The exon junction complex is required for DMD gene splicing fidelity and myogenic differentiation.

Deposition of the exon junction complex (EJC) upstream of exon-exon junctions helps maintain transcriptome integrity by preventing spurious re-splicing events in already spliced mRNAs. Here we investigate the importance of EJC for the correct splicing of the 2.2-megabase-long human DMD pre-mRNA, which encodes dystrophin, an essential protein involved in cytoskeletal organization and cell signaling. Using targeted RNA-seq, we show that knock-down of the eIF4A3 and Y14 core components of EJC in a human muscle cell line causes an accumulation of mis-splicing events clustered towards the 3' end of the DMD transcript (Dp427m). This deregulation is conserved in the short Dp71 isoform expressed ubiquitously except in adult skeletal muscle and is rescued with wild-type eIF4A3 and Y14 proteins but not with an EJC assembly-defective mutant eIF4A3. MLN51 protein and EJC-associated ASAP/PSAP complexes independently modulate the inclusion of the regulated exons 71 and 78. Our data confirm the protective role of EJC in maintaining splicing fidelity, which in the DMD gene is necessary to preserve the function of the critical C-terminal protein-protein interaction domain of dystrophin present in all tissue-specific isoforms. Given the role of the EJC in maintaining the integrity of dystrophin, we asked whether the EJC could also be involved in the regulation of a mechanism as complex as skeletal muscle differentiation. We found that eIF4A3 knockdown impairs myogenic differentiation by blocking myotube formation. Collectively, our data provide new insights into the functional roles of EJC in human skeletal muscle.

miR-6893-3p is a bonafide negative regulator of splicing activator, RNPS1.

RNA-binding protein with serine-rich domain 1, RNPS1, is a global guardian of splicing fidelity and has implications in cervical cancer cell progression. We previously observed elevated RNPS1 expression in cervical cancer cells compared to normal cells. To understand the mechanisms that lead to the dysregulation of RNPS1 expression in cervical cancer cells, we focused on microRNAs. Using an in silico approach, we predicted potential miRNA candidates targeting RNPS1. Among the candidate miRNAs, we found miR-6893-3p as a potential regulator of RNPS1 expression. Interestingly, the expression of miR-6893-3p is downregulated in cervical cancer cells compared to normal cells and its level is negatively correlated with the expression of RNPS1. Further, qPCR, Western blot analysis, and luciferase reporter assay confirmed that miR-6893-3p negatively regulates RNPS1 in HeLa cells. In this regard, overexpression of miR-6893-3p suppresses the endogenous mRNA and protein levels of RNPS1 in HeLa cells. Further investigation revealed that miR-6893-3p mediated regulation of RNPS1 is dependent on the binding of miR-6893-3p to a microRNA response element in the 3'UTR of RNPS1 mRNA. Furthermore, mechanistic analysis showed that targeted negative regulation of RNPS1 by miR-6893-3p occurs via enhanced mRNA degradation. Collectively, our findings establish miR-6893-3p as an important player in the post-transcriptional regulation of RNPS1 in HeLa cells. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03761-2.

Serine-rich domain of RNPS1 functions in activation of alternative splicing.

RNA-binding protein with serine-rich domain 1 (RNPS1) gets deposited on the mRNA during the process of splicing and concomitantly associates with the exon junction complex (EJC). RNPS1 participates in post-transcriptional gene regulation, including constitutive and alternative splicing, transcriptional regulation and nonsense-mediated mRNA decay. In this study, we found that the tethering of RNPS1 or its isolated serine-rich domain (S domain) causes exon inclusion of an HIV-1 splicing substrate. In contrast, overexpressing the RRM domain of RNPS1 acts in a dominant negative manner and leads to the exon skipping of endogenous apoptotic pre-mRNAs (Bcl-X and MCL-1). Further, tethering of core EJC proteins, eIF4A3, MAGOH, or Y14, does not lead to exon inclusion of an HIV substrate. Together, our results demonstrate how RNPS1 and its domains are differentially involved in alternative splicing activity.

A molecular brake that modulates spliceosome pausing at detained introns contributes to neurodegeneration.

Emerging evidence suggests that intron-detaining transcripts (IDTs) are a nucleus-detained and polyadenylated mRNA pool for cell to quickly and effectively respond to environmental stimuli and stress. However, the underlying mechanisms of detained intron (DI) splicing are still largely unknown. Here, we suggest that post-transcriptional DI splicing is paused at the Bact state, an active spliceosome but not catalytically primed, which depends on Smad Nuclear Interacting Protein 1 (SNIP1) and RNPS1 (a serine-rich RNA binding protein) interaction. RNPS1 and Bact components preferentially dock at DIs and the RNPS1 docking is sufficient to trigger spliceosome pausing. Haploinsufficiency of Snip1 attenuates neurodegeneration and globally rescues IDT accumulation caused by a previously reported mutant U2 snRNA, a basal spliceosomal component. Snip1 conditional knockout in the cerebellum decreases DI splicing efficiency and causes neurodegeneration. Therefore, we suggest that SNIP1 and RNPS1 form a molecular brake to promote spliceosome pausing, and that its misregulation contributes to neurodegeneration.

RNPS1 functions as an oncogenic splicing factor in cervical cancer cells.

Numerous recent studies suggest that cancer-specific splicing alteration is a critical contributor to the pathogenesis of cancer. RNA-binding protein with serine-rich domain 1, RNPS1, is an essential regulator of the splicing process. However, the defined role of RNPS1 in tumorigenesis still remains elusive. We report here that the expression of RNPS1 is higher in cervical carcinoma samples from The Cancer Genome Atlas (TCGA-cervical squamous cell carcinoma and endocervical adenocarcinoma) compared to the normal tissues. Consistently, the expression of RNPS1 was high in cervical cancer cells compared to a normal cell line. This study shows for the first time that RNPS1 promotes cell proliferation and colony-forming ability of cervical cancer cells. Importantly, RNPS1 positively regulates migration-invasion of cervical cancer cells. Intriguingly, depletion of RNPS1 increases the chemosensitivity against the chemotherapeutic drug doxorubicin in cervical cancer cells. Further, we characterized the genome-wide isoform switching stimulated by RNPS1 in cervical cancer cell. Mechanistically, RNA-sequencing analysis showed that RNPS1 regulates the generation of tumor-associated isoforms of key genes, particularly Rac1b, RhoA, MDM4, and WDR1, through alternative splicing. RNPS1 regulates the splicing of Rac1 pre-mRNA via a specific alternative splicing switch and promotes the formation of its tumorigenic splice variant, Rac1b. While the transcriptional regulation of RhoA has been well studied, the role of alternative splicing in RhoA upregulation in cancer cells is largely unknown. Here, we have shown that the knockdown of RNPS1 in cervical cancer cells leads to the skipping of exons encoding the RAS domain of RhoA, consequently causing decreased expression of RhoA. Collectively, we conclude that the gain of RNPS1 expression may be associated with tumor progression in cervical carcinoma. RNPS1-mediated alternative splicing favors an active Rac1b/RhoA signaling axis that could contribute to cervical cancer cell invasion and metastasis. Thus, our work unveils a novel role of RNPS1 in the development of cervical cancer.

Molecular cloning, expression and generation of a polyclonal antibody specific for RNPS1.

BACKGROUND: RNA-binding protein with serine-rich domain 1 (RNPS1) is a member of a splicing-dependent mega Dalton protein complex or exon junction complex (EJC). During splicing, RNPS1 acts as a protector of global transcriptome integrity by suppressing the usage of cryptic splice sites. Additionally, RNPS1 functions in almost all stages of mRNA metabolism, including constitutive splicing, alternative splicing, translation and nonsense-mediated mRNA decay (NMD). The aim of the present study was to generate a highly specific polyclonal antibody against human RNPS1. METHODS AND RESULTS: A plasmid, pHis-TEV-RNPS1, has been constructed to overexpress recombinant RNPS1 (22-305 amino acids) by cloning the nucleotide sequence downstream of an N-terminal His-tag in the parent plasmid pHis-TEV. The recombinant plasmid was then transformed into Rosetta and expression was induced using IPTG. The His-tagged RNPS1 protein was purified using Ni-NTA affinity chromatography. The rabbit antiserum was then obtained by immunizing rabbits with the purified recombinant RNPS1 protein. The antiserum was further purified by antigen-immunoaffinity chromatography. The sensitivity and the specificity of the polyclonal antibody were assessed by enzyme-linked immunosorbent assay (ELISA) and knockdown assay. ELISA demonstrated that the antibody has a high binding affinity for RNPS1 and the usable titre is 1:2000. CONCLUSION: The antibody detected RNPS1 in human, mouse cell lines and rat tissue in Western blot. Importantly, the antibody efficiently detected the decrease in RNPS1 expression in siRNA induced knockdown assay, indicating the specificity of the antibody. The polyclonal antibody against RNPS1 will be a useful tool for performing further functional studies on RNPS1.

RNA-binding protein with serine-rich domain 1 regulates microsatellite instability of uterine corpus endometrial adenocarcinoma

OBJECTIVE: To determine the role of RNA-binding protein with serine-rich domain 1 (RNPS1) in uterine corpus endometrial carcinoma (UCEC), the role of RNPS1 knockdown in UCEC development in vitro and in vivo, and the relationship between RNPS1 and mismatch repair (MMR) in UCEC. METHODS: We predicted the potential function of RNPS1 using bioinformatics systems. The expression of RNPS1 in tissues and cell lines was analyzed by western blotting and immunohistochemistry. The expression of RNPS1 in MMR was assessed using bioinformatics and western blotting. The proliferation and apoptosis of UCEC cells were assessed under RNPS1 knockdown conditions, and RNPS1 regulation in MMR was detected by suppressing Notch signaling. Associations between RNPS1 and gene mutations in UCEC and prognosis were analyzed. RESULTS: The RNPS1 level was higher in UCEC tumors than in normal tissues and tumors or RL952 cells. Prognostic outcomes were worse when UCEC showed abundant RNPS1 expression. Lentiviral RNPS1 knockdown weakened tumor cell proliferation and suppressed biomarker expression, reduced the tumor volume, promoted apoptosis in vitro and in vivo, and inhibited UCEC development. Increased MutS homolog 2 (MSH2) and MutS homolog 6 (MSH6) levels in MMR after RNPS1 knockdown were reversed by inhibiting Notch signaling. Furthermore, RNPS1 was associated with mutations in NAA11, C2orf57, NUPR1, and other genes involved in UCEC prognosis. CONCLUSION: RNPS1 may regulate the expression levels of MSH2 and MSH6 in MMR, enhancing the proliferation, development, and prognosis of UCEC through a Notch signaling pathway in UCEC. Our study offers a new method and strategy for delaying UCEC development through modulating MMR.

RNPS1 inhibition aggravates ischemic brain injury and promotes neuronal death.

RNA-binding protein with serine-rich domain 1 (RNPS1) is essential for modulating mRNA metabolism, but its role in ischemic stroke is unknown. In this study, we found that RNPS1 expression was significantly up-regulated in the brains of ischemic stroke mice and primary cortical neurons after oxygen-glucose deprivation (OGD) treatment. Knockdown of RNPS1 significantly aggravated ischemic brain injury after middle cerebral artery occlusion (MCAO) and promoted neuronal death. In addition, knockdown of RNPS1 exacerbated ischemia induced neuronal apoptosis, and downregulated the expression of anti-apoptotic proteins Bcl-xL and Mcl-1. Our study suggested that RNPS1 might be a potential therapeutic target for alleviating neuronal death in ischemic stroke.

Genome-wide CRISPR-Cas9 Interrogation of Splicing Networks Reveals a Mechanism for Recognition of Autism-Misregulated Neuronal Microexons.

Alternative splicing is crucial for diverse cellular, developmental, and pathological processes. However, the full networks of factors that control individual splicing events are not known. Here, we describe a CRISPR-based strategy for the genome-wide elucidation of pathways that control splicing and apply it to microexons with important functions in nervous system development and that are commonly misregulated in autism. Approximately 200 genes associated with functionally diverse regulatory layers and enriched in genetic links to autism control neuronal microexons. Remarkably, the widely expressed RNA binding proteins Srsf11 and Rnps1 directly, preferentially, and frequently co-activate these microexons. These factors form critical interactions with the neuronal splicing regulator Srrm4 and a bi-partite intronic splicing enhancer element to promote spliceosome formation. Our study thus presents a versatile system for the identification of entire splicing regulatory pathways and further reveals a common mechanism for the definition of neuronal microexons that is disrupted in autism.

Splicing activator RNPS1 suppresses errors in pre-mRNA splicing: A key factor for mRNA quality control.

Human RNPS1 protein was first identified as a pre-mRNA splicing activator in vitro and RNPS1 regulates alternative splicing in cellulo. RNPS1 was also known as a peripheral factor of the exon junction complex (EJC). Here we show that cellular knockdown of RNPS1 induced a reduction of the wild-type aurora kinase B (AURKB) protein due to the induced aberrant pre-mRNA splicing events, indicating that the fidelity of AURKB pre-mRNA splicing was reduced. The major aberrant AURKB mRNA was derived from the upstream pseudo 5' and 3' splice sites in intron 5, which resulted in the production of the non-functional truncated AURKB protein. AURKB, is an essential mitotic factor, whose absence is known to cause multiple nuclei, and this multinucleation phenotype was recapitulated in RNPS1-knockdown cells. Importantly this RNPS1-knockdown phenotype was rescued by ectopic expression of AURKB, implying it is a major functional target of RNPS1. We found RNPS1 protein, not as a component of the EJC, binds directly to a specific element in the AURKB exon upstream of the authentic 5' splice site, and this binding is required for normal splicing. RNPS1-knockdown induces a parallel aberrant splicing pattern in a fully distinct pre-mRNA, MDM2, suggesting that RNPS1 is a global guardian of splicing fidelity. We conclude that RNPS1 is a key factor for the quality control of mRNAs that is essential for the phenotypes including cell division.

The exon junction complex is required for definition and excision of neighboring introns in Drosophila.

Splicing of pre-mRNAs results in the deposition of the exon junction complex (EJC) upstream of exon-exon boundaries. The EJC plays crucial post-splicing roles in export, translation, localization, and nonsense-mediated decay of mRNAs. It also aids faithful splicing of pre-mRNAs containing large introns, albeit via an unknown mechanism. Here, we show that the core EJC plus the accessory factors RnpS1 and Acinus aid in definition and efficient splicing of neighboring introns. This requires prior deposition of the EJC in close proximity to either an upstream or downstream splicing event. If present in isolation, EJC-dependent introns are splicing-defective also in wild-type cells. Interestingly, the most affected intron belongs to the piwi locus, which explains the reported transposon desilencing in EJC-depleted Drosophila ovaries. Based on a transcriptome-wide analysis, we propose that the dependency of splicing on the EJC is exploited as a means to control the temporal order of splicing events.