Alternative Splicing-coupled Nonsense-mediated Decay Research Articles

Downregulated RBM5 Enhances CARM1 Expression and Activates the PRKACA/GSK3β Signaling Pathway through Alternative Splicing-Coupled Nonsense-Mediated Decay.

Downregulated RNA-binding motif protein 5 (RBM5) promotes the development and progression of various tumors, including bladder cancer (BC). Alternative splicing (AS) plays a crucial role in the progression of cancer by producing protein isomers with different functions or by promoting nonsense-mediated mRNA decay (NMD). However, whether RBM5 modulates the progression of BC through AS-NMD remains unexplored. In this study, we revealed that the downregulation of RBM5 expression promoted the expression of coactivator-associated arginine methyltransferase 1 (CARM1) in BC cells and tissues. Increased expression of CARM1 facilitated the activation of the Wnt/β-catenin axis and cell proliferation, which then contributed to the poor prognosis of patients with BC. Interestingly, RBM5 bound directly to CARM1 mRNA and participated in AS-NMD, downregulating the expression of CARM1. In addition, we revealed that protein kinase catalytic subunit alpha (PRKACA) functioned as a phosphorylated kinase of GSK3β, was regulated by CARM1 at the transcription level, and promoted the growth and progression of BC cells. Furthermore, in this study, we demonstrated a regulatory mechanism of Wnt/β-catenin activation through the RBM5/CARM1/PRKACA axis and identified a novel potential target for treating BC.

Altered expressions and splicing profiles of Acin1 transcripts differentially modulate brown adipogenesis through an alternative splicing mechanism

Apoptotic chromatin condensation inducer in the nucleus (also referred as Acin1) was first characterized as an RNA-binding protein involved in apoptosis. In later reports, Acin1 was identified as an auxiliary component of the exon junction complex (EJC) which is assembled throughout pre-messenger RNA splicing. In this study, results of whole-transcriptome analyses revealed reduced expressions and reprogrammed splicing profiles of Acin1 transcripts throughout development of brown adipose tissues (BATs) that execute non-shivering thermogenesis in small rodents and infants by consuming lipids. Depletion of endogenous Acin1 isoforms led to activation of brown adipogenic signatures in mouse C3H10T1/2 fibroblasts. Nevertheless, overexpressions of the Acin1-L or Acin1-S isoform exerted discriminative influences on brown adipogenesis and reprogramming of the expression of serine/arginine-rich splicing factor 3 (SRSF3) through an alternative splicing-coupled nonsense-mediated decay mechanism in a sequence-specific manner. Moreover, the Acin1-SRSF3 axis constitutes a regulatory pathway that participates in the brown adipocyte-related splicing network. Taken together, the interplay between accessory EJC components and splicing regulators constitutes an emerging mechanism for differentially manipulating the activity of brown adipogenesis via alternative splicing network.

Autoregulation of RBM10 and cross-regulation of RBM10/RBM5 via alternative splicing-coupled nonsense-mediated decay.

Mutations in the spliceosomal RNA binding protein RBM10 cause TARP syndrome and are frequently observed in lung adenocarcinoma (LUAD). We have previously shown that RBM10 enhances exon skipping of its target genes, including its paralog RBM5. Here, we report that RBM10 negatively regulates its own mRNA and protein expression and that of RBM5 by promoting alternative splicing-coupled nonsense-mediated mRNA decay (AS-NMD). Through computational analysis and experimental validation, we identified RBM10-promoted skipping of exon 6 or 12 in RBM10 and exon 6 or 16 in RBM5 as the underlying AS-NMD events. Importantly, we showed that LUAD-associated mutations affecting splice sites of RBM10 exons 6 or 12 abolished exon inclusion and correlated with reduced expression of RBM10 RNA. Together, our investigations have revealed novel molecular mechanisms underlying RBM10 autoregulation and cross-regulation of RBM5, thereby providing insights concerning the functions of RBM10 under various physiological and pathological conditions. Our combined computational and experimental approach should be useful for elucidating the role of AS-NMD in auto- and cross-regulation by other splicing regulators.

The impact of the RBM4-initiated splicing cascade on modulating the carcinogenic signature of colorectal cancer cells

A growing body of studies has demonstrated that dysregulated splicing profiles constitute pivotal mechanisms for carcinogenesis. In this study, we identified discriminative splicing profiles of colorectal cancer (CRC) cells compared to adjacent normal tissues using deep RNA-sequencing (RNA-seq). The RNA-seq results and cohort studies indicated a relatively high ratio of exon 4-excluded neuro-oncological ventral antigen 1 (Nova1−4) and intron 2-retained SRSF6 (SRSF6+intron 2) transcripts in CRC tissues and cell lines. Nova1 variants exhibited differential effects on eliminating SRSF6 expression in CRC cells by inducing SRSF6+intron 2 transcripts which were considered to be the putative target of alternative splicing-coupled nonsense-mediated decay mechanism. Moreover, the splicing profile of vascular endothelial growth factor (VEGF)165/VEGF165b transcripts was relevant to SRSF6 expression, which manipulates the progression of CRC calls. These results highlight the novel and hierarchical role of an alternative splicing cascade that is involved in the development of CRC.

RBM4a-regulated splicing cascade modulates the differentiation and metabolic activities of brown adipocytes

RNA-binding motif protein 4a (RBM4a) reportedly reprograms splicing profiles of the insulin receptor (IR) and myocyte enhancer factor 2C (MEF2C) genes, facilitating the differentiation of brown adipocytes. Using an RNA-sequencing analysis, we first compared the gene expressing profiles between wild-type and RBM4a−/− brown adipocytes. The ablation of RBM4a led to increases in the PTBP1, PTBP2 (nPTB), and Nova1 proteins, whereas elevated RBM4a reduced the expression of PTBP1 and PTBP2 proteins in brown adipocytes through an alternative splicing-coupled nonsense-mediated decay mechanism. Subsequently, RBM4a indirectly shortened the half-life of the Nova1 transcript which was comparatively stable in the presence of PTBP2. RBM4a diminished the influence of PTBP2 in adipogenic development by reprogramming the splicing profiles of the FGFR2 and PKM genes. These results constitute a mechanistic understanding of the RBM4a-modulated splicing cascade during the brown adipogenesis.

Alternative splicing coupled nonsense-mediated decay generates neuronal cell type-specific expression of SLM proteins.

The unique physiological and morphological properties of neuronal populations are crucial for the appropriate functioning of neuronal circuits. Alternative splicing represents an attractive mechanism for generating cell type-specific molecular repertoires that steer neuronal development and function. However, the mechanisms that link neuronal identity to alternative splicing programs are poorly understood. We report that cell type-specific, mutually exclusive expression of two alternative splicing regulators, SLM1 and SLM2, in the mouse hippocampus is achieved by a cross-repression mechanism. Deletion of SLM2 in vivo modifies alternative splicing of its paralog Slm1 and stabilizes its mRNA, resulting in expression of SLM1 in previously SLM2-expressing cells. Despite this ectopic upregulation of SLM1, loss of SLM2 severely disrupts the alternative splicing regulation of Nrxn1, Nrxn2, and Nrxn3, highlighting that the two SLM paralogs have partially divergent functions. Our study uncovers a hierarchical, SLM2-dependent mechanism for establishing cell type-specific expression of neuronal splicing regulators in vivo.

Rbfox2 controls autoregulation in RNA-binding protein networks

The tight regulation of splicing networks is critical for organismal development. To maintain robust splicing patterns, many splicing factors autoregulate their expression through alternative splicing-coupled nonsense-mediated decay (AS-NMD). However, as negative autoregulation results in a self-limiting window of splicing factor expression, it is unknown how variations in steady-state protein levels can arise in different physiological contexts. Here, we demonstrate that Rbfox2 cross-regulates AS-NMD events within RNA-binding proteins to alter their expression. Using individual nucleotide-resolution cross-linking immunoprecipitation coupled to high-throughput sequencing (iCLIP) and mRNA sequencing, we identified >200 AS-NMD splicing events that are bound by Rbfox2 in mouse embryonic stem cells. These "silent" events are characterized by minimal apparent splicing changes but appreciable changes in gene expression upon Rbfox2 knockdown due to degradation of the NMD-inducing isoform. Nearly 70 of these AS-NMD events fall within genes encoding RNA-binding proteins, many of which are autoregulated. As with the coding splicing events that we found to be regulated by Rbfox2, silent splicing events are evolutionarily conserved and frequently contain the Rbfox2 consensus UGCAUG. Our findings uncover an unexpectedly broad and multilayer regulatory network controlled by Rbfox2 and offer an explanation for how autoregulatory splicing networks are tuned.

A dynamic alternative splicing program regulates gene expression during terminal erythropoiesis

Alternative pre-messenger RNA splicing remodels the human transcriptome in a spatiotemporal manner during normal development and differentiation. Here we explored the landscape of transcript diversity in the erythroid lineage by RNA-seq analysis of five highly purified populations of morphologically distinct human erythroblasts, representing the last four cell divisions before enucleation. In this unique differentiation system, we found evidence of an extensive and dynamic alternative splicing program encompassing genes with many diverse functions. Alternative splicing was particularly enriched in genes controlling cell cycle, organelle organization, chromatin function and RNA processing. Many alternative exons exhibited differentiation-associated switches in splicing efficiency, mostly in late-stage polychromatophilic and orthochromatophilic erythroblasts, in concert with extensive cellular remodeling that precedes enucleation. A subset of alternative splicing switches introduces premature translation termination codons into selected transcripts in a differentiation stage-specific manner, supporting the hypothesis that alternative splicing-coupled nonsense-mediated decay contributes to regulation of erythroid-expressed genes as a novel part of the overall differentiation program. We conclude that a highly dynamic alternative splicing program in terminally differentiating erythroblasts plays a major role in regulating gene expression to ensure synthesis of appropriate proteome at each stage as the cells remodel in preparation for production of mature red cells.

A Dynamic Alternative Splicing Program Regulates Gene Expression In A Differentiation Stage-Specific Manner During Terminal Erythropoiesis

Spatio-temporal regulation of switches in alternative pre-mRNA splicing modulate exon usage, critically remodeling the transcriptome during development and differentiation of many tissues, while aberrant regulation of alternative splicing disrupts these processes and plays a role in numerous human diseases. Recently, the discovery of splicing factor mutations in myelodysplasia has increased interest in splicing regulation in hematology. Previously, a functionally critical erythroid splicing switch in protein 4.1 pre-mRNA has been reported, in which activation of alternative exon 16 splicing in late erythroblasts is required for assembly of a mechanically stable red cell membrane. To explore globally the landscape of important alternative splicing events in the erythroid lineage, we applied RNA-seq analysis to five highly FACS-purified populations of human erythroblasts, cultured from CD34+ cord blood progenitors, representing proerythroblasts, early and late basophilic erythroblasts, polychromatophilic erythroblasts, and orthochromatophilic erythroblasts. Alternative splicing events predicted by computational analysis were filtered to remove low expression genes and low frequency splicing events, to derive a list of >3000 ‘major' alternative splicing events of potential importance in erythroid biology. Many of these were validated by inspection of RNA-seq reads mapped on the human genome, and/or by RT-PCR analysis. In this unique differentiation system we found an extensive and dynamic alternative splicing program enriched in genes that function in cell cycle regulation, organelle organization, chromatin structure and function, and RNA processing. For example, we identified alternative splicing events in ∼25 genes encoding chromatin modifying enzymes that methylate, demethylate, or acetylate specific lysine or arginine residues in histones; in transcription modulators such as ATRX and BCL11A that regulate normal globin gene expression; and in ∼50 RNA binding proteins with various roles in post-transcriptional gene regulation. Comparison of PSI (percent spliced in) values across the differentiation series revealed that dozens of alternative exons exhibit substantial switches in splicing efficiency during terminal erythropoiesis. The majority of splicing switches occur in late-stage polychromatophilic and orthochromatophilic erythroblasts, temporally correlated with changes in transcript abundance for many splicing factors and with substantial cell remodeling prior to enucleation. One of the biggest switches in late erythroblasts involves inclusion of a 35nt exon in the NDEL1 (nuclear distribution factor E-homolog-like1) gene, which alters C-terminal structure of a protein that functions in nuclear migration and nucleokinesis in nonerythroid cells and may have a role in erythroblast enucleation. Most of the regulated splicing events insert or delete sequences predicted to modulate protein structure and function in late erythroblasts. However, a subset of altered splicing events have a different effect on gene expression by introducing premature translation termination codons (PTCs), leading us to hypothesize that alternative splicing-coupled nonsense-mediated-decay (AS-NMD) contributes to stage-specific down-regulation of numerous erythroid transcripts. Consistent with such a model, most genes that up-regulate PTC exons in late erythroblasts exhibit reduction in overall expression levels, and inhibition of NMD increases the apparent expression of PTC isoforms. In contrast, genes that up-regulate coding exons are not preferentially down-regulated in late erythroblasts. We conclude that a dynamically regulated alternative splicing program in terminally differentiating erythroblasts plays a major post-transcriptional role in shaping gene expression as the cells transition from proliferation to differentiation, ensuring synthesis of the appropriate constellation of proteins as the cells prepare for enucleation and production of mature red cells. Disclosures:No relevant conflicts of interest to declare.