Biology subjects

Miura, P.

Publications and source records attributed to Miura, P..

7 recordsLinked to original sources

bioRxiv2025

The RNA-binding protein NOVA-1 regulates circRNA expression, alternative splicing, and aging in C. elegans

Circular RNA (circRNA) biogenesis is regulated by RNA-binding proteins (RBPs) that alter back-splicing of exons in protein coding genes. However, few in vivo roles for RBPs in the regulation of circRNA biogenesis have been characterized. We previously showed that many circRNAs increase with age in C. elegans, and that loss of circ-crh-1, an abundant age-accumulated circRNA, extends mean lifespan. Given the established role of the mammalian RBP NOVA2 in promoting circRNA biogenesis, we investigated whether nova-1, the sole C. elegans homolog of NOVA1/2, similarly regulates circRNA expression and function in vivo. RNA-sequencing of nova-1 mutants compared to wild-type identified 686 circRNAs. Of these, 103 were differentially expressed in nova-1 mutants compared to wild-type, with 76 upregulated and 27 downregulated circRNAs, suggesting NOVA-1 acts as a negative regulator of a subset of circRNAs. nova-1 mutants also exhibited linear alternative splicing changes, primarily in alternative 3' splice site usage and exon skipping, and showed minimal overlap with circRNA loci. Notably, circ-crh-1 represented a shared regulatory target, suggesting NOVA-1 may coordinate splicing regulation with the production of crh-1 circRNAs. Motif analysis further revealed that over half of the NOVA-1-regulated splicing events contained YCAY motif sites, with crh-1 harboring a high density of sites, consistent with its alternative 3' splice site usage and circRNA production. Finally, nova-1 mutants exhibited an extended mean lifespan and enhanced heat stress recovery. Together, these findings identify NOVA-1 as a key regulator of circRNA expression and alternative splicing in C. elegans, with likely downstream consequences for organismal lifespan and stress resilience.

genomics↗

bioRxiv2025

APALORD: An R-based tool for differential alternative polyadenylation analysis of long-read RNA-seq data

Alternative polyadenylation (APA) is a critical co/post-transcriptional process that enhances RNA isoform diversity, regulating mRNA stability, localization and translation in a spatiotemporal manner. Over the past decade, long-read (LR) RNA sequencing techniques have advanced rapidly, producing datasets that could offer insights into APA mechanisms. Here we introduce APALORD (Alternative Polyadenylation Analysis of LOng-ReaDs), an R-based analysis tool for APA analysis of LR RNA-seq data. Leveraging precise 3 end information from 3-primed LR RNA-seq data, APALORD identifies polyadenylation sites (PASs) and quantifies PAS usage (PAU) at individual sites for each sample. It conducts APA analysis at the gene level (using Kolmogorov-Smirnov test) and at the level of individual PAS (using DEXSeq) across sample conditions. APALORD was applied to direct RNA-seq data from human embryonic stem cells (hESCs) and hESC-derived neurons. PASs were identified with high accuracy and a transcriptome-wide 3UTR lengthening trend was found, consistent with previous studies. APALORD analysis of PacBio cDNA data from human tissues confirmed a 3UTR lengthening trend in cortex compared to liver. R2C2 libraries generated on the Nanopore platform were analyzed with APALORD and it revealed APA change associated with polysome fractions in human neural progenitor cells. In summary, APALORD offers a comprehensive framework for differential APA analysis using LR RNA-Seq data, empowering researchers to investigate 3 end dynamics across diverse biological contexts.

bioinformatics↗

bioRxiv2023

Coordination of Alternative Splicing and Alternative Polyadenylation revealed by Targeted Long Read Sequencing

Nervous system development is associated with extensive regulation of alternative splicing (AS) and alternative polyadenylation (APA). AS and APA have been extensively studied in isolation, but little is known about how these processes are coordinated. Here, the coordination of cassette exon (CE) splicing and APA in Drosophila was investigated using a targeted long-read sequencing approach we call Pull-a-Long-Seq (PL-Seq). This cost-effective method uses cDNA pulldown and Nanopore sequencing combined with an analysis pipeline to resolve the connectivity of alternative exons to alternative 3 ends. Using PL-Seq, we identified genes that exhibit significant differences in CE splicing depending on connectivity to short versus long 3UTRs. Genomic long 3UTR deletion was found to alter upstream CE splicing in short 3UTR isoforms and ELAV loss differentially affected CE splicing depending on connectivity to alternative 3UTRs. This work highlights the importance of considering connectivity to alternative 3UTRs when monitoring AS events.

molecular biology↗

bioRxiv2021

NOVA2 regulates neural circRNA biogenesis

Circular RNAs (circRNAs) are highly expressed in the brain and their expression increases during neuronal differentiation. The factors regulating circRNAs in the developing mouse brain are unknown. NOVA1 and NOVA2 are neural-enriched RNA-binding proteins with well-characterized roles in alternative splicing. Profiling of circRNAs from RNA-seq data revealed that global circRNA levels were reduced in embryonic cortex of Nova2 but not Nova1 knockout mice. Analysis of isolated inhibitory and excitatory cortical neurons lacking NOVA2 revealed an even more dramatic reduction of circRNAs and establish a widespread role for NOVA2 in enhancing circRNA biogenesis. To investigate the cis-elements controlling NOVA2-regulation of circRNA biogenesis, we generated a backsplicing reporter based on the Efnb2 gene. We found that NOVA2-mediated backsplicing of circ-Efnb2 was impaired when YCAY clusters located in flanking introns were removed. CLIP and additional reporter analysis demonstrated the importance of NOVA2 binding sites located in both flanking introns of circRNA loci. NOVA2 is the first RNA-binding protein identified to globally promote circRNA biogenesis in the developing brain.

genomics↗

bioRxiv2019

Elav-mediated exon skipping and alternative polyadenylation of the Dscam1 gene is required for axon outgrowth

Many metazoan genes express alternative long 3' UTR isoforms in the nervous system, but their functions remain largely unclear. In Drosophila melanogaster, the Dscam1 gene generates short and long (Dscam1-L) 3' UTR isoforms due to alternative polyadenylation (APA). Here, we found that the RNA-binding protein Embryonic Lethal Abnormal Visual System (Elav) impacts Dscam1 biogenesis at two levels, including regulation of long 3' UTR biogenesis and skipping of an upstream exon (exon 19). MinION long-read sequencing confirmed the connectivity of this alternative splicing event to the long 3' UTR. Knockdown or CRISPR deletion of Dscam1-L impaired axon growth in Drosophila. The Dscam1 long 3' UTR was found to be required for correct Elav-mediated skipping of exon 19. Elav thus co-regulates APA and alternative splicing to generate specific Dscam1 transcripts that are essential for neural development. This coupling of APA to alternative splicing might represent a new class of regulated RNA processing.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC=\"FIGDIR/small/613059_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (45K):\norg.highwire.dtl.DTLVardef@1a23558org.highwire.dtl.DTLVardef@5c3dddorg.highwire.dtl.DTLVardef@1e1808forg.highwire.dtl.DTLVardef@1bf3888_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIElav regulates Dscam1 long 3' UTR (Dscam1-L) biogenesis\nC_LIO_LILong-read sequencing reveals connectivity of long 3' UTR to skipping of upstream exon 19\nC_LIO_LILoss of Dscam1-L impairs axon outgrowth\nC_LIO_LIDscam1 long 3' UTR is required for correct splicing of exon 19\nC_LI

molecular biology↗

bioRxiv2019

Precise removal of Calm1 long 3′ UTR isoform by CRISPR-Cas9 genome editing impairs dorsal root ganglion development in mice.

Most mammalian genes are subject to Alternative cleavage and PolyAdenylation (APA), often resulting in alternative length 3' UTR isoforms. Thousands of extended or long 3' UTR variants are preferentially expressed in neuron-enriched tissues of metazoans. However, the in vivo functions of these long 3' UTR isoforms are largely unknown. Calmodulin 1 (Calm1) is a key integrator of calcium signaling that is required for correct neural development. Calm1 generates short (Calm1-S) and long 3' UTR (Calm1-L) mRNA isoforms via APA. We found Calm1-S to be broadly expressed across mouse tissues, whereas Calm1-L expression was largely restricted to neural tissues, including the dorsal root ganglion (DRG). Using CRISPR-Cas9 genome editing, a series of mouse deletion lines were generated that successfully eliminated expression of Calm1-L while maintaining expression of Calm1-S. One of these lines, Calm1{Delta}3' UTR, carried a 163 bp deletion surrounding the distal polyA site. Examination of Calm1{Delta}3' UTR embryos revealed disrupted development of the DRG. In Calm1{Delta}3' UTR DRG explant cultures undergoing axon outgrowth, we observed a dramatic increase in axon fasciculation. These results demonstrate a physiological role for Calm1-L in DRG development, and more generally, establish a genome-editing strategy to study in vivo functions of long 3' UTR isoforms. Author SummaryMore than half of all human genes generate alternative mRNA isoforms which differ in the length of their 3 Untranslated regions (3 UTRs). Through a process called Alternative Cleavage and Polyadenylation thousands of broadly expressed genes preferentially express long 3 UTR variants in brain tissues whereas their short 3 UTR counterparts are more broadly expressed. A challenge to study the functions of these transcripts has been to generate loss of function mutant animals that lack a long 3 UTR isoform but maintain expression of the corresponding short 3 UTR isoform. Here, we used the precise, rapid, and efficient approach of CRISPR genome-editing to generate long 3 UTR mutant mice. These mice, which do not express the long 3 UTR of the Calmodulin 1 (Calm1) gene, exhibit impairment in the development of sensory neurons, including increased fasciculation of axons and aberrant cell body migration. This finding is important because it provides conclusive genetic evidence for a neural function of a long 3 UTR isoform in an animal. The CRISPR genome-editing approach used here can be applied to the study of neuron-enriched long 3 UTR isoforms, which number in the thousands and have largely unexplored functions.

genetics↗

bioRxiv2017

Global accumulation of circRNAs during aging in Caenorhabditis elegans

Circular RNAs (CircRNAs) are a newly appreciated class of RNAs that lack free 5{acute} and 3{acute} ends, are expressed by the thousands in diverse forms of life, and are mostly of enigmatic function. Ostensibly due to their resistance to exonucleases, circRNAs are known to be exceptionally stable. Here, we examined the global profile of circRNAs in C. elegans during aging by performing ribo-depleted total RNA-seq from the fourth larval stage (L4) through 10-day old adults. Using stringent bioinformatic criteria and experimental validation, we annotated 1,166 circRNAs, including 575 newly discovered circRNAs. These circRNAs were derived from 797 genes with diverse functions, including genes involved in the determination of lifespan. A massive accumulation of circRNAs during aging was uncovered. Many hundreds of circRNAs were significantly increased among the aging time-points and increases of select circRNAs by over 40-fold during aging were quantified by qRT-PCR. The age-accumulation of circRNAs was not accompanied by increased expression of linear RNAs from the same host genes. We attribute the global scale of circRNA age-accumulation to the high composition of postmitotic cells in adult C. elegans, coupled with the high resistance of circRNAs to decay. These findings suggest that the exceptional stability of circRNAs might explain age-accumulation trends observed from neural tissues of other organisms, which also have a high composition of post-mitotic cells. Given the suitability of C. elegans for aging research, it is now poised as an excellent model system to determine if there are functional consequences of circRNA accumulation during aging.

genomics↗