Splicing Components Research Articles

The Many Roads from Alternative Splicing to Cancer: Molecular Mechanisms Involving Driver Genes.

Cancer driver genes are either oncogenes or tumour suppressor genes that are classically activated or inactivated, respectively, by driver mutations. Alternative splicing-which produces various mature mRNAs and, eventually, protein variants from a single gene-may also result in driving neoplastic transformation because of the different and often opposed functions of the variants of driver genes. The present review analyses the different alternative splicing events that result in driving neoplastic transformation, with an emphasis on their molecular mechanisms. To do this, we collected a list of 568 gene drivers of cancer and revised the literature to select those involved in the alternative splicing of other genes as well as those in which its pre-mRNA is subject to alternative splicing, with the result, in both cases, of producing an oncogenic isoform. Thirty-one genes fall into the first category, which includes splicing factors and components of the spliceosome and splicing regulators. In the second category, namely that comprising driver genes in which alternative splicing produces the oncogenic isoform, 168 genes were found. Then, we grouped them according to the molecular mechanisms responsible for alternative splicing yielding oncogenic isoforms, namely, mutations in cis splicing-determining elements, other causes involving non-mutated cis elements, changes in splicing factors, and epigenetic and chromatin-related changes. The data given in the present review substantiate the idea that aberrant splicing may regulate the activation of proto-oncogenes or inactivation of tumour suppressor genes and details on the mechanisms involved are given for more than 40 driver genes.

On homology planes and contractible 4‐manifolds

AbstractWe call a non‐trivial homology sphere a Kirby–Ramanujam sphere if it bounds both a homology plane and a Mazur or Poénaru manifold. In 1980, Kirby found the first example by proving that the boundary of the Ramanujam surface bounds a Mazur manifold and it has remained a single example since then. By tracing their initial step, we provide the first additional examples and we present three infinite families of Kirby–Ramanujam spheres. Also, we show that one of our families of Kirby–Ramanujam spheres is diffeomorphic to the splice of two certain families of Brieskorn spheres. Since this family of Kirby–Ramanujam spheres bound contractible 4‐manifolds, they lie in the class of the trivial element in the homology cobordism group; however, both splice components are separately linearly independent in that group.

An ATP-independent role for Prp16 in promoting aberrant splicing.

The spliceosome is assembled through a step-wise process of binding and release of its components to and from the pre-mRNA. The remodeling process is facilitated by eight DExD/H-box RNA helicases, some of which have also been implicated in splicing fidelity control. In this study, we unveil a contrasting role for the prototypic splicing proofreader, Prp16, in promoting the utilization of aberrant 5' splice sites and mutated branchpoints. Prp16 is not essential for the branching reaction in wild-type pre-mRNA. However, when a mutation is present at the 5' splice site or if Cwc24 is absent, Prp16 facilitates the reaction and encourages aberrant 5' splice site usage independently of ATP. Prp16 also promotes the utilization of mutated branchpoints while preventing the use of nearby cryptic branch sites. Our study demonstrates that Prp16 can either enhance or impede the utilization of faulty splice sites by stabilizing or destabilizing interactions with other splicing components. Thus, Prp16 exerts dual roles in 5' splice site and branch site selection, via ATP-dependent and ATP-independent activities. Furthermore, we provide evidence that these functions of Prp16 are mediated through the step-one factor Cwc25.

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.

Elongation factor-specific capture of RNA polymerase II complexes

Transcription of protein-coding genes is regulated by dynamic association of co-factors with RNA polymerase II (RNAPII). The function of these factors and their relationship with RNAPII is often poorly understood. Here, we present an approach for elongation-factor-specific mNET capture (ELCAP) of RNAPII complexes for sequencing and mass spectrometry analysis aimed at investigating the function of such RNAPII regulatory proteins. As proof of principle, we apply ELCAP to the RNAPII-associated proteins SCAF4 and SCAF8, which share an essential role as mRNA anti-terminators but have individual roles at the 3' end of genes. Mass spectrometry analysis shows that both SCAF4 and SCAF8 are part of RNAPII elongation complexes containing 3' end processing factors but depleted of splicing components. Importantly, the ELCAP sequencing (ELCAP-seq) profiles of SCAF4- and SCAF8-RNAPII complexes nicely reflect their function as mRNA-anti-terminators and their competing functions at the end of genes, where they prevent or promote transcriptional readthrough.

Histone Marks-Dependent Effect on Alternative Splicing: New Perspectives for Targeted Splicing Modulation in Cancer?

Alternative splicing (AS) is a tightly regulated mechanism that generates the complex human proteome from a small number of genes. Cis-regulatory RNA motifs in exons and introns control AS, recruiting positive and negative trans-acting splicing regulators. At a higher level, chromatin affects splicing events. Growing evidence indicates that the popular histone code hypothesis can be extended to RNA-level processes, such as AS. In addition to nucleosome positioning, which can generate transcriptional barriers to shape the final splicing outcome, histone post-translational modifications can contribute to the detailed regulation of single exon inclusion/exclusion. A histone-based system can identify alternatively spliced chromatin stretches, affecting RNAPII elongation locally or recruiting splicing components via adaptor complexes. In tumor cells, several mechanisms trigger misregulated AS events and produce cancer-associated transcripts. On a genome-wide level, aberrant AS can be the consequence of dysfunctional epigenetic splicing code, including altered enrichment in histone post-translational modifications. This review describes the main findings related to the effect of histone modifications and variants on splicing outcome and how a dysfunctional epigenetic splicing code triggers aberrant AS in cancer. In addition, it highlights recent advances in programmable DNA-targeting technologies and their possible application for AS targeted epigenetic modulation.

ALX1-transcribed LncRNA AC132217.4 promotes osteogenesis and bone healing via IGF-AKT signaling in mesenchymal stem cells.

The osteogenic potential of bone marrow mesenchymal stem cells (BMSCs) is critical for bone formation and regeneration. A high non-/delayed-union rate of fracture healing still occurs in specific populations, implying an urgent need to discover novel targets for promoting osteogenesis and bone regeneration. Long non-coding (lnc)RNAs are emerging regulators of multiple physiological processes, including osteogenesis. Based on differential expression analysis of RNA sequencing data, we found that lncRNA AC132217.4, a 3'UTR-overlapping lncRNA of insulin growth factor 2 (IGF2), was highly induced during osteogenic differentiation of BMSCs. Afterward, both gain-of-function and loss-of-function experiments proved that AC132217.4 promotes osteoblast development from BMSCs. As for its molecular mechanism, we found that AC132217.4 binds with IGF2 mRNA to regulate its expression and downstream AKT activation to control osteoblast maturation and function. Furthermore, we identified two splicing factors, splicing component 35 KDa (SC35) and heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), which regulate the biogenesis of AC132217.4 at the post-transcriptional level. We also identified a transcription factor, ALX1, which regulates AC132217.7 expression at the transcriptional level to promote osteogenesis. Importantly, in-vivo over-expression of AC132217.4 essentially promotes the bone healing process in a murine tibial drill-hole model. Our study demonstrates that lncRNA AC132217.4 is a novel anabolic regulator of BMSC osteogenesis and could be a plausible therapeutic target for improving bone regeneration.

Covalent narlaprevir- and boceprevir-derived hybrid inhibitors of SARS-CoV-2 main protease

Emerging SARS-CoV-2 variants continue to threaten the effectiveness of COVID-19 vaccines, and small-molecule antivirals can provide an important therapeutic treatment option. The viral main protease (Mpro) is critical for virus replication and thus is considered an attractive drug target. We performed the design and characterization of three covalent hybrid inhibitors BBH-1, BBH-2 and NBH-2 created by splicing components of hepatitis C protease inhibitors boceprevir and narlaprevir, and known SARS-CoV-1 protease inhibitors. A joint X-ray/neutron structure of the Mpro/BBH-1 complex demonstrates that a Cys145 thiolate reaction with the inhibitor’s keto-warhead creates a negatively charged oxyanion. Protonation states of the ionizable residues in the Mpro active site adapt to the inhibitor, which appears to be an intrinsic property of Mpro. Structural comparisons of the hybrid inhibitors with PF-07321332 reveal unconventional F···O interactions of PF-07321332 with Mpro which may explain its more favorable enthalpy of binding. BBH-1, BBH-2 and NBH-2 exhibit comparable antiviral properties in vitro relative to PF-07321332, making them good candidates for further design of improved antivirals.

High Temperature In Situ Optical Observation and Structural Optimization Numerical Simulation of High Nitrogen Steel (Cr18Mn18N)

As a steel with high strength, good plasticity and fracture toughness, high temperature resistance, and corrosion resistance, Cr18Mn18N is widely used in industrial engineering and military fields. However, in a high temperature environment, Cr18Mn18N needs to be subjected to higher temperature, resulting in excessive expansion deformation and larger stress, which will greatly damage the stability and service life of the material structure. In this paper, the high temperature arc wind tunnel is used to heat the high nitrogen steel material with prefabricated round structure, and the surface images of the material are collected at the temperature of 1500 K. After comparison, it is found that the material is well preserved in a high temperature environment, indicating that the circular structure has better thermal protection ability. Based on the experiment, the thermal-fluid-solid coupling model is established, and the surface temperature field, deformation field, and stress field are analyzed. Different surface structures are designed, and numerical models of horizontal and vertical splicing components are established. Through numerical simulation, the surface structure is optimized, the surface temperature of the material is reduced, and the gap change trend of the splicing component is displayed. This work has theoretical significance for the application of materials in a high temperature environment and the optimization and improvement of material surface structure.

SF3B1 homeostasis is critical for survival and therapeutic response in T cell leukemia.

The production of noncanonical mRNA transcripts is associated with cell transformation. Driven by our previous findings on the sensitivity of T cell acute lymphoblastic leukemia (T-ALL) cells to SF3B1 inhibitors, we identified that SF3B1 inhibition blocks T-ALL growth in vivo with no notable associated toxicity. We also revealed protein stabilization of the U2 complex component SF3B1 via deubiquitination. Our studies showed that SF3B1 inhibition perturbs exon skipping, leading to nonsense-mediated decay and diminished levels of DNA damage response–related transcripts, such as the serine/threonine kinase CHEK2, and impaired DNA damage response. We also identified that SF3B1 inhibition leads to a general decrease in R-loop formation. We further demonstrate that clinically used SF3B1 inhibitors synergize with CHEK2 inhibitors and chemotherapeutic drugs to block leukemia growth. Our study provides the proof of principle for posttranslational regulation of splicing components and associated roles and therapeutic implications for the U2 complex in T cell leukemia.

Galectin-3-U1 snRNP Complexes Initiate Splicing Activity in U1-Depleted Nuclear Extracts.

Fractionation of HeLa cell nuclear extracts by glycerol gradient centrifugation separates endogenous uracil-rich small nuclear ribonucleoprotein complexes (U snRNP) into numerous particles sedimenting from 7S to greater than 60S. Complexes sedimenting at 10S contain a single U snRNP (U1 snRNP) and galectin-3. Addition of antibodies specific for galectin-3 to fractions containing these 10S complexes coprecipitates U1 snRNP, indicating that a fraction of the U1 snRNP is associated with this galectin. Galectin-3 has been shown by depletion-reconstitution studies to be an integral splicing component involved both in spliceosome assembly and splicing activity. The first step in initiation of spliceosome assembly is binding of U1 snRNP to the 5' splice site of the premessenger RNA substrate. The finding that U1 snRNP and galectin-3 are associated in splicing extracts hints that this complex affords a potential entry point for galectin-3 into the splicing pathway. Addition of U1 snRNP-galectin-3 complexes immunoselected from the 10S region of glycerol gradients to a U1-depleted nuclear extract initiates splicing activity with the formation of splicing intermediates and mature mRNA. This chapter describes the materials and methods for these experiments that document galectin-3-U1 snRNP complexes initiate the splicing reaction in a U1-depleted nuclear extract.

Phylogeny and conservation of plant U2A/U2A', a core splicing component in U2 spliceosomal complex.

This study systematically identifies 112 U2A genes from 80 plant species by combinatory bioinformatics analysis, which is important for understanding their phylogenetic history, expression profiles and for predicting specific functions. In eukaryotes, a pre-mRNA can generate multiple transcripts by removing certain introns and joining corresponding exons, thus greatly expanding the transcriptome and proteome diversity. The spliceosome is a mega-Dalton ribonucleoprotein (RNP) complex that is essential for the process of splicing. In spliceosome components, the U2 small nuclear ribonucleoprotein (U2 snRNP) forms the pre-spliceosome by association with the branch site. An essential component that promotes U2 snRNP assembly, named U2A, has been extensively identified in humans, yeast and nematodes. However, studies examining U2A genes in plants are scarce. In this study, we performed a comprehensive analysis and identified a total of 112 U2A genes from 80 plant species representing dicots, monocots, mosses and algae. Comparisons of the gene structures, protein domains, and expression patterns of 112 U2A genes indicated that the conserved functions were likely retained by plant U2A genes and important for responses to internal and external stimuli. In addition, analysis of alternative transcripts and splice sites of U2A genes indicated that the fifth intron contained a conserved alternative splicing event that might be important for its molecular function. Our work provides a general understanding of this splicing factor family in terms of genes and proteins, and it will serve as a fundamental resource that will contribute to further mechanistic characterization in plants.

Study on improving interface bonding performance of magnesium potassium phosphate cement mortar

To enhance the interfacial bonding performance between magnesium potassium phosphate cement (MKPC) repair mortar and matrix concrete, in this study, MKPC modified mortar was used as the repair material to splice long prismatic test pieces. The four-point bending test was used to determine the flexural bearing capacity of the long prism, and the influence of changing the interface conditions and the modifying the MKPC repair mortar on the improvement of the basic performance of the splicing component is investigated.The research results show that, when the matrix concrete section is in a natural state, applying silica-fume modified MKPC interface agents on the interface with a repair thickness of 3 cm can improve the interface bonding performance. Furthermore, the working performance and mechanical properties of the MKPC repair mortar modified using nickel-iron slag and steel fibers have been significantly improved.

Abstract 202: Identification of survival associated hub genes in prostate cancer patients from the TCGA database

Abstract Background: Prostate cancer is the most frequently diagnosed malignancy and the fourth leading cause of cancer-related death in the global male population. Although the disease has a relatively low mortality rate with some patients surviving for 10-20 years after treatment, others respond poorly to treatment and die of metastatic disease within 2-3 years. Therefore, there is an urgent need to develop strategies to identify patients with clinically significant prostate cancer requiring aggressive treatment to improve survival, while sparing others unnecessary side effects. The purpose of this study was to identify survival associated genes in prostate cancer patients from the TCGA database using bioinformatics tools. Methods: Data from prostate cancer patients in the TCGA database were divided into two study groups: a high and a low expression group, relative to the median expression. The Gene Expression Profiling Interactive Analysis (GEPIA2) tool was used for the identification of the most differential survival genes. Metascape bioinformatics tool was subsequently used for clustering of genes based on processes, pathway enrichment analysis, and construction of Protein-Protein Interaction (PPI) network. Metascape was also used for molecular Complex Detection (MCODE) to identify the genes with the highest degree of connection, known as hub genes, and to screen modules of the PPI network. Results: Bioinformatics analysis allowed the identification of 361 genes whose expression levels were significantly associated with overall survival in prostate cancer patients from the TCGA. Survival associated genes were primarily enriched in mRNA processing, DNA repair, ncRNA processing, DNA replication, macromolecule methylation, among others. The 12 most connected genes were selected as hub genes and Kaplan-Meier analysis was used to verify survival associated with this set of genes. Hub genes included several splicing factors and components of the processing machinery of cellular pre-mRNAs. Conclusions: These hub genes may reveal basic mechanisms underlying the development of clinically relevant prostate cancer and contribute to the identification of novel markers for prognosis of this cancer. Citation Format: Niradiz Reyes, Raj Tiwari, Jan Geliebter. Identification of survival associated hub genes in prostate cancer patients from the TCGA database [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 202.

Splicing regulation in hematopoiesis.

Splicing mutations are among the most recurrent genetic perturbations in hematological malignancies, highlighting an important impact of splicing regulation in hematopoietic development. However, compared to our understanding of splicing factor mutations in hematological malignancies, studies of splicing components and alternative splicing in normal hematopoiesis have been less well investigated. Here, we outline the most recent findings on splicing regulation in normal hematopoiesis and discuss the important questions in the field. Recent studies have highlighted the critical role of splicing regulation in hematopoiesis, including characterization of splicing components in normal hematopoiesis, investigation of transcriptional alterations on splicing, and identification of stage-specific alternative splicing events during hematopoietic development. These interesting findings provide insights on hematopoietic regulation at a co-transcriptional level. More high-throughput RNA ribonucleic acid (RNA) sequencing and functional genomic screens are needed to advance our knowledge of critical alternative splicing patterns in shaping hematopoiesis.

Therapeutic approaches targeting splicing factor mutations in myelodysplastic syndromes and acute myeloid leukemia.

Mutations in components of the spliceosome are the most common acquired lesions in myelodysplastic syndromes (MDS) and are frequently identified in other myeloid malignancies with a high rate of progression to acute myeloid leukemia (AML) including chronic myelomonocytic leukemia and primary myelofibrosis. The only curative option for these disorders remains allogeneic stem-cell transplantation, which is associated with high morbidity and mortality in these patients. The purpose of this review is to highlight the recent therapeutic developments and strategies being pursued for clinical benefit in splicing factor mutant myeloid malignancies. Cells harboring splicing factor mutations have increased aberrant splicing leading to R-loop formation and cell cycle stalling that create dependencies on Checkpoint kinase 1 (CHK1) activation and canonical splicing maintained by protein arginine methyltransferase activity. Both targeting of the spliceosome and targeting of the downstream consequences of splicing factor mutation expression show promise as selective strategies for the treatment of splicing factor-mutant myeloid malignancies. An improved understanding of the therapeutic vulnerabilities in splicing factor-mutant MDS and AML has led to the development of clinical trials of small molecule inhibitors that target the spliceosome, ataxia telangectasia and Rad3 related (ATR)-CHK1 pathway, and methylation of splicing components.

Selective inhibition of CDK7 reveals high-confidence targets and new models for TFIIH function in transcription

CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e., distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a "master regulator" role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g., SPT5 and SF3B1) are phosphorylated by the three-subunit CDK-activating kinase (CAK; CCNH, MAT1, and CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.

A supervised machine learning-based methodology for analyzing dysregulation in splicing machinery: An application in cancer diagnosis.

Deregulated splicing machinery components have shown to be associated with the development of several types of cancer and, therefore, the determination of such alterations can help the development of tumor-specific molecular targets for early prognosis and therapy. Determining such splicing components, however, is not a straightforward task mainly due to the heterogeneity of tumors, the variability across samples, and the fat-short characteristic of genomic datasets. In this work, a supervised machine learning-based methodology is proposed, allowing the determination of subsets of relevant splicing components that best discriminate samples. The methodology comprises three main phases: first, a ranking of features is determined by means of applying feature weighting algorithms that compute the importance of each splicing component; second, the best subset of features that allows the induction of an accurate classifier is determined by means of conducting an effective heuristic search; then the confidence over the induced classifier is assessed by means of explaining the individual predictions and its global behavior. At the end, an extensive experimental study was conducted on a large collection of transcript-based datasets, illustrating the utility and benefit of the proposed methodology for analyzing dysregulation in splicing machinery.

Phylogenetic comparison of 5' splice site determination in central spliceosomal proteins of the U1-70K gene family, in response to developmental cues and stress conditions.

Intron-containing genes have the ability to generate multiple transcript isoforms by splicing, thereby greatly expanding the eukaryotic transcriptome and proteome. In eukaryotic cells, precursor mRNA (pre-mRNA) splicing is performed by a mega-macromolecular complex defined as a spliceosome. Among its splicing components, U1 small nuclear ribonucleoprotein (U1 snRNP) is the smallest subcomplex involved in early spliceosome assembly and 5'-splice site recognition. Its central component, named U1-70K, has been extensively characterized in animals and yeast. Very few investigations on U1-70K genes have been conducted in plants, however. To this end, we performed a comprehensive study to systematically identify 115 U1-70K genes from 67 plant species, ranging from algae to angiosperms. Phylogenetic analysis suggested that the expansion of the plant U1-70K gene family was likely to have been driven by whole-genome duplications. Subsequent comparisons of gene structures, protein domains, promoter regions and conserved splicing patterns indicated that plant U1-70Ks are likely to preserve their conserved molecular function across plant lineages and play an important functional role in response to environmental stresses. Furthermore, genetic analysis using T-DNA insertion mutants suggested that Arabidopsis U1-70K may be involved in response to osmotic stress. Our results provide a general overview of this gene family in Viridiplantae and will act as a reference source for future mechanistic studies on this U1 snRNP-specific splicing factor.

Blastocladiella emersonii spliceosome is regulated in response to the splicing inhibition caused by the metals cadmium, cobalt and manganese

Blastocladiella emersonii is an aquatic fungus of the phylum Blastocladiomycota, localized near the base of the fungal tree. Previous studies have shown that B. emersonii responds to heat shock and cadmium exposure inducing the transcription of a high number of genes. EST sequencing from heat shocked and cadmium exposed B. emersonii cells has shown that exposure to cadmium causes strong splicing inhibition. Despite the knowledge about splicing inhibition by cadmium, it is still unclear if other metal contaminants can cause the same response. In the present study, we have demonstrated that the effect of cadmium exposure on splicing inhibition is much stronger than that of other divalent metals such as cobalt and manganese. Data presented here also indicate that intron retention occurs randomly among the fungal transcripts, as verified by analyzing differently affected transcripts. In addition, we identified in the genome of B. emersonii the genes encoding the snRNA splicing components U1, U2, U4, U5 and U6 and observed that spliceosome snRNAs are upregulated in the presence of metals, in particular snRNA U1 in cells under cadmium exposure. This observation suggests that snRNA upregulation might be a defense of the fungal cell against the metal stress condition.