RBFOX2 is Critical for Maintaining Alternative Polyadenylation Patterns and Mitochondrial Health in Rat Myoblasts

Abstract

The RNA binding protein RBFOX2 is linked to heart and skeletal muscle diseases; yet, RBFOX2-regulated RNA networks have not been systematically identified.Although RBFOX2 has a well-known function in alternative splicing (AS), it is unclear whether RBFOX2 has other roles in RNA metabolism that affect gene expression and function. Utilizing state of the art techniques Poly(A)-ClickSeq (PAC-seq) and nanopore cDNA sequencing, we revealed a new role for RBFOX2 in fine tuning alternative polyadenylation (APA) of pre-mRNAs in myoblasts. We found that depletion of RBFOX2 altered expression of mitochondrial genes. We identified the mitochondrial gene Slc25a4 gene that transports ATP/ADP across inner mitochondrial membrane as a target of RBFOX2. Dissecting how RBFOX2 affects Slc25a4 APA uncovered that RBFOX2 binding motifs near the distal polyadenylation site (PAS) are critical for expression of Slc25a4. Consistent with changes in expression of mitochondrial genes, loss of RBFOX2 altered mitochondrial membrane potential and induced mitochondrial swelling. Our results unveiled a novel role for RBFOX2 in maintaining APA decisions and expression of mitochondrial genes in myoblasts relevant to heart diseases. Keywords: alternative polyadenylation/mitochondria/nanopore sequencing/ poly(A) sequencing/RBFOX2 Non-standard Abbreviations and Acronyms: AS alternative splicing PAC-seq poly(A)-ClickSeq APA alternative polyadenylation KD knock down 3´UTR 3´untranslated region PAS poly(A)-site PAC poly(A)-cluster DPAC differential-poly(A)-clustering dPAS distal poly(A)-site pPAS proximal poly(A)-site ANT1 (Slc25a4) ATP/ADP translocator (mitochondrial) MFN1 Mitofusin

The landscape of alternative polyadenylation in single cells of the developing mouse embryo

“…6D ). These expression patterns are broadly consistent with the known brain-specific and ubiquitous expression associated with ELAV-like 39–41 and Nova 42 family members, and support the growing functional evidence for Elavl2-4 43,44 , Rbfox2 45 and Nova1-2 42 in the regulation of APA. Additionally, we found that ELAVL2-4 , CELF2-6 , and RBFOX1-3 form an experimentally supported network of protein-protein interactions 46 ( Supplementary Fig.…”

“…Prior work provides functional evidence that fly orthologs of the ELAV-like induce neural-specific 3′-UTR lengthening through competition with CstF 43,44,49–51 , and that mammalian Elavl2-4 can also regulate APA 41,52 . Moreover, Nova1-2 42 and Rbfox2 45 have been directly implicated as regulators of APA in mouse and rat cells, respectively. Although only Celf2 has been shown to regulate APA 53 , evidence for the roles of CELF family proteins in this process include: i) enriched expression in neurons and later developmental stages, ii) direct interaction with RBFOX and ELAVL family members, iii) nuclear localization 39 , iv) enriched binding to the 3′-UTR terminus 54 , v) interaction with U2 snRNP 39 , which is known to promote distal isoform usage 55 , and vi) roles in splicing 39,40 .…”

Nanopore sequencing reveals full-length Tropomyosin 1 isoforms and their regulation by RNA binding proteins during rat heart development

“…We showed that RBFOX2 controls AS of muscle specific terminal exons and alters the polyadenylation pattern of Tpm1, generating a shorter isoform with a different 3` end. We observed the same change in Tpm1 polyadenylation pattern in RBFOX2 depleted cells by analyzing our polyA sequencing data (45,46). Our results both sequencing methods combined with RT-PCR validations demonstrate that RBFOX2 is an important determinant of Tpm1 AS and TPM1 protein levels.…”

“…Strikingly, muscle specific short Tpm1 isoforms that end with exon 9b disappeared upon RBFOX2 depletion (Figure 2A, bottom panel). To determine how RBFOX2-mediated AS of terminal exons affects polyadenylation of Tpm1, we analyzed our poly(A)-ClickSeq data from RBFOX2 KD H9c2 cells that identified genome wide alternative polyadenylation changes (45,46). We found that polyA (p(A)) usage in exon 9b was abolished in RBFOX2 KD cells compared to control cells ( Figure 2B), in agreement with exclusion of this exon in full length Tpm1 transcripts (Figure 2A, bottom panel), validating our nanopore sequencing data.…”