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Wiley Interdisciplinary Reviews. RNA Nov 2022Alternative RNA splicing increases transcript diversity in different cell types and under varying conditions. It is executed with the help of RNA splicing regulators... (Review)
Review
Alternative RNA splicing increases transcript diversity in different cell types and under varying conditions. It is executed with the help of RNA splicing regulators (RSRs), which are operationally defined as RNA-binding proteins (RBPs) that regulate alternative splicing, but not directly catalyzing the chemical reactions of splicing. By systematically searching for RBPs and manually identifying those that regulate splicing, we curated 305 RSRs in the human genome. Surprisingly, most of the RSRs are involved in neurogenesis. Among these RSRs, we focus on nine families (PTBP, NOVA, RBFOX, ELAVL, CELF, DBHS, MSI, PCBP, and MBNL) that play essential roles in the neurogenic pathway. A better understanding of their functions will provide novel insights into the role of splicing in brain development, health, and disease. This comprehensive review serves as a stepping-stone to explore the diverse and complex set of RSRs as fundamental regulators of neural development. This article is categorized under: RNA-Based Catalysis > RNA Catalysis in Splicing and Translation RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA Processing > Splicing Regulation/Alternative Splicing.
Topics: Humans; RNA Splicing; Alternative Splicing; RNA-Binding Proteins; Neurogenesis; RNA
PubMed: 35388651
DOI: 10.1002/wrna.1728 -
International Journal of Biological... Sep 2021Atypical S1 and S11 split inteins have been used for N-terminal or C-terminal protein labeling. Here we reported a novel site-specific internal protein labeling method...
Atypical S1 and S11 split inteins have been used for N-terminal or C-terminal protein labeling. Here we reported a novel site-specific internal protein labeling method based on two atypical split inteins, Ter DnaE3 S11 and Rma DnaB S1. Protein-peptide trans-splicing activity was first demonstrated in vitro between a short peptide (Flag tag, FLAG) and two recombinant proteins (Maltose binding protein, MBP, and Thioredoxin, Trx) by trans-splicing between MBP-TE3S11N (MBP-N fragment of Ter DnaE3 S11), TE3S11C-FLAG-RBS1N (C fragment of Ter DnaE3 S11-FLAG-N fragment of Rma DnaB S1), and RBS1C-Trx (C fragment of Rma DnaB S1-Trx). To minimize the middle synthetic peptide (TE3S11C-linker-RBS1N), we reduced the number of native extein amino acids, which may play a role in protein trans-splicing. The results showed at least 3 (CKG) native extein amino acids were required for detectable trans-splicing activity. This method was further demonstrated to be effective in facilitating the incorporation of fluorescent probe (FITC) to the internal site of recombinant protein, generating the FITC-labeled protein. Besides the fluorescent group, these two split inteins can also be useful for adding any desirable chemical groups into a protein of interest, which may include biotin, modified and unnatural amino acids, or drug molecules.
Topics: Fluorescein-5-isothiocyanate; Inteins; Maltose-Binding Proteins; Oligopeptides; Protein Engineering; Protein Splicing; Thioredoxins; Trans-Splicing
PubMed: 34246886
DOI: 10.1016/j.ijbiomac.2021.07.009 -
Cancer Cell International Dec 2023As aberrant alternative splicing by either dysregulation or mutations of splicing factors contributes to cancer initiation and progression, splicing factors are emerging... (Review)
Review
As aberrant alternative splicing by either dysregulation or mutations of splicing factors contributes to cancer initiation and progression, splicing factors are emerging as potential therapeutic targets for cancer therapy. Therefore, pharmacological modulators targeting splicing factors have been under development. Epithelial splicing regulatory protein 1 (ESRP1) is an epithelial cell-specific splicing factor, whose downregulation is associated with epithelial-mesenchymal transition (EMT) by regulating alternative splicing of multiple genes, such as CD44, CTNND1, ENAH, and FGFR2. Consistent with the downregulation of ESRP1 during EMT, it has been initially revealed that high ESRP1 expression is associated with favorable prognosis and ESRP1 plays a tumor-suppressive role in cancer progression. However, ESRP1 has been found to promote cancer progression in some cancers, such as breast and ovarian cancers, indicating that it plays a dual role in cancer progression depending on the type of cancer. Furthermore, recent studies have reported that ESRP1 affects tumor growth by regulating the metabolism of tumor cells or immune cell infiltration in the tumor microenvironment, suggesting the novel roles of ESRP1 in addition to EMT. ESRP1 expression was also associated with response to anticancer drugs. This review describes current understanding of the roles and mechanisms of ESRP1 in cancer progression, and further discusses the emerging novel roles of ESRP1 in cancer and recent attempts to target splicing factors for cancer therapy.
PubMed: 38110955
DOI: 10.1186/s12935-023-03180-6 -
Cell Reports. Medicine Mar 2023Pediatric acute myeloid leukemia (pAML) is typified by high relapse rates and a relative paucity of somatic DNA mutations. Although seminal studies show that splicing...
Pediatric acute myeloid leukemia (pAML) is typified by high relapse rates and a relative paucity of somatic DNA mutations. Although seminal studies show that splicing factor mutations and mis-splicing fuel therapy-resistant leukemia stem cell (LSC) generation in adults, splicing deregulation has not been extensively studied in pAML. Herein, we describe single-cell proteogenomics analyses, transcriptome-wide analyses of FACS-purified hematopoietic stem and progenitor cells followed by differential splicing analyses, dual-fluorescence lentiviral splicing reporter assays, and the potential of a selective splicing modulator, Rebecsinib, in pAML. Using these methods, we discover transcriptomic splicing deregulation typified by differential exon usage. In addition, we discover downregulation of splicing regulator RBFOX2 and CD47 splice isoform upregulation. Importantly, splicing deregulation in pAML induces a therapeutic vulnerability to Rebecsinib in survival, self-renewal, and lentiviral splicing reporter assays. Taken together, the detection and targeting of splicing deregulation represent a potentially clinically tractable strategy for pAML therapy.
Topics: Adult; Child; Humans; Stem Cells; RNA Splicing; Leukemia, Myeloid, Acute; Protein Isoforms; Mutation; RNA Splicing Factors; Repressor Proteins
PubMed: 36889320
DOI: 10.1016/j.xcrm.2023.100962 -
Cell Reports Oct 2023Pre-mRNA splicing is surveilled at different stages by quality control (QC) mechanisms. The leukemia-associated DExH-box family helicase hDHX15/scPrp43 is known to...
Pre-mRNA splicing is surveilled at different stages by quality control (QC) mechanisms. The leukemia-associated DExH-box family helicase hDHX15/scPrp43 is known to disassemble spliceosomes after splicing. Here, using rapid protein depletion and analysis of nascent and mature RNA to enrich for direct effects, we identify a widespread splicing QC function for DHX15 in human cells, consistent with recent in vitro studies. We find that suboptimal introns with weak splice sites, multiple branch points, and cryptic introns are repressed by DHX15, suggesting a general role in promoting splicing fidelity. We identify SUGP1 as a G-patch factor that activates DHX15's splicing QC function. This interaction is dependent on both DHX15's ATPase activity and on SUGP1's U2AF ligand motif (ULM) domain. Together, our results support a model in which DHX15 plays a major role in splicing QC when recruited and activated by SUGP1.
Topics: Humans; RNA; RNA Helicases; RNA Precursors; RNA Splicing; RNA Splicing Factors; Spliceosomes; Splicing Factor U2AF
PubMed: 37805921
DOI: 10.1016/j.celrep.2023.113223 -
Biochimie Jun 2021Inteins are intervening polypeptides that interrupt the functional domains of several important proteins across the three domains of life. Inteins excise themselves from... (Review)
Review
Inteins are intervening polypeptides that interrupt the functional domains of several important proteins across the three domains of life. Inteins excise themselves from the precursor protein, ligating concomitant extein residues in a process called protein splicing. Post-translational auto-removal of inteins remain critical for the generation of active proteins. The perspective of inteins in science is a robust field of research, however fundamental studies centralized upon splicing regulatory mechanism are imperative for addressing more intricate issues. Controlled engineering of intein splicing has many applications; intein inhibition can facilitate novel drug design, while activation of intein splicing is exploited in protein purification. This paper provides a comprehensive review of the past and recent advances in the splicing regulation via metal-intein interaction. We compare the behavior of different metal ions on diverse intein systems. Though metals such as Zn, Cu, Pt, Cd, Co, Ni exhibit intein inhibitory effect heterogeneously on different inteins, divalent metal ions such as Ca and Mg fail to do so. The observed diversity in the metal-intein interaction arises mostly due to intein polymorphism and variations in atomic structure of metals. A mechanistic understanding of intein regulation by metals in native as well as synthetically engineered intein systems may yield potent intein inhibitors via direct or indirect approach.
Topics: Inteins; Metals; Protein Splicing
PubMed: 33727137
DOI: 10.1016/j.biochi.2021.03.006 -
Biochemical and Biophysical Research... May 2023Conditional protein splicing is a powerful biotechnological tool that can be used to post-translationally control the activity of target proteins. Here we demonstrated a...
Conditional protein splicing is a powerful biotechnological tool that can be used to post-translationally control the activity of target proteins. Here we demonstrated a novel conditional protein splicing approach in which the small ubiquitin-like modifier (SUMO) protease induced the splicing of an atypical split intein. The engineered Ter DnaE-3 S11 split intein which has a small C-intein segment with only 6 amino acids was used in this study. A SUMO tag was fused to the N-terminus of the C-intein to inhibit the protein trans-splicing in vitro. The splicing products could be detected in 15 min with the addition of SUMO protease by western blotting and the splicing efficiency was ∼4-fold higher than the control without SUMO protease for overnight reaction. This engineered Ter DnaE-3 S11 split intein-mediated protein trans-splicing had been further shown to be triggered by SUMO protease in different exteins in vitro. Our study provides new insights into the regulation of protein splicing and is a promising tool for the control of protein structure and function in vitro.
Topics: Protein Splicing; Peptide Hydrolases; Ubiquitin; Inteins; Proteins; Endopeptidases
PubMed: 36924678
DOI: 10.1016/j.bbrc.2023.03.023 -
Recent Patents on Anti-cancer Drug... 2020RNA splicing, a fundamental step in gene expression, is aimed at intron removal and ordering of exons to form the protein's reading frame. (Review)
Review
BACKGROUND
RNA splicing, a fundamental step in gene expression, is aimed at intron removal and ordering of exons to form the protein's reading frame.
OBJECTIVE
This review is focused on the role of RNA splicing in cancer biology; the splicing abnormalities that lead to tumor progression emerge as targets for therapeutic intervention.
METHODS
We discuss the role of aberrant mRNA splicing in carcinogenesis and drug response.
RESULTS AND CONCLUSION
Pharmacological modulation of RNA splicing sets the stage for treatment approaches in situations where mRNA splicing is a clinically meaningful mechanism of the disease.
Topics: Drug Resistance, Neoplasm; Exons; Humans; Introns; Mutation; Neoplasms; Patents as Topic; Phosphotransferases; RNA Splicing; RNA Splicing Factors; RNA, Messenger
PubMed: 32900350
DOI: 10.2174/1574892815666200908122402 -
Methods in Molecular Biology (Clifton,... 2020Expressed protein ligation is a simple and powerful method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and... (Review)
Review
Expressed protein ligation is a simple and powerful method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and biophysical probes into proteins of any size. This methodology has been developed based on the knowledge obtained from protein splicing. Protein splicing is a multistep biochemical reaction that includes the concomitant cleavage and formation of peptide bonds carried out by self-processing domains named inteins. The natural substrates of protein splicing are essential proteins found in intein-containing organisms; inteins are also functional in nonnative frameworks and can be used to alter nearly any protein's primary amino acid sequence. Accordingly, different reactivity features of inteins have been largely exploited to manipulate proteins in countless methods encompassing fields from biochemical research to the development of biotechnological applications including the study of disease progression and validation of potential drug candidates. Here, we review almost three decades of research to uncover the chemical and biochemical enigmas of protein splicing and the development of inteins as potent protein engineering tools.
Topics: Biotechnology; Isotope Labeling; Peptides, Cyclic; Protein Engineering; Protein Splicing; Recombinant Proteins
PubMed: 32144661
DOI: 10.1007/978-1-0716-0434-2_2 -
BMC Biology Oct 2023Through alternative splicing, most human genes produce multiple isoforms in a cell-, tissue-, and disease-specific manner. Numerous studies show that alternative...
BACKGROUND
Through alternative splicing, most human genes produce multiple isoforms in a cell-, tissue-, and disease-specific manner. Numerous studies show that alternative splicing is essential for development, diseases, and their treatments. Despite these important examples, the extent and biological relevance of splicing are currently unknown.
RESULTS
To solve this problem, we developed pairedGSEA and used it to profile transcriptional changes in 100 representative RNA-seq datasets. Our systematic analysis demonstrates that changes in splicing, on average, contribute to 48.1% of the biological signal in expression analyses. Gene-set enrichment analysis furthermore indicates that expression and splicing both convey shared and distinct biological signals.
CONCLUSIONS
These findings establish alternative splicing as a major regulator of the human condition and suggest that most contemporary RNA-seq studies likely miss out on critical biological insights. We anticipate our results will contribute to the transition from a gene-centric to an isoform-centric research paradigm.
Topics: Humans; Gene Expression Profiling; RNA Splicing; Alternative Splicing; RNA-Seq; Sequence Analysis, RNA; Protein Isoforms
PubMed: 37858135
DOI: 10.1186/s12915-023-01724-w