Interaction of CDK12 with NXF1 is a new node for the linking mechanism between transcription and transportation of mRNA.

The transcription and transportation of mRNA are coupled processes; however, the mechanisms linking these processes remain unclear. Additionally, the significance of this connection in cancer drug development is poorly understood. To address these issues, we investigated the role of CDK12 kinase, which regulates RNA transcription through the phosphorylation of RNA polymerase II (Pol II) and has a repeated serine-arginine dipeptide (RS domain) involved in mRNA transport. Despite the anticipated uniqueness of CDK12 function, the mechanism by which CDK12 bridges and manages mRNA transcription and transport has not been fully analyzed. Our study revealed that CDK12 interacts with NXF1, a key molecule involved in the export of mRNA from the nucleus to the cytosol. Although CDK12 does not phosphorylate NXF1, we found that NXF1 unexpectedly stabilized the CDK12 protein, suggesting that NXF1 mRNA export activity indirectly affects mRNA transcriptional activity by modifying the protein level of CDK12. Furthermore, CDK12 recruited other essential RNA transporters, specifically the exon junction complex (EJC) and THO complexes, into the CDK12-NXF1 axis through its kinase activity. These observations provide insights into the mechanisms linking mRNA transcription and transport through the formation of a novel CDK12-NXF1 complex that involves EJC and THO. Importantly, the expression level of NXF1 influences sensitivity to CDK12 inhibitors, which are emerging as novel anti-cancer drug candidates. This highlights the importance of considering the relationship between mRNA transcription and transport when targeting RNA transcription in cancer therapy.

Epistatic interactions between NMD and TRP53 control progenitor cell maintenance and brain size.

Mutations in human nonsense-mediated mRNA decay (NMD) factors are enriched in neurodevelopmental disorders. We show that deletion of key NMD factor Upf2 in mouse embryonic neural progenitor cells causes perinatal microcephaly but deletion in immature neurons does not, indicating NMD's critical roles in progenitors. Upf2 knockout (KO) prolongs the cell cycle of radial glia progenitor cells, promotes their transition into intermediate progenitors, and leads to reduced upper-layer neurons. CRISPRi screening identified Trp53 knockdown rescuing Upf2KO progenitors without globally reversing NMD inhibition, implying marginal contributions of most NMD targets to the cell cycle defect. Integrated functional genomics shows that NMD degrades selective TRP53 downstream targets, including Cdkn1a, which, without NMD suppression, slow the cell cycle. Trp53KO restores the progenitor cell pool and rescues the microcephaly of Upf2KO mice. Therefore, one physiological role of NMD in the developing brain is to degrade selective TRP53 targets to control progenitor cell cycle and brain size.

The Arabidopsis SR45 splicing factor bridges the splicing machinery and the exon-exon junction complex.

The Arabidopsis splicing factor serine/arginine-rich 45 (SR45) contributes to several biological processes. The sr45-1 loss-of-function mutant exhibits delayed root development, late flowering, unusual numbers of floral organs, shorter siliques with decreased seed sets, narrower leaves and petals, and altered metal distribution. SR45 bears a unique RNA recognition motif (RRM) flanked by one serine/arginine-rich (RS) domain on both sides. Here, we studied the function of each SR45 domains by examining their involvement in: (i) the spatial distribution of SR45; (ii) the establishment of a protein-protein interaction network including spliceosomal and exon-exon junction complex (EJC) components; and (iii) the RNA binding specificity. We report that the endogenous SR45 promoter is active during vegetative and reproductive growth, and that the SR45 protein localizes in the nucleus. We demonstrate that the C-terminal arginine/serine-rich domain is a determinant of nuclear localization. We show that the SR45 RRM domain specifically binds purine-rich RNA motifs via three residues (H101, H141, and Y143), and is also involved in protein-protein interactions. We further show that SR45 bridges both mRNA splicing and surveillance machineries as a partner of EJC core components and peripheral factors, which requires phosphoresidues probably phosphorylated by kinases from both the CLK and SRPK families. Our findings provide insights into the contribution of each SR45 domain to both spliceosome and EJC assemblies.

mRNA accessibility within mRNPs as a determinant of gene expression.

Gene expression is a complex process requiring many control mechanisms to achieve a desired phenotype. DNA accessibility within chromatin is well established as an important determinant of gene expression. By contrast, while mRNA also associates with a complement of proteins, the exact nature of messenger ribonucleoprotein (mRNP) packaging and its functional relevance is not as clear. Recent reports indicate that exon junction complex (EJC)-mediated mRNP packaging renders exon junction-proximal regions inaccessible for m6A methylation, and that EJCs reside within the inaccessible interior of globular transcription and export (TREX) complex-associated nuclear mRNPs. We propose that 'mRNA accessibility' within mRNPs is an important determinant of gene expression that may modulate the specificity of a broad array of regulatory processes including but not limited to m6A methylation.

Functional role of SAP18 protein: From transcriptional repression to splicing regulation.

Sin3 associated protein 18 (SAP18) is an evolutionary conserved protein, originally discovered in a complex with the transcriptional regulatory protein, Sin3. Subsequent investigations revealed SAP18 as an integral splicing component of the exon junction complex (EJC)-associated apoptosis-and splicing-associated protein (ASAP)/PNN-RNPS1-SAP18 (PSAP) complex. In association with Sin3, SAP18 contributes toward transcriptional repression of genes implicated in embryonic development, stress response, human immunodeficiency virus type 1 replication, and tumorigenesis. As a part of EJC, SAP18 mediates alternative splicing events and suppresses the cryptic splice sites present within flanking regions of exon-exon junctions. In this review, we provide a thorough discussion on SAP18, focussing on its conserved dual role in transcriptional regulation and messenger RNA splicing. Recent research on the involvement of SAP18 in the emergence of cancer and human disorders has also been highlighted. The potential of SAP18 as a therapeutic target is also discussed in these recent studies, particularly related to malignancies of the myeloid lineage.

Nuclear mRNPs are compact particles packaged with a network of proteins promoting RNA-RNA interactions.

Messenger RNAs (mRNAs) are at the center of the central dogma of molecular biology. In eukaryotic cells, these long ribonucleic acid polymers do not exist as naked transcripts; rather, they associate with mRNA-binding proteins to form messenger ribonucleoprotein (mRNP) complexes. Recently, global proteomic and transcriptomic studies have provided comprehensive inventories of mRNP components. However, knowledge of the molecular features of distinct mRNP populations has remained elusive. We purified endogenous nuclear mRNPs from Saccharomyces cerevisiae by harnessing the mRNP biogenesis factors THO and Sub2 in biochemical procedures optimized to preserve the integrity of these transient ribonucleoprotein assemblies. We found that these mRNPs are compact particles that contain multiple copies of Yra1, an essential protein with RNA-annealing properties. To investigate their molecular and architectural organization, we used a combination of proteomics, RNA sequencing, cryo-electron microscopy, cross-linking mass spectrometry, structural models, and biochemical assays. Our findings indicate that yeast nuclear mRNPs are packaged around an intricate network of interconnected proteins capable of promoting RNA-RNA interactions via their positively charged intrinsically disordered regions. The evolutionary conservation of the major mRNA-packaging factor (yeast Yra1 and Aly/REF in metazoans) points toward a general paradigm governing nuclear mRNP packaging.

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.

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.

Multifaceted roles of MAGOH Proteins.

MAGOH and MAGOHB are paralog proteins that can substitute each other in the exon junction complex (EJC). The EJC is formed of core components EIF4A3, RBM8A, and MAGOH/MAGOHB. As a part of the EJC, MAGOH proteins are required for mRNA splicing, export, translation and nonsense-mediated mRNA decay (NMD). MAGOH is also essential for embryonic development and normal cellular functioning. The haploinsufficiency of MAGOH results in disorders such as microcephaly and cancer. The present review discusses the discovery of MAGOH, its paralog MAGOHB, their roles in cellular function as part of the EJC, and other cellular roles that are not directly associated with mRNA processing. We also discuss how MAGOH haploinsufficiency in cancer cells can be exploited to develop a novel targeted cancer treatment.

FRG1 is a direct transcriptional regulator of nonsense-mediated mRNA decay genes.

FRG1 is the primary candidate gene for Fascioscapulohumeral Muscular Dystrophy. So far, its role has been reported in muscle development, vasculogenesis, angiogenesis, and tumorigenesis. Mechanistically studies suggest FRG1's role in RNA biogenesis which may have implications in multiple physiological processes and diseases, including tumorigenesis. Its probable role as hnRNP and association with NMD-related genes prompted us to look into FRG1's effect on NMD gene expression and the mechanism. Using microarray profiling in cell lines, we found that FRG1 altered the mRNA surveillance pathway and associated pathways, such as RNA transport and spliceosome machinery molecules. Multiple sequence alignment of core factors, namely, UPF1, UPF3B, and SMG1, showed conserved stretches of nucleotide sequence 'CTGGG'. Structural modeling followed by EMSA, ChIP-qPCR, and luciferase reporter assays showed 'CTGGG' as a FRG1 binding site. Analysis of the publicly available datasets showed that the expression of FRG1 correlates with NMD genes in different tissue types. We validated the effect of FRG1 on NMD gene transcription by qRT-PCR. Overall, FRG1 might be a transcriptional regulator of NMD genes.

Diverse Roles of the Exon Junction Complex Factors in the Cell Cycle, Cancer, and Neurodevelopmental Disorders-Potential for Therapeutic Targeting.

The exon junction complex (EJC) plays a crucial role in regulating gene expression at the levels of alternative splicing, translation, mRNA localization, and nonsense-mediated decay (NMD). The EJC is comprised of three core proteins: RNA-binding motif 8A (RBM8A), Mago homolog (MAGOH), eukaryotic initiation factor 4A3 (eIF4A3), and a peripheral EJC factor, metastatic lymph node 51 (MLN51), in addition to other peripheral factors whose structural integration is activity-dependent. The physiological and mechanistic roles of the EJC in contribution to molecular, cellular, and organismal level function continue to be explored for potential insights into genetic or pathological dysfunction. The EJC's specific role in the cell cycle and its implications in cancer and neurodevelopmental disorders prompt enhanced investigation of the EJC as a potential target for these diseases. In this review, we highlight the current understanding of the EJC's position in the cell cycle, its relation to cancer and developmental diseases, and potential avenues for therapeutic targeting.

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.

The Exon Junction Complex Core Represses Cancer-Specific Mature mRNA Re-splicing: A Potential Key Role in Terminating Splicing.

Using TSG101 pre-mRNA, we previously discovered cancer-specific re-splicing of mature mRNA that generates aberrant transcripts/proteins. The fact that mRNA is aberrantly re-spliced in various cancer cells implies there must be an important mechanism to prevent deleterious re-splicing on the spliced mRNA in normal cells. We thus postulated that mRNA re-splicing is controlled by specific repressors, and we searched for repressor candidates by siRNA-based screening for mRNA re-splicing activity. We found that knock-down of EIF4A3, which is a core component of the exon junction complex (EJC), significantly promoted mRNA re-splicing. Remarkably, we could recapitulate cancer-specific mRNA re-splicing in normal cells by knock-down of any of the core EJC proteins, EIF4A3, MAGOH, or RBM8A (Y14), implicating the EJC core as the repressor of mRNA re-splicing often observed in cancer cells. We propose that the EJC core is a critical mRNA quality control factor to prevent over-splicing of mature mRNA.

Revealing molecular pathways for cancer cell fitness through a genetic screen of the cancer translatome.

The major cap-binding protein eukaryotic translation initiation factor 4E (eIF4E), an ancient protein required for translation of all eukaryotic genomes, is a surprising yet potent oncogenic driver. The genetic interactions that maintain the oncogenic activity of this key translation factor remain unknown. In this study, we carry out a genome-wide CRISPRi screen wherein we identify more than 600 genetic interactions that sustain eIF4E oncogenic activity. Our data show that eIF4E controls the translation of Tfeb, a key executer of the autophagy response. This autophagy survival response is triggered by mitochondrial proteotoxic stress, which allows cancer cell survival. Our screen also reveals a functional interaction between eIF4E and a single anti-apoptotic factor, Bcl-xL, in tumor growth. Furthermore, we show that eIF4E and the exon-junction complex (EJC), which is involved in many steps of RNA metabolism, interact to control the migratory properties of cancer cells. Overall, we uncover several cancer-specific vulnerabilities that provide further resolution of the cancer translatome.

Effect of eukaryotic translation initiation factor 4A3 in malignant tumors.

Eukaryotic translation initiation factor 4A3 (EIF4A3), a key component of the exon junction complex, is widely involved in RNA splicing and nonsense-mediated mRNA decay. EIF4A3 has also been reported to be involved in cell cycle regulation and apoptosis. Thus, EIF4A3 may serve as a pivotal regulatory factor involved in the occurrence and development of multiple diseases. Previous studies have demonstrated that EIF4A3 is mutated in neuromuscular degenerative lesions and is differentially expressed in several tumors, serving as a non-coding RNA binding protein to regulate its expression. In addition, studies have reported that inhibiting EIF4A3 can prevent tumor cell proliferation, thus, several researchers are trying to design and synthesize potent and selective EIF4A3 inhibitors. The present review summarizes the function of EIF4A3 in cell cycle and discusses it underlying molecular mechanisms that contribute to the occurrence of malignant diseases. In addition, EIF4A3 selective inhibitors, and bioinformatics analyses performed to analyze the expression and mutations of EIF4A3 in gynecological tumors and breast cancer, are also discussed.

Long noncoding RNA VCAN-AS1 contributes to the progression of gastric cancer via regulating p53 expression.

Gastric cancer (GC) is one of the most frequent malignancies worldwide. Long noncoding RNAs (lncRNAs) are found to be largely implicated in various cancers, including GC. However, the function of lncRNA VCAN antisense RNA 1 (VCAN-AS1) in GC remains unclear. Herein, we observed a low level of VCAN-AS1 in normal gastric tissues through NCBI and UCSC, and that VCAN-AS1 upregulation in GC tissues was related to poor prognosis by TCGA. Furthermore, VCAN-AS1 was found markedly enhanced in GC tissues and cell lines, while its upregulation was related with clinical outcomes of GC patients. Besides this, silencing VCAN-AS1 represses cell proliferation, migration, and invasion but enhances apoptosis. More important, we discovered that VCAN-AS1 expression was negatively correlated with wild-type p53 levels in GC tissues and that p53 was negatively modulated by VCAN-AS1 in GC cells. Furthermore, p53 suppression reversed the repression of VCAN-AS1 silence on the biological processes of AGS cells. Intriguingly, we identified that both VCAN-AS1 and TP53 can bind with eIF4A3, one of the core proteins in the exon junction complex. Also, we confirmed that VCAN-AS1 negatively regulates TP53 expression by competitively binding with eIF4A3. Our findings disclosed that VCAN-AS1 contributes to GC progression through interacting with eIF4A3 to downregulate TP53 expression, indicating that VCAN-AS1 is a novel therapeutic strategy for GC treatment.

The exon junction complex is required for stem and progenitor cell maintenance in planarians.

Named for its assembly near exon-exon junctions during pre-mRNA splicing, the exon junction complex (EJC) regulates multiple aspects of RNA biochemistry, including export of spliced mRNAs from the nucleus and translation. Transcriptome analyses have revealed broad EJC occupancy of spliced metazoan transcripts, yet inhibition of core subunits has been linked to surprisingly specific phenotypes and a growing number of studies support gene-specific regulatory roles. Here we report results from a classroom-based RNAi screen revealing the EJC is necessary for regeneration in the planarian flatworm Schmidtea mediterranea. RNAi animals rapidly lost the stem and progenitor cells that drive formation of new tissue during both regeneration and cell turnover, but exhibited normal amputation-induced changes in gene expression in differentiated tissues. Together with previous reports that partial loss of EJC function causes stem cell defects in Drosophila and mice, our observations implicate the EJC as a conserved, posttranscriptional regulator of gene expression in stem cell lineages. This work also highlights the combined educational and scientific impacts of discovery-based research in the undergraduate biology curriculum.

The Transcriptome-wide Landscape and Modalities of EJC Binding in Adult Drosophila.

Exon junction complex (EJC) assembles after splicing at specific positions upstream of exon-exon junctions in mRNAs of all higher eukaryotes, affecting major regulatory events. In mammalian cell cytoplasm, EJC is essential for efficient RNA surveillance, while in Drosophila, EJC is essential for localization of oskar mRNA. Here we developed a method for isolation of protein complexes and associated RNA targets (ipaRt) to explore the EJC RNA-binding landscape in a transcriptome-wide manner in adult Drosophila. We find the EJC at canonical positions, preferably on mRNAs from genes comprising multiple splice sites and long introns. Moreover, EJC occupancy is highest at junctions adjacent to strong splice sites, CG-rich hexamers, and RNA structures. Highly occupied mRNAs tend to be maternally localized and derive from genes involved in differentiation or development. These modalities, which have not been reported in mammals, specify EJC assembly on a biologically coherent set of transcripts in Drosophila.

Co-transcriptional Loading of RNA Export Factors Shapes the Human Transcriptome.

During gene expression, RNA export factors are mainly known for driving nucleo-cytoplasmic transport. While early studies suggested that the exon junction complex (EJC) provides a binding platform for them, subsequent work proposed that they are only recruited by the cap binding complex to the 5' end of RNAs, as part of TREX. Using iCLIP, we show that the export receptor Nxf1 and two TREX subunits, Alyref and Chtop, are recruited to the whole mRNA co-transcriptionally via splicing but before 3' end processing. Consequently, Alyref alters splicing decisions and Chtop regulates alternative polyadenylation. Alyref is recruited to the 5' end of RNAs by CBC, and our data reveal subsequent binding to RNAs near EJCs. We demonstrate that eIF4A3 stimulates Alyref deposition not only on spliced RNAs close to EJC sites but also on single-exon transcripts. Our study reveals mechanistic insights into the co-transcriptional recruitment of mRNA export factors and how this shapes the human transcriptome.

The arginine and serine-rich domains of Acinus modulate splicing.

Apoptotic chromatin condensation inducer in the nucleus (Acinus) is an RNA-binding protein that has a functional role in inducing apoptotic chromatin condensation and regulating messenger RNA (mRNA) processing. Acinus interacts with the spliceosomal machinery and is a member of the ASAP (apoptosis and splicing-associated protein complex) as well as the EJC (exon junction complex), which gets deposited onto mRNA during splicing. In this study, we have used in vivo splicing assays to characterize the function of Acinus in pre-mRNA splicing more closely. We show that full-length Acinus-S', an isoform of Acinus, does not have a role in modulating splice site selection in human immunodeficiency virus 1 minigene reporter system. In contrast, we observed that the tethering of arginine/serine (RS) and RNPS1-SAP18-binding (RSB) domains of Acinus could regulate the selection of alternative splice sites, thereby revealing the potential of Acinus in stimulating alternative splicing. Altogether, our data suggest that the RS and RSB domains play a critical role in regulating splicing activity via selection of distinct splice sites during pre-mRNA splicing.