ALG-1 Influences Accurate mRNA Splicing Patterns in the Caenorhabditis elegans Intestine and Body Muscle Tissues by Modulating Splicing Factor Activities

Abstract

MicroRNAs (miRNAs) are known to modulate gene expression, but their activity at the tissue-specific level remains largely uncharacterized. To study their contribution to tissue-specific gene expression, we developed novel tools to profile putative miRNA targets in the Caenorhabditis elegans intestine and body muscle. We validated many previously described interactions and identified ∼3500 novel targets. Many of the candidate miRNA targets curated are known to modulate the functions of their respective tissues. Within our data sets we observed a disparity in the use of miRNA-based gene regulation between the intestine and body muscle. The intestine contained significantly more putative miRNA targets than the body muscle highlighting its transcriptional complexity. We detected an unexpected enrichment of RNA-binding proteins targeted by miRNA in both tissues, with a notable abundance of RNA splicing factors. We developed in vivo genetic tools to validate and further study three RNA splicing factors identified as putative miRNA targets in our study (asd-2, hrp-2, and smu-2), and show that these factors indeed contain functional miRNA regulatory elements in their 3′UTRs that are able to repress their expression in the intestine. In addition, the alternative splicing pattern of their respective downstream targets (unc-60, unc-52, lin-10, and ret-1) is dysregulated when the miRNA pathway is disrupted. A reannotation of the transcriptome data in C. elegans strains that are deficient in the miRNA pathway from past studies supports and expands on our results. This study highlights an unexpected role for miRNAs in modulating tissue-specific gene isoforms, where post-transcriptional regulation of RNA splicing factors associates with tissue-specific alternative splicing.

Global regulatory features of alternative splicing across tissues and within the nervous system of C. elegans

“…Moreover, C. elegans has served as an excellent model system to study the mechanisms governing alternative splicing regulation (Wani and Kuroyanagi 2017;Zahler 2012;Gracida et al 2016). Recent investigations harnessing the tractability of this organism have focused on whole animal or tissue-enriched transcriptome sequencing approaches to explore changes in post-transcriptional gene regulation during development and aging (Roach et al 2020;Kaletsky et al 2018;Warner et al 2019;Blazie et al 2017Blazie et al , 2015Kotagama et al 2019;Li et al 2020;Barberan-Soler and Zahler 2008;Barberan-Soler et al 2009;Ragle et al 2015;Gerstein et al 2010;Ramani et al 2011). However, it still remains unclear how splicing in C.…”

miRNA-Based Regulation of Alternative RNA Splicing in Metazoans

“…The RNA splicing factor RBFOX1 is implicated in mammalian neuronal development, myoblast fusion, and skeletal muscle and heart function [ 108 ]. The C. elegans RBFOX-1 homolog ( fox-1 ), which has been shown to control male or hermaphrodite development by splicing several genes including xol-1 pre-mRNA [ 109 ], is expressed in body muscle tissue and is targeted by miRNAs in intestinal tissue [ 19 ]. While the mammalian homologs of RBFOX1 may have distinct or unrelated functions, RBFOX1 is also expressed in muscle tissue and neurons [ 110 ], and is needed for proper neuronal development [ 111 ], and its absence or alteration has been implicated in a variety of neurological processes and disorders [ 112 ].…”

“…The ability of genes to express different 3′UTR lengths is possible through the mechanism of alternative polyadenylation (APA). APA has been shown by several groups as a potent mechanism to bypass miRNA regulation [ 19 , 113 , 114 , 115 ]. In addition, another miRNA, miR-129-5p, has been shown to directly inhibit the expression of Rbfox1 and Rbfox3 transcripts through targeting their 3′UTRs, which both have multiple miR-129-5p binding sites [ 53 ].…”

“…miRNAs and their regulatory mechanisms are conserved from humans [ 17 ] to Cnidarians [ 18 ]. In the past years, several groups produced tissue-specific localization data for many miRNAs in worm, mouse, rat, and human somatic tissues [ 19 , 20 , 21 ] and in cancers [ 22 ]. These results unequivocally show distinct functional miRNA populations in tissues, which can reshape transcriptomes and contribute to cell identity acquisition and maintenance.…”

“…In this view, miRNAs do not only regulate gene expression at the post-transcriptional level, as canonically described, but also indirectly at the co-transcriptional level, throughout the modulation of these RNA splicing factors. In C. elegans , for example, virtually all SR and hnRNP orthologs are targeted by miRNAs, and the depletion of the miRISC leads to widespread changes in splice junction usage [ 19 ]. These broad splicing errors have also been observed in transcriptomes from Dicer knockout mice [ 23 ], suggesting that this form of gene regulation is not only present in vertebrates but may have evolved earlier in the tree of life.…”