Small nuclear RNAs (snRNAs) combine with specific proteins to generate small nuclear ribonucleoproteins (snRNPs), the building blocks of the spliceosome. U4 snRNA forms a duplex with U6 and, together with U5, contributes to the tri-snRNP spliceosomal complex. Variants in RNU4-2, which encodes U4, have recently been implicated in neurodevelopmental disorders. Here we show that heterozygous inherited and de novo variants in RNU4-2 and in four RNU6 paralogs (RNU6-1, RNU6-2, RNU6-8 and RNU6-9), which encode U6, recur in individuals with nonsyndromic retinitis pigmentosa (RP), a genetic disorder causing progressive blindness. These variants cluster within the three-way junction of the U4/U6 duplex, a site that interacts with tri-snRNP splicing factors also known to cause RP (PRPF3, PRPF8, PRPF31), and seem to affect snRNP biogenesis. Based on our cohort, deleterious variants in RNU4-2 and RNU6 paralogs may explain up to ~1.4% of otherwise undiagnosed RP cases. This study highlights the contribution of noncoding RNA genes to Mendelian disease and reveals pleiotropy in RNU4-2, where distinct variants underlie neurodevelopmental disorder and retinal degeneration.

MicroRNAs (miRNAs) are small non-protein-coding RNAs that regulate gene expression in many eukaryotes. Next-generation sequencing of small RNAs (small RNA-seq) is central to the discovery and annotation of miRNAs. Newly annotated miRNAs and their longer precursors encoded by MIRNA loci are typically submitted to databases such as the miRBase microRNA registry following the publication of a peer-reviewed study. However, genome-wide scans using small RNA-seq data often yield high rates of false-positive MIRNA annotations, highlighting the need for more robust validation methods. miRScore was developed as an independent and efficient tool for evaluating new MIRNA annotations using sRNA-seq data. miRScore combines structural and expression-based analyses to provide rapid and reliable validation of new MIRNA annotations. By providing users with detailed metrics and visualization, miRScore enhances the ability to assess confidence in MIRNA annotations. miRScore has the potential to advance the overall quality of MIRNA annotations by improving accuracy of new submissions to miRNA databases and serving as a resource for re-evaluating existing annotations.

Vertebrate branchial arches (BAs) are a developmental paradigm, undergoing coordinated differentiation and morphogenesis to form various adult derivative tissues. MicroRNAs can strengthen gene regulatory networks (GRNs) to promote developmental stability. To investigate microRNA-mediated regulation in BA development, we generated a novel microRNA-sequencing dataset from mouse BAs. We identified 550 expressed microRNAs, of which approximately 20% demonstrate significant differential expression across BA domains. The three most posterior BAs and the connecting outflow tract (PBA/OFT) express genes important for cardiovascular development. We predicted microRNA-target interactions with PBA/OFT-expressed cardiovascular genes and found target sites for miR-92b-3p to be enriched. We used a dual luciferase assay to validate miR-92b-3p interactions with two transcripts encoding the fundamental cardiac transcription factors (TFs), Gata6 and Tbx20. Furthermore, we demonstrated that miR-92b-3p mimic can downregulate endogenous GATA6 and TBX20 in human embryonic stem cells (hESCs) undergoing cardiomyocyte differentiation, confirming microRNA-target binding can occur in a cardiac cell type. miR-92b-3p has previously been shown to target transcripts encoding for two other cardiac TFs, Hand2 and Mef2D. Therefore, we hypothesise that miR-92b-3p acts to stabilise cardiovascular GRNs during PBA/OFT development, through multiple microRNA-mediated regulatory networks.

Curation of literature in life sciences is a growing challenge. The continued increase in the rate of publication, coupled with the relatively fixed number of curators worldwide, presents a major challenge to developers of biomedical knowledgebases. Very few knowledgebases have resources to scale to the whole relevant literature and all have to prioritize their efforts. In this work, we take a first step to alleviating the lack of curator time in RNA science by generating summaries of literature for noncoding RNAs using large language models (LLMs). We demonstrate that high-quality, factually accurate summaries with accurate references can be automatically generated from the literature using a commercial LLM and a chain of prompts and checks. Manual assessment was carried out for a subset of summaries, with the majority being rated extremely high quality. We apply our tool to a selection of >4600 ncRNAs and make the generated summaries available via the RNAcentral resource. We conclude that automated literature summarization is feasible with the current generation of LLMs, provided that careful prompting and automated checking are applied. Database URL: https://rnacentral.org/

RNAcentral was founded in 2014 to serve as a comprehensive database of non-coding RNA sequences. It began by providing a single unified interface to more specialised resources, and now contains 45 million sequences. It has grown beyond providing a single interface to many specialised resources and now provides several services and analyses. These include secondary structure prediction with R2DT, sequence search, and analysis with Rfam. Since its last publication in 2021, RNAcentral has developed two major features. First, literature integration with the development of LitScan and LitSumm. LitScan automatically identifies and links relevant publications to RNA entries, while LitSumm uses natural language processing to generate functional summaries from the literature. Together, these tools address the critical challenge of connecting sequence data with scattered functional knowledge across thousands of publications. Secondly, RNAcentral has created gene level entries. Gene level entries represent a large structural change to RNAcentral. While RNAcentral previously organized data exclusively at the sequence level, we now group related transcripts into gene-centric views. This allows researchers to explore all isoforms, splice variants, and related sequences for a gene in a unified interface, better reflecting biological organization and facilitating comparative analyses. RNAcentral is freely available at: https://rnacentral.org .

The U4 small nuclear RNA (snRNA) forms a duplex with the U6 snRNA and, together with U5 and ~30 proteins, is part of the U4/U6.U5 tri-snRNP complex, located at the core of the major spliceosome. Recently, recurrent de novo variants in the U4 RNA, transcribed from the RNU4-2 gene, and in at least two other RNU genes were discovered to cause neurodevelopmental disorder. We detected inherited and de novo heterozygous variants in RNU4-2 (n.18_19insA and n.56T>C) and in four out of the five RNU6 paralogues (n.55_56insG and n.56_57insG) in 135 individuals from 62 families with non-syndromic retinitis pigmentosa (RP), a rare form of hereditary blindness. We show that these variants are recurrent among RP families and invariably cluster in close proximity within the three-way junction (between stem-I, the 5' stem-loop and stem-II) of the U4/U6 duplex, affecting its natural conformation. Interestingly, this region binds to numerous splicing factors of the tri-snRNP complex including PRPF3, PRPF8 and PRPF31, previously associated with RP as well. The U4 and U6 variants identified seem to affect snRNP biogenesis, namely the U4/U6 di-snRNP, which is an assembly intermediate of the tri-snRNP. Based on the number of positive cases observed, deleterious variants in RNU4-2 and in RNU6 paralogues could be a significant cause of isolated or dominant RP, accounting for up to 1.2% of all undiagnosed RP cases. This study highlights the role of non-coding genes in rare Mendelian disorders and uncovers pleiotropy in RNU4-2, where different variants underlie neurodevelopmental disorder and RP.

The intestine is a site of diverse functions including digestion, nutrient absorption, immune surveillance, and microbial symbiosis. Intestinal microRNAs (miRNAs) are detectable in faeces and regulate barrier integrity, host-microbe interactions and the immune response, potentially offering valuable non-invasive tools to study intestinal health. However, current experimental methods are suboptimal and heterogeneity in study design limits the utility of faecal miRNA data. Here, we develop an optimised protocol for faecal miRNA detection and report a reproducible murine faecal miRNA profile in healthy mice. We use this pipeline to study faecal miRNAs during infection with the gastrointestinal helminth, Trichuris muris, revealing roles for miRNAs in fibrosis and wound healing. Intestinal fibrosis was confirmed in vivo using Hyperion (R) imaging mass cytometry, demonstrating the efficacy of this approach. Further applications of this optimised pipeline to study host-microbe interactions and intestinal disease will enable the generation of hypotheses and therapeutic strategies in diverse contexts.

PIWI-interacting RNAs (piRNAs) are a group of short (∼21-31 nucleotide) non-coding RNAs that protect the germline of metazoans against the activity of genomic parasites known as transposable elements (TEs). Although originally discovered in Drosophila where they are germ-line restricted, recent studies in arthropods have shown piRNAs are often expressed also in somatic cells suggesting they might have originally evolved as a somatic immune system protecting against the detrimental action of TEs in the last common ancestor. Beyond their classic TE-silencing roles piRNAs functions range from sex determination in moths, to degradation of maternal mRNAs during the maternal-to-zygotic transition, thus highlighting the need for more sampling across the tree of life, particularly in underrepresented speciose rich subphyla such as the crustaceans were to date only two representatives have been analysed. In this study we sequenced and analysed putative piRNAs in the amphipod Parhyale hawaiensis , throughout a time-course of embryogenesis. Initially, during early embryogenesis piRNAs-mapping reads were abundant, maternally loaded piRNAs and showed the hallmark signatures of piRNA processing suggesting active targeting of TEs. Interestingly, this initial high content was followed by a dramatic loss of piRNA-mapping transcripts and signatures during mid-embryogenesis shortly after germ cell specification. This was confirmed by in-situ hybridization of key mRNAs coding for piRNA pathway proteins which revealed their expression becomes restricted specifically to the germ cells by early germband formation, providing an explanation for the observed reduction in piRNA abundance at later stages, and suggesting that piRNAs are absent in Parhyale post embryonic somatic cells. ### Competing Interest Statement The authors have declared no competing interest. Wellcome Trust, https://ror.org/029chgv08, 203990/Z/16/A

Current models of microRNA (miRNA) silencing posit that RNA-sequence rules are sufficient for canonical targeting of mRNAs by Argonaute 2 (AGO2), the central protein of the miRNA-induced silencing complex (miRISC). Using chimeric eCLIP in CRISPR-edited LIMD1+/+, LIMD1+/−, and LIMD1−/− human small airway epithelial cells (hSAECs), we reveal a transcriptome-wide dependency on LIMD1, an AGO2 adaptor, for effective miRNA targeting and repression. In LIMD1-deficient cells, miRNA loading is uncoupled from productive targeting: despite increased AGO2–miRNA interactions, complexes engage fewer transcripts and sites, reducing occupancy and more than halving both the breadth and depth of targeting. We also observe altered AGO2 positional footprints across targets in LIMD1-deficient cells. LIMD1 dependence is most pronounced at defined RNA contexts: weak (GC-poor) seed pairings, interactions involving evolutionarily young miRNAs or sites that nonetheless form thermodynamically stable duplexes, with these losses particularly enriched in coding sequences of rapidly evolving C2H2-zinc-finger genes. Even within canonical seed repertoires of individual AGO2–miRNAs, LIMD1 is most critical at poorly conserved sites, indicating that LIMD1 broadens miRNA regulation beyond ancient, deeply conserved targets. In culture, LIMD1 deficiency de-represses oncogenic proteins that, in vivo , inversely correlate with LIMD1 levels in normal lung and adenocarcinoma, where LIMD1 is characteristically reduced, and whose dysregulation predicts poor survival. Thus, LIMD1 emerges as a key determinant of miRISC architecture, targeting, and potency, challenging RNA-centric models of miRNA function and exemplifying how adaptor proteins diversify post-transcriptional regulation. ![Figure][1] LIMD1 defines the scope of miRNA-mediated targeting and repression ### Competing Interest Statement K.S. and D.C. are employees of Eclipse BioInnovations. The other authors declare no competing interests. [1]: pending:yes

R-loops are DNA-RNA hybrid structures that may promote mutagenesis. However, their contribution to human Mendelian disorders is unexplored. Here we show excess de novo variants in genomic regions that form R-loops (henceforth, 'R-loop regions') and demonstrate enrichment of R-loop region variants (RRVs) in ribozyme, snoRNA and snRNA genes, specifically in rare disease cohorts. Using this insight, we report neurodevelopmental disorders (NDDs) caused by rare variants in two major spliceosomal RNA encoding genes, RNU2-2 and RNU5B-1. These, along with the recently described RNU4-2-related ReNU syndrome, provide a genetic explanation for a substantial proportion of individuals with NDDs.

The Rfam database, a widely-used repository of non-coding RNA (ncRNA) families, has undergone significant updates in release 15.0. This paper introduces major improvements, including the expansion of Rfamseq to 26,106 genomes, a 76% increase, incorporating the latest UniProt reference proteomes and additional viral genomes. Sixty-five RNA families were enhanced using experimentally determined 3D structures, improving the accuracy of consensus secondary structures and annotations. R-scape covariation analysis was used to refine structural predictions in 26 families. Gene Ontology and Sequence Ontology annotations were comprehensively updated, increasing GO term coverage to 75% of families. The release adds 14 new Hepatitis C Virus RNA families and completes microRNA family synchronisation with miRBase, resulting in 1,603 microRNA families. New data types, including FULL alignments, have been implemented. Integration with APICURON for improved curator attribution and multiple website enhancements further improve user experience. These updates significantly expand Rfam's coverage and improve annotation quality, reinforcing its critical role in RNA research, genome annotation, and the development of machine learning models. Rfam is freely available at https://rfam.org.

Vertebrate branchial arches (BAs) are a developmental paradigm, undergoing coordinated differentiation and morphogenesis to form various adult derivative tissues. MicroRNAs can strengthen gene regulatory networks (GRNs) to promote developmental stability. To interrogate the contribution of microRNAs to BA development, we generated a novel microRNA-sequencing dataset from mouse BAs. We identified 550 expressed microRNAs, of which approximately 20% demonstrate significant differential expression across BA domains. The three most posterior BAs and the connecting outflow tract (PBA/OFT) are enriched in biological processes linked to cardiovascular development. We identified enriched predicted microRNA-target interactions with PBA/OFT upregulated cardiovascular genes and validated transcripts encoding for two fundamental cardiac transcription factors (TFs), Gata6 and Tbx20, as targets of miR-92b-3p. Furthermore, we demonstrated that miR-92b-3p can downregulate endogenous GATA6 and TBX20 in human embryonic stem cells (hESCs) undergoing cardiomyocyte differentiation, consistent with conservation of these microRNA-target interactions in a cardiogenic setting. miR-92b-3p has previously been shown to target two other cardiac TFs, Hand2 and Mef2D. Therefore, we hypothesise that miR-92b-3p acts to stabilise cardiovascular GRNs during PBA/OFT development, through acting in multiple microRNA-mediated coherent feedforward loops. ### Competing Interest Statement The authors have declared no competing interest.

Motivation: Curation of literature in life sciences is a growing challenge. The continued increase in the rate of publication, coupled with the relatively fixed number of curators worldwide presents a major challenge to developers of biomedical knowledgebases. Very few knowledgebases have resources to scale to the whole relevant literature and all have to prioritise their efforts. Results: In this work, we take a first step to alleviating the lack of curator time in RNA science by generating summaries of literature for non-coding RNAs using large language models (LLMs). We demonstrate that high-quality, factually accurate summaries with accurate references can be automatically generated from the literature using a commercial LLM and a chain of prompts and checks. Manual assessment was carried out for a subset of summaries, with the majority being rated extremely high quality. We also applied the most commonly used automated evaluation approaches, finding that they do not correlate with human assessment. Finally, we apply our tool to a selection of over 4,600 ncRNAs and make the generated summaries available via the RNAcentral resource. We conclude that automated literature summarization is feasible with the current generation of LLMs, provided careful prompting and automated checking are applied. Availability: Code used to produce these summaries can be found here: https://github.com/RNAcentral/litscan-summarization and the dataset of contexts and summaries can be found here: https://huggingface.co/datasets/RNAcentral/litsumm-v1. Summaries are also displayed on the RNA report pages in RNAcentral (https://rnacentral.org/)

AIMS:To identify differential expression of shorter non-coding RNA (ncRNA) genes associated with autism spectrum disorders (ASD). BACKGROUND:ncRNA are functional molecules that derive from non-translated DNA sequence. The HUGO Gene Nomenclature Committee (HGNC) have approved ncRNA gene classes with alignment to the reference human genome. One subset is microRNA (miRNA), which are highly conserved, short RNA molecules that regulate gene expression by direct post-transcriptional repression of messenger RNA. Several miRNA genes are implicated in the development and regulation of the nervous system. Expression of miRNA genes in ASD cohorts have been examined by multiple research groups. Other shorter classes of ncRNA have been examined less. A comprehensive systematic review examining expression of shorter ncRNA gene classes in ASD is timely to inform the direction of research. METHODS:We extracted data from studies examining ncRNA gene expression in ASD compared with non-ASD controls. We included studies on miRNA, piwi-interacting RNA (piRNA), small NF90 (ILF3) associated RNA (snaR), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), transfer RNA (tRNA), vault RNA (vtRNA) and Y RNA. The following electronic databases were searched: Cochrane Library, EMBASE, PubMed, Web of Science, PsycINFO, ERIC, AMED and CINAHL for papers published from January 2000 to May 2022. Studies were screened by two independent investigators with a third resolving discrepancies. Data was extracted from eligible papers. RESULTS:Forty-eight eligible studies were included in our systematic review with the majority examining miRNA gene expression alone. Sixty-four miRNA genes had differential expression in ASD compared to controls as reported in two or more studies, but often in opposing directions. Four miRNA genes had differential expression in the same direction in the same tissue type in at least 3 separate studies. Increased expression was reported in miR-106b-5p, miR-155-5p and miR-146a-5p in blood, post-mortem brain, and across several tissue types, respectively. Decreased expression was reported in miR-328-3p in bloods samples. Seven studies examined differential expression from other classes of ncRNA, including piRNA, snRNA, snoRNA and Y RNA. No individual ncRNA genes were reported in more than one study. Six studies reported differentially expressed snoRNA genes in ASD. A meta-analysis was not possible because of inconsistent methodologies, disparate tissue types examined, and varying forms of data presented. CONCLUSION:There is limited but promising evidence associating the expression of certain miRNA genes and ASD, although the studies are of variable methodological quality and the results are largely inconsistent. There is emerging evidence associating differential expression of snoRNA genes in ASD. It is not currently possible to say whether the reports of differential expression in ncRNA may relate to ASD aetiology, a response to shared environmental factors linked to ASD such as sleep and nutrition, other molecular functions, human diversity, or chance findings. To improve our understanding of any potential association, we recommend improved and standardised methodologies and reporting of raw data. Further high-quality research is required to shine a light on possible associations, which may yet yield important information.

ABSTRACT The homeostasis of the human body is protected by the skin, where the process of keratinocyte differentiation in outer layers has a crucial role. Cessation of proliferation in the basal layer of keratinocytes and initiation of their subrabasal functions are tightly controlled at the level of gene transcription and message translation. A subset of mRNAs has to be repressed during differentiation, and microRNAs are known to contribute to this by directly binding mRNAs at the 3’UTRs. Using results of RNA sequencing from human primary keratinocytes during induced differentiation, we evaluated the predicted binding of highly, moderately, and lowly expressed miRs to their target mRNAs. We found that moderately expressed miRs can regulate more mRNAs, and that they do so using both conserved and non-conserved canonical binding. The cumulative score for the majority of repressed mRNAs revealed a surprisingly weak binding to miRs, and we found a significant contribution of non-conserved sites to the repression of the targets. While the presence of at least one conserved site was necessary for the miR function, its weak binding may be reinforced by a non-conserved site. Together, we found that the combination of conserved and non-conserved sites lower the binding threshold for miR-mRNA interactions to assume a tighter repression of the mRNA target during cell differentiation.

Parhyale hawaiensis has emerged as the crustacean model of choice due to its tractability, ease of imaging, sequenced genome, and development of CRISPR/Cas9 genome editing tools. However, transcriptomic datasets spanning embryonic development are lacking, and there is almost no annotation of non-protein-coding RNAs, including microRNAs. We have sequenced microRNAs, together with mRNAs and long non-coding RNAs, in Parhyale using paired size-selected RNA-seq libraries at seven time-points covering important transitions in embryonic development. Focussing on microRNAs, we annotate 175 loci in Parhyale, 88 of which have no known homologs. We use these data to annotate the microRNAome of 37 crustacean genomes, and suggest a core crustacean microRNA set of around 61 sequence families. We examine the dynamic expression of microRNAs and mRNAs during the maternal-zygotic transition. Our data suggest that zygotic genome activation occurs in two waves in Parhyale with microRNAs transcribed almost exclusively in the second wave. Contrary to findings in other arthropods, we do not predict a general role for microRNAs in clearing maternal transcripts. These data significantly expand the available transcriptomics resources for Parhyale, and facilitate its use as a model organism for the study of small RNAs in processes ranging from embryonic development to regeneration.

MicroRNAs can have subtle and combinatorial effects on the levels of the targets and pathways they act on. Studying the consequences of a single microRNA knockout often proves difficult as many such knockouts exhibit phenotypes only under stress conditions. This has often led to the hypothesis that microRNAs buffer the effects of intrinsic and environmental stochasticity on gene expression. Observing and understanding this buffering effect entails quantitative analysis of microRNA and target expression in single cells. To this end, we have employed single-molecule fluorescence in situ hybridization, immunofluorescence, and high-resolution confocal microscopy to investigate the effects of miR-9a loss on the expression of the serine-protease Rhomboid in Drosophila melanogaster early embryos. Our single-cell quantitative approach shows that spatially, the rhomboid mRNA pattern is identical in WT and miR-9a knockout embryos. However, we find that the number of mRNA molecules per cell is higher when miR-9a is absent, and the level and temporal accumulation of rhomboid protein shows a more dramatic increase in the miR-9a knockout. Specifically, we see accumulation of rhomboid protein in miR-9a mutants by stage 5, much earlier than in WT. The data, therefore, show that miR-9a functions in the regulation of rhomboid mRNA and protein levels. While further work is required to establish whether rhomboid is a direct target of miR-9 in Drosophila, our results further establish the miR-9 family microRNAs as conserved regulators of timing in neurogenic processes. This study shows the power of single-cell quantification as an experimental tool to study phenotypic consequences of microRNA mis-regulation.

Recent studies identified non-coding RNAs (ncRNAs) with unknown function that are responsible for major fitness changes in yeast. To understand ncRNA interplay and aid their functional assignment, the synthetic genetic array (SGA) methodology was employed to create >15,000 double mutants and to score their epistasis in different environments. Unlike the protein network, ncRNAs mostly displayed positive epistasis in rich medium. Interestingly, the negative interactions significantly increased under stressors, showing environmental-dependent functions for ncRNAs. No correlation was found between the network of ncRNAs and that of their neighbouring genes, suggesting functional independence. The U3 paralogs, SNR17A and SNR17B, share the majority of genetic interactions in rich medium as expected. For example, SUT480 interacted with both paralogs and its function was linked to 18S rRNA processing. However, under stressors, a large number of unique epistatic interactions were observed, supporting the notion that SNR17A and SNR17B have diverged and sub-functionalised after genome duplication.