Exon Junction Complexes Suppress Spurious Splice Sites to Safeguard Transcriptome Integrity.

Productive splicing of human precursor messenger RNAs (pre-mRNAs) requires the correct selection of authentic splice sites (SS) from the large pool of potential SS. Although SS consensus sequence and splicing regulatory proteins are known to influence SS usage, the mechanisms ensuring the effective suppression of cryptic SS are insufficiently explored. Here, we find that many aberrant exonic SS are efficiently silenced by the exon junction complex (EJC), a multi-protein complex that is deposited on spliced mRNA near the exon-exon junction. Upon depletion of EJC proteins, cryptic SS are de-repressed, leading to the mis-splicing of a broad set of mRNAs. Mechanistically, the EJC-mediated recruitment of the splicing regulator RNPS1 inhibits cryptic 5'SS usage, while the deposition of the EJC core directly masks reconstituted 3'SS, thereby precluding transcript disintegration. Thus, the EJC protects the transcriptome of mammalian cells from inadvertent loss of exonic sequences and safeguards the expression of intact, full-length mRNAs.

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.

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.

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.

A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs.

The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs.

DS6: anticandidal, antibiofilm peptide against Candida tropicalis and exhibit synergy with commercial drug.

Antifungal peptides have gained interest as therapeutic agents in recent years because of increased multidrug resistance against present antifungal drugs. This study designed, synthesized and characterized antifungal activity of a small peptide analogue, DS6. This peptide was designed using the template from the N-terminal part of the antifungal protein, Aspergillus giganteous. DS6 inhibited Candida tropicalis (ATCC 13803), as well as its clinical isolates. DS6 was found to be a fungicidal, killing the fungus very rapidly. DS6 is also non-toxic to human cells. Synergistic interactions of DS6 with amphotericin B and fluconazole were also evident. DS6 is membrane lytic and exhibits antibiofilm activity against C. tropicalis. In conclusion, DS6 may have utility as an alternative antifungal therapy for C. tropicalis. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

A novel antimicrobial peptide derived from modified N-terminal domain of bovine lactoferrin: design, synthesis, activity against multidrug-resistant bacteria and Candida.

Lactoferrin (LF) is believed to contribute to the host's defense against microbial infections. This work focuses on the antibacterial and antifungal activities of a designed peptide, L10 (WFRKQLKW) by modifying the first eight N-terminal residues of bovine LF by selective homologous substitution of amino acids on the basis of hydrophobicity, L10 has shown potent antibacterial and antifungal properties against clinically isolated extended spectrum beta lactamases (ESBL), producing gram-negative bacteria as well as Candida strains with minimal inhibitory concentrations (MIC) ranging from 1 to 8 µg/mL and 6.5 µg/mL, respectively. The peptide was found to be least hemolytic at a concentration of 800 µg/mL. Interaction with lipopolysaccharide (LPS) and lipid A (LA) suggests that the peptide targets the membrane of gram-negative bacteria. The membrane interactive nature of the peptide, both antibacterial and antifungal, was further confirmed by visual observations employing electron microscopy. Further analyses, by means of propidium iodide based flow cytometry, also supported the membrane permeabilization of Candida cells. The peptide was also found to possess anti-inflammatory properties, by virtue of its ability to inhibit cyclooxygenase-2 (COX-2). L10 therefore emerges as a potential therapeutic remedial solution for infections caused by ESBL positive, gram-negative bacteria and multidrug-resistant (MDR) fungal strains, on account of its multifunctional activities. This study may open up new approach to develop and design novel antimicrobials.

MicroRNA-mediated regulation of nonsense-mediated mRNA decay factors: Insights into microRNA prediction tools and profiling techniques.

Nonsense-mediated mRNA decay (NMD) stands out as a prominent RNA surveillance mechanism within eukaryotes, meticulously overseeing both RNA abundance and integrity by eliminating aberrant transcripts. These defective transcripts are discerned through the concerted efforts of translating ribosomes, eukaryotic release factors (eRFs), and trans-acting NMD factors, with Up-Frameshift 3 (UPF3) serving as a noteworthy component. Remarkably, in humans, UPF3 exists in two paralogous forms, UPF3A (UPF3) and UPF3B (UPF3X). Beyond its role in quality control, UPF3 wields significant influence over critical cellular processes, including neural development, synaptic plasticity, and axon guidance. However, the precise regulatory mechanisms governing UPF3 remain elusive. MicroRNAs (miRNAs) emerge as pivotal post-transcriptional gene regulators, exerting substantial impact on diverse pathological and physiological pathways. This comprehensive review encapsulates our current understanding of the intricate regulatory nexus between NMD and miRNAs, with particular emphasis on the essential role played by UPF3B in neurodevelopment. Additionally, we bring out the significance of the 3'-untranslated region (3'-UTR) as the molecular bridge connecting NMD and miRNA-mediated gene regulation. Furthermore, we provide an in-depth exploration of diverse computational tools tailored for the prediction of potential miRNA targets. To complement these computational approaches, we delineate experimental techniques designed to validate predicted miRNA-mRNA interactions, empowering readers with the knowledge necessary to select the most appropriate methodology for their specific research objectives.

Two mammalian MAGOH genes contribute to exon junction complex composition and nonsense-mediated decay.

The exon junction complex (EJC) participates in the regulation of many post-transcriptional steps of gene expression. EJCs are deposited on messenger RNAs (mRNAs) during splicing and their core consists of eIF4A3, MLN51, Y14, and MAGOH. Here, we show that two genes encoding MAGOH paralogs (referred to as MAGOH and MAGOHB) are expressed in mammals. In macrophages, the expression of MAGOHB, but not MAGOH mRNA, increases rapidly after LPS stimulation. Both MAGOH proteins interact with other EJC components, incorporate into mRNA-bound EJCs, and activate nonsense-mediated decay. Furthermore, the simultaneous depletion of MAGOH and MAGOHB, but not individual depletions, impair nonsense-mediated decay in human cells. Hence, our results establish that the core composition of mammalian EJCs is more complex than previously recognized.

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.

Genomic analyses of the RNA-binding protein Hu antigen R (HuR) identify a complex network of target genes and novel characteristics of its binding sites.

The ubiquitously expressed RNA-binding protein Hu antigen R (HuR) or ELAVL1 is implicated in a variety of biological processes as well as being linked with a number of diseases, including cancer. Despite a great deal of prior investigation into HuR, there is still much to learn about its function. We take an important step in this direction by conducting cross-linking and immunoprecipitation and RNA sequencing experiments followed by an extensive computational analysis to determine the characteristics of the HuR binding site and impact on the transcriptome. We reveal that HuR targets predominantly uracil-rich single-stranded stretches of varying size, with a strong conservation of structure and sequence composition. Despite the fact that HuR sites are observed in intronic regions, our data do not support a role for HuR in regulating splicing. HuR sites in 3'-UTRs overlap extensively with predicted microRNA target sites, suggesting interplay between the functions of HuR and microRNAs. Network analysis showed that identified targets containing HuR binding sites in the 3' UTR are highly interconnected.

Human SAP18 mediates assembly of a splicing regulatory multiprotein complex via its ubiquitin-like fold.

RNPS1, Acinus, and SAP18 form the apoptosis- and splicing-associated protein (ASAP) complex, which is also part of the exon junction complex. Whereas RNPS1 was originally identified as a general activator of mRNA processing, all three proteins have been found within functional spliceosomes. Both RNPS1 and Acinus contain typical motifs of splicing regulatory proteins including arginine/serine-rich domains. Due to the absence of such structural features, however, a function of SAP18 in splicing regulation is completely unknown. Here we have investigated splicing regulatory activities of the ASAP components. Whereas a full-length Acinus isoform displayed only limited splicing regulatory activity, both RNPS1 and, surprisingly, SAP18 strongly modulated splicing regulation. Detailed mutational analysis and three-dimensional modeling data revealed that the ubiquitin-like fold of SAP18 was required for efficient splicing regulatory activity. Coimmunoprecipitation and immunofluorescence experiments demonstrated that SAP18 assembles a nuclear speckle-localized splicing regulatory multiprotein complex including RNPS1 and Acinus via its ubiquitin-like fold. Our results therefore suggest a novel function of SAP18 in splicing regulation.

A single nucleotide polymorphism determines protein isoform production of the human c-FLIP protein.

The cellular FLICE-inhibitory protein (c-FLIP) is a modulator of death receptor-mediated apoptosis and plays a major role in T- and B-cell homeostasis. Three different isoforms have been described on the protein level, including the long form c-FLIP(L) as well as 2 short forms, c-FLIP(S) and the recently identified c-FLIP(R). The mechanisms controlling c-FLIP isoform production are largely unknown. Here, we identified by sequence comparison in several mammals that c-FLIP(R) and not the widely studied c-FLIP(S) is the evolutionary ancestral short c-FLIP protein. Unexpectedly, the decision for production of either c-FLIP(S) or c-FLIP(R) in humans is defined by a single nucleotide polymorphism in a 3' splice site of the c-FLIP gene (rs10190751A/G). Whereas an intact splice site directs production of c-FLIP(S), the splice-dead variant causes production of c-FLIP(R). Interestingly, due to differences in protein translation rates, higher amounts of c-FLIP(S) protein compared with c-FLIP(R) are produced. Investigation of diverse human cell lines points to an increased frequency of c-FLIP(R) in transformed B-cell lines. A comparison of 183 patients with follicular lymphoma and 233 population controls revealed an increased lymphoma risk associated with the rs10190751 A genotype causing c-FLIP(R) expression.

A comparison of structural equation and multidimensional Rasch modeling approaches to Confirmatory Factor Analysis.

This paper compares the results of applications of the Multidimensional Random Coefficients Multinomial Logit Model (MRCMLM) to comparable Structural Equation Model (SEM) applications for the purpose of conducting a Confirmatory Factor Analysis (CFA). We review SEM as it is applied to CFA, identify some parallels between the MRCMLM approach to CFA and that utilized in a standard SEM CFA, and illustrate the comparability of MRCMLM and SEM CFA results for three datasets. Results indicate that the two approaches tend to identify similar dimensional models as exhibiting best fit and provide comparable depictions of latent variable correlations, but the two procedures depict the reliability of measures differently.

SpliceViNCI: Visualizing the splicing of non-canonical introns through recurrent neural networks.

Most of the current computational models for splice junction prediction are based on the identification of canonical splice junctions. However, it is observed that the junctions lacking the consensus dimers GT and AG also undergo splicing. Identification of such splice junctions, called the non-canonical splice junctions, is also essential for a comprehensive understanding of the splicing phenomenon. This work focuses on the identification of non-canonical splice junctions through the application of a bidirectional long short-term memory (BLSTM) network. Furthermore, we apply a back-propagation-based (integrated gradient) and a perturbation-based (occlusion) visualization techniques to extract the non-canonical splicing features learned by the model. The features obtained are validated with the existing knowledge from the literature. Integrated gradient extracts features that comprise contiguous nucleotides, whereas occlusion extracts features that are individual nucleotides distributed across the sequence.

Functional roles of human Up-frameshift suppressor 3 (UPF3) proteins: From nonsense-mediated mRNA decay to neurodevelopmental disorders.

Nonsense-mediated mRNA decay (NMD) is a post-transcriptional quality control mechanism that eradicates aberrant transcripts from cells. Aberrant transcripts are recognized by translating ribosomes, eRFs, and trans-acting NMD factors leading to their degradation. The trans-factors are conserved among eukaryotes and consist of UPF1, UPF2, and UPF3 proteins. Intriguingly, in humans, UPF3 exists as paralog proteins, UPF3A, and UPF3B. While UPF3 paralogs are traditionally known to be involved in the NMD pathway, there is a growing consensus that there are other critical cellular functions beyond quality control that are dictated by the UPF3 proteins. This review presents the current knowledge on the biochemical functions of UPF3 paralogs in diverse cellular processes, including NMD, translation, and genetic compensation response. We also discuss the contribution of the UPF3 paralogs in development and function of the central nervous system and germ cells. Furthermore, significant advances in the past decade have provided new perspectives on the implications of UPF3 paralogs in neurodevelopmental diseases. In this regard, genome- and transcriptome-wide sequencing analysis of patient samples revealed that loss of UPF3B is associated with brain disorders such as intellectual disability, autism, attention deficit hyperactivity disorder, and schizophrenia. Therefore, we further aim to provide an insight into the brain diseases associated with loss-of-function mutations of UPF3B.

Using the Chou's 5-steps rule to predict splice junctions with interpretable bidirectional long short-term memory networks.

Neural models have been able to obtain state-of-the-art performances on several genome sequence-based prediction tasks. Such models take only nucleotide sequences as input and learn relevant features on their own. However, extracting the interpretable motifs from the model remains a challenge. This work explores various existing visualization techniques in their ability to infer relevant sequence information learnt by a recurrent neural network (RNN) on the task of splice junction identification. The visualization techniques have been modulated to suit the genome sequences as input. The visualizations inspect genomic regions at the level of a single nucleotide as well as a span of consecutive nucleotides. This inspection is performed based on the modification of input sequences (perturbation based) or the embedding space (back-propagation based). We infer features pertaining to both canonical and non-canonical splicing from a single neural model. Results indicate that the visualization techniques produce comparable performances for branchpoint detection. However, in the case of canonical donor and acceptor junction motifs, perturbation based visualizations perform better than back-propagation based visualizations, and vice-versa for non-canonical motifs. The source code of our stand-alone SpliceVisuL tool is available at https://github.com/aaiitggrp/SpliceVisuL.

SpliceVec: Distributed feature representations for splice junction prediction.

Identification of intron boundaries, called splice junctions, is an important part of delineating gene structure and functions. This also provides valuable insights into the role of alternative splicing in increasing functional diversity of genes. Identification of splice junctions through RNA-seq is by mapping short reads to the reference genome which is prone to errors due to random sequence matches. This encourages identification of splicing junctions through computational methods based on machine learning. Existing models are dependent on feature extraction and selection for capturing splicing signals lying in the vicinity of splice junctions. But such manually extracted features are not exhaustive. We introduce distributed feature representation, SpliceVec, to avoid explicit and biased feature extraction generally adopted for such tasks. SpliceVec is based on two widely used distributed representation models in natural language processing. Learned feature representation in form of SpliceVec is fed to multilayer perceptron for splice junction classification task. An intrinsic evaluation of SpliceVec indicates that it is able to group true and false sites distinctly. Our study on optimal context to be considered for feature extraction indicates inclusion of entire intronic sequence to be better than flanking upstream and downstream region around splice junctions. Further, SpliceVec is invariant to canonical and non-canonical splice junction detection. The proposed model is consistent in its performance even with reduced dataset and class-imbalanced dataset. SpliceVec is computationally efficient and can be trained with user-defined data as well.