an evolutionarily conserved role for CAPRIN1 condensation. With this quantitative kinetic analysis, we describe the multifaceted mechanism behind regulation of CNOT7 deadenylation by a condensate environment.

The regnase family of endoribonucleases regulates immune gene expression through degradation of cellular mRNAs. Regnase-1 is known to also target viral RNA, but whether antiviral activity is a conserved property of all human regnases remains unknown. Here, we systematically compare the antiviral properties of all four human regnases. Regnases-1-4 expression inhibited HIV-1, HIV-2, MLV, RSV and hCoVs OC43 and SARS-CoV-2, but not the DNA virus HSV-1. Endogenous knockout and knockdown experiments demonstrated that physiological regnase expression restricts HIV-1 replication in a cell-type-dependent manner. Regnase-1 and regnase-4 were induced by interferons in macrophages, and all four regnases displayed signatures of positive selection during mammalian evolution, consistent with their potential roles as antiviral restriction factors. Mechanistically, antiviral activity of regnases required intact catalytic core and CCCH zinc finger domains, while nuclear shuttling and dimerisation site conservation were not shared features of all family members. Domain-swap and reporter analyses further showed that differences in antiviral potency between regnases primarily reflect differential RNA target recognition rather than catalytic activity. Regnase-1 exhibited broad RNA targeting, whereas regnases-2-4 displayed more selective targeting profiles. Collectively, our findings establish the human regnase family as evolutionarily diversified antiviral RNA restriction factors with distinct substrate specificities.

Although glycosylated small non-coding RNAs are emerging players in cancer, their functions in lung adenocarcinoma (LUAD) are largely unknown. We identify SNORA73B as a glycosylated small nucleolar RNA (glycol-snoRNA) that carries sialic acid-capped O-glycans in both normal lung epithelial and LUAD cells. SNORA73B is markedly elevated in LUAD, and its plasma levels distinguish early-stage LUAD from healthy controls with an area under the curve (AUC) of 0.7903. Subcellular fractionation reveals predominant nuclear localization. Functional assays demonstrate that SNORA73B depletion curbs LUAD cell proliferation, migration, and invasion, whereas its overexpression fosters these malignant phenotypes and accelerates tumor growth. Mechanistically, SNORA73B directly binds the T-cell-restricted intracellular antigen-related protein (TIAR), thereby enhancing TIAR protein abundance without affecting its mRNA levels. TIAR then recognizes the 3-untranslated region (3-UTR) of MYC mRNA to upregulate c-Myc, which subsequently augments AKT phosphorylation. Importantly, c-Myc knockdown largely rescues the oncogenic phenotypes and tumorigenesis induced by SNORA73B overexpression. Collectively, our data unveil a glycoRNA-dependent oncogenic axis SNORA73B-TIAR-c-Myc-AKT that drives LUAD progression. These findings position SNORA73B as a promising early diagnostic biomarker and a candidate therapeutic target in LUAD.

Background: Long non-coding RNAs (lncRNAs) exhibit rapid evolutionary turnover, often driven by genomic duplication. How paralogous lncRNAs maintain, partition, or diverge in function across distinct genomic contexts remains poorly understood. The evolutionarily conserved lncRNA MORRBID and its primate-specific paralog CYTOR provide a natural framework to interrogate the functional consequences of lncRNA duplication. Results: Although CYTOR and MORRBID have acquired distinct transcript variants influenced by their divergent genomic environments, we demonstrate that they maintain a robust, shared core function encoded by near-identical dominant two-exon transcripts. Using SNP-based paralog-specific quantification, we found that CYTOR contributes more strongly to the shared transcript pool, while both transcripts localize predominantly to the cytoplasm, consistent with a shared trans-acting function. Simultaneous repression of CYTOR and MORRBID consistently impairs cell adhesion and migration across multiple cancer models. Mechanistically, the shared CYTOR/MORRBID transcript pool associates with MEK2 and sustains MEK-ERK signaling. This signaling axis promotes FOSL1 expression and AP-1-linked transcriptional output, including expression of the downstream effector EPHA4, whose role was supported by rescue experiments. Patient tumor transcriptomes and healthy single-cell datasets further supported the associated mesenchymal, adhesion, and epithelial-mesenchymal transition program. Conclusions: Our findings establish that paralogous lncRNAs can retain a conserved mechanistic core despite context-dependent transcriptional divergence. The CYTOR/MORRBID transcript pool defines a shared lncRNA signaling module that supports MAPK-ERK signaling and adhesion-migration programs across cancer and mesenchymal-like cellular contexts. This defines a unified mechanistic framework for the shared core function of these widely studied paralogous lncRNAs.

BACKGROUNDTAR DNA binding protein - 43 (TDP-43) nuclear loss is a pathological hallmark of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and related neurodegenerative disorders. While the consequences of TDP-43 dysfunction have been well-characterized, the mechanisms driving TDP-43 mislocalization remain poorly understood. Previous observations of altered localization and function of the adenosine-to-inosine (A-to-I) RNA editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) in ALS/FTD tissue prompted us to investigate whether dysregulated RNA editing contributes to pathological TDP-43 nucleocytoplasmic trafficking. METHODSTDP-43 cytoplasmic mislocalization was assessed following ADAR2 and TDP-43 co-overexpression in HEK293T cells and a Drosophila model co-overexpressing human TDP-43 and dADAR in motor neurons. We further evaluated TDP-43 mislocalization through both HeLa cell assays and interspecies heterokaryon assays. Next, we assessed TDP-43 binding to A-to-I edited RNA oligomers through electrophoretic mobility shift assays (EMSAs), and investigated inosine-containing RNAs in vivo via TDP-43 RNA immunoprecipitation followed by sequencing (RIP-seq) datasets from human TDP-43-expressing Drosophila. Finally, RNAseq and enhanced cross-linking and immunoprecipitation (eCLIP-seq) were performed in SH-SY5Y cells overexpressing three ADAR2 variants with differing editing activity to identify editing-related transcriptional alterations and RNAs differentially bound to TDP-43. RESULTSADAR2 overexpression reduced the nucleocytoplasmic (N:C) ratio of TDP-43 in HEK293T cells in a ADAR2 catalytic activity- and TDP-43 RNA-binding capacity-dependent manner. Drosophila motor neurons overexpressing dADAR also exhibited decreased nuclear TDP-43. Interspecies heterokaryons and permeabilized HeLa cell assays demonstrated that catalytically active ADAR2 and synthetic inosine-containing RNA oligomers, respectively, enhance nuclear export of endogenous TDP-43. EMSAs revealed preferential binding of TDP-43 to inosine-containing RNAs relative to unedited RNAs, and analysis of Drosophila RIP-seq datasets demonstrated enrichment of edited transcripts within TDP-43-bound RNAs. Finally, RNAseq and eCLIP-seq analyses identified editing-dependent alterations in gene expression and TDP-43 RNA-binding profiles in SH-SY5Y cells overexpressing active ADAR2 variants. CONCLUSIONSTogether, our findings identify A-to-I RNA editing as a previously unrecognized regulator of TDP-43 localization and RNA interactions. These results support a model where altered RNA editing modifies TDP-43-RNA interactions, promoting increased nuclear export of TDP-43. Broadly, our work highlights RNA editing dysregulation as a potential contributor to early pathogenic mechanisms underlying TDP-43 proteinopathies.

Long noncoding RNAs (lncRNAs) play roles in gene regulation across kingdoms of life. However, lncRNAs with related functions often lack linear sequence similarity, making it difficult to leverage studies of one lncRNA to inform the understanding of others. We describe a k-mer-based hidden Markov model, hmSEEKR, that enables the scanning of transcriptomes for regions of non-linear sequence similarity to a query domain, without prior knowledge of where within the transcriptome the similarities may be located. When individual lncRNA domains were used as search features, hmSEEKR successfully identified regions in other RNAs that harbor non-linear sequence similarity and bind similar sets of proteins. Applying hmSEEKR to transcriptome-wide searches, we found that certain domains within the lncRNAs XIST, NEAT1, and MALAT1 exhibited widespread regional similarity to both lncRNA and protein-coding genes, while others were more unique, exhibiting similarity to ~100 genes or fewer. Combinatorial searches uncovered RNAs containing sequential matches to core functional domains of XIST and NEAT1, and eCLIP-inferred protein-interaction networks within these RNAs more closely resembled those of XIST and NEAT1, respectively, than would be expected by chance, suggesting the searches recovered RNAs with similar biological properties. Finally, within annotated sets of cis-activating and cis-repressive lncRNAs, we observed opposing enrichments for similarity to domains associated with transcription-promoting complexes and heterogeneous nuclear ribonucleoprotein (hnRNP) binding, respectively, suggesting the enriched sequences may contribute to regulatory functions. hmSEEKR can be applied with minimal training data and enables the a priori discovery of RNA domains that share nonlinear similarity, offering a sequence-informed approach to discover functional elements within noncoding transcriptomes.