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Signal Transduction and Targeted Therapy Mar 2021Alternative splicing is a critical process to generate protein diversity. However, whether and how alternative splicing regulates autophagy remains largely elusive. Here...
Alternative splicing is a critical process to generate protein diversity. However, whether and how alternative splicing regulates autophagy remains largely elusive. Here we systematically identify the splicing factor SRSF1 as an autophagy suppressor. Specifically, SRSF1 inhibits autophagosome formation by reducing the accumulation of LC3-II and numbers of autophagosomes in different cell lines. Mechanistically, SRSF1 promotes the splicing of the long isoform of Bcl-x that interacts with Beclin1, thereby dissociating the Beclin1-PIK3C3 complex. In addition, SRSF1 also directly interacts with PIK3C3 to disrupt the interaction between Beclin1 and PIK3C3. Consequently, the decrease of SRSF1 stabilizes the Beclin1 and PIK3C3 complex and activates autophagy. Interestingly, SRSF1 can be degraded by starvation- and oxidative stresses-induced autophagy through interacting with LC3-II, whereas reduced SRSF1 further promotes autophagy. This positive feedback is critical to inhibiting Gefitinib-resistant cancer cell progression both in vitro and in vivo. Consistently, the expression level of SRSF1 is inversely correlated to LC3 level in clinical cancer samples. Our study not only provides mechanistic insights of alternative splicing in autophagy regulation but also discovers a new regulatory role of SRSF1 in tumorigenesis, thereby offering a novel avenue for potential cancer therapeutics.
Topics: A549 Cells; Alternative Splicing; Animals; Autophagosomes; Autophagy; Carcinogenesis; Class III Phosphatidylinositol 3-Kinases; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Lung Neoplasms; Mice; Microtubule-Associated Proteins; Serine-Arginine Splicing Factors; bcl-X Protein
PubMed: 33664238
DOI: 10.1038/s41392-021-00495-6 -
Nature Communications Oct 2019Selectable markers are widely used in transgenesis and genome editing for selecting engineered cells with a desired genotype but the variety of markers is limited. Here...
Selectable markers are widely used in transgenesis and genome editing for selecting engineered cells with a desired genotype but the variety of markers is limited. Here we present split selectable markers that each allow for selection of multiple "unlinked" transgenes in the context of lentivirus-mediated transgenesis as well as CRISPR-Cas-mediated knock-ins. Split marker gene segments fused to protein splicing elements called "inteins" can be separately co-segregated with different transgenic vectors, and rejoin via protein trans-splicing to reconstitute a full-length marker protein in host cells receiving all intended vectors. Using a lentiviral system, we create and validate 2-split Hygromycin, Puromycin, Neomycin and Blasticidin resistance genes as well as mScarlet fluorescent proteins. By combining split points, we create 3- and 6-split Hygromycin resistance genes, demonstrating that higher-degree split markers can be generated by a "chaining" design. We adapt the split marker system for selecting biallelically engineered cells after CRISPR gene editing. Future engineering of split markers may allow selection of a higher number of genetic modifications in target cells.
Topics: CRISPR-Cas Systems; Cell Line, Tumor; Cinnamates; Drug Resistance, Bacterial; Gene Editing; Gene Transfer Techniques; Genetic Engineering; Genetic Vectors; HEK293 Cells; HeLa Cells; Humans; Hygromycin B; Induced Pluripotent Stem Cells; Inteins; Lentivirus; Luminescent Proteins; Neomycin; Nucleosides; Protein Splicing; Puromycin; Trans-Splicing; Transgenes
PubMed: 31672965
DOI: 10.1038/s41467-019-12891-2 -
Molecular Cell Apr 2023RNA-binding proteins (RBPs) bind at different positions of the pre-mRNA molecules to promote or reduce the usage of a particular exon. Seeking to understand the working...
RNA-binding proteins (RBPs) bind at different positions of the pre-mRNA molecules to promote or reduce the usage of a particular exon. Seeking to understand the working principle of these positional effects, we develop a capture RIC-seq (CRIC-seq) method to enrich specific RBP-associated in situ proximal RNA-RNA fragments for deep sequencing. We determine hnRNPA1-, SRSF1-, and PTBP1-associated proximal RNA-RNA contacts and regulatory mechanisms in HeLa cells. Unexpectedly, the 3D RNA map analysis shows that PTBP1-associated loops in individual introns preferentially promote cassette exon splicing by accelerating asymmetric intron removal, whereas the loops spanning across cassette exon primarily repress splicing. These "positional rules" can faithfully predict PTBP1-regulated splicing outcomes. We further demonstrate that cancer-related splicing quantitative trait loci can disrupt RNA loops by reducing PTBP1 binding on pre-mRNAs to cause aberrant splicing in tumors. Our study presents a powerful method for exploring the functions of RBP-associated RNA-RNA proximal contacts in gene regulation and disease.
Topics: Humans; RNA; HeLa Cells; Polypyrimidine Tract-Binding Protein; RNA Splicing; RNA-Binding Proteins; RNA Precursors; Alternative Splicing; Heterogeneous-Nuclear Ribonucleoproteins; Serine-Arginine Splicing Factors
PubMed: 36958328
DOI: 10.1016/j.molcel.2023.03.001 -
International Journal of Biological... 2023Serine/arginine rich splicing factor 3 (SRSF3) is an important multi-functional splicing factor, and has attracted increasing attentions in the past thirty years. The... (Review)
Review
Serine/arginine rich splicing factor 3 (SRSF3) is an important multi-functional splicing factor, and has attracted increasing attentions in the past thirty years. The importance of SRSF3 is evidenced by its impressively conserved protein sequences in all animals and alternative exon 4 which represents an autoregulatory mechanism to maintain its proper cellular expression level. New functions of SRSF3 have been continuously discovered recently, especially its oncogenic function. SRSF3 plays essential roles in many cellular processes by regulating almost all aspects of RNA biogenesis and processing of many target genes, and thus, contributes to tumorigenesis when overexpressed or disregulated. This review updates and highlights the gene, mRNA, and protein structure of SRSF3, the regulatory mechanisms of SRSF3 expression, and the characteristics of SRSF3 targets and binding sequences that contribute to SRSF3's diverse molecular and cellular functions in tumorigenesis and human diseases.
Topics: Animals; Humans; Cell Line, Tumor; Exons; RNA Splicing Factors; Carcinogenesis; Serine-Arginine Splicing Factors; Alternative Splicing
PubMed: 37416784
DOI: 10.7150/ijbs.83368 -
Trends in Biochemical Sciences Apr 2020The collection of chemically different protein variants, or proteoforms, by far exceeds the number of protein-coding genes in the human genome. Major contributors are... (Review)
Review
The collection of chemically different protein variants, or proteoforms, by far exceeds the number of protein-coding genes in the human genome. Major contributors are alternative splicing and protein modifications. In this review, we focus on those proteoforms that differ at their N termini with a molecular link to disease. We describe the main underlying mechanisms that give rise to such N-terminal proteoforms, these being splicing, initiation of protein translation, and protein modifications. Given their role in several human diseases, it is becoming increasingly clear that several of these N-terminal proteoforms may have potential as therapeutic interventions and/or for diagnosing and prognosing their associated disease.
Topics: Alternative Splicing; Humans; Protein Biosynthesis; Protein Processing, Post-Translational
PubMed: 32001092
DOI: 10.1016/j.tibs.2019.12.009 -
Frontiers in Microbiology 2023Protein splicing is a posttranslational process in which an intein segment excises itself from two flanking peptides, referred to as exteins. In the native context,... (Review)
Review
Protein splicing is a posttranslational process in which an intein segment excises itself from two flanking peptides, referred to as exteins. In the native context, protein splicing results in two separate protein products coupled to the activation of the intein-containing host protein. Inteins are generally described as either full-length inteins, mini-inteins or split inteins, which are differentiated by their genetic structure and features. Inteins can also be divided into three classes based on their splicing mechanisms, which differ in the location of conserved residues that mediate the splicing pathway. Although inteins were once thought to be selfish genetic elements, recent evidence suggests that inteins may confer a genetic advantage to their host cells through posttranslational regulation of their host proteins. Finally, the ability of modified inteins to splice and cleave their fused exteins has enabled many new applications in protein science and synthetic biology. In this review, we briefly cover the mechanisms of protein splicing, evidence for some inteins as environmental sensors, and intein-based applications in protein engineering.
PubMed: 38029209
DOI: 10.3389/fmicb.2023.1305848 -
British Medical Bulletin Dec 2020RNA trans-splicing joins exons from different pre-mRNA transcripts to generate a chimeric product. Trans-splicing can also occur at the protein level, with split inteins...
BACKGROUND
RNA trans-splicing joins exons from different pre-mRNA transcripts to generate a chimeric product. Trans-splicing can also occur at the protein level, with split inteins mediating the ligation of separate gene products to generate a mature protein.
SOURCES OF DATA
Comprehensive literature search of published research papers and reviews using Pubmed.
AREAS OF AGREEMENT
Trans-splicing techniques have been used to target a wide range of diseases in both in vitro and in vivo models, resulting in RNA, protein and functional correction.
AREAS OF CONTROVERSY
Off-target effects can lead to therapeutically undesirable consequences. In vivo efficacy is typically low, and delivery issues remain a challenge.
GROWING POINTS
Trans-splicing provides a promising avenue for developing novel therapeutic approaches. However, much more research needs to be done before developing towards preclinical studies.
AREAS TIMELY FOR DEVELOPING RESEARCH
Increasing trans-splicing efficacy and specificity by rational design, screening and competitive inhibition of endogenous cis-splicing.
Topics: Humans; Inteins; Proteins; Trans-Splicing
PubMed: 33010155
DOI: 10.1093/bmb/ldaa028 -
PLoS Pathogens Apr 2023N6-methyladenosine (m6A) modification on viral RNAs has a profound impact on infectivity. m6A is also a highly pervasive modification for influenza viral RNAs. However,...
N6-methyladenosine (m6A) modification on viral RNAs has a profound impact on infectivity. m6A is also a highly pervasive modification for influenza viral RNAs. However, its role in virus mRNA splicing is largely unknown. Here, we identify the m6A reader protein YTHDC1 as a host factor that associates with influenza A virus NS1 protein and modulates viral mRNA splicing. YTHDC1 levels are enhanced by IAV infection. We demonstrate that YTHDC1 inhibits NS splicing by binding to an NS 3' splicing site and promotes IAV replication and pathogenicity in vitro and in vivo. Our results provide a mechanistic understanding of IAV-host interactions, a potential therapeutic target for blocking influenza virus infection, and a new avenue for the development of attenuated vaccines.
Topics: Humans; Influenza A virus; Influenza, Human; Virus Replication; RNA, Messenger; RNA Splicing Factors; Nerve Tissue Proteins
PubMed: 37053288
DOI: 10.1371/journal.ppat.1011305 -
BioRxiv : the Preprint Server For... Dec 2023Despite significant interest in therapeutic targeting of splicing, few chemical probes are available for the proteins involved in splicing. Here, we show that elaborated...
Despite significant interest in therapeutic targeting of splicing, few chemical probes are available for the proteins involved in splicing. Here, we show that elaborated stereoisomeric acrylamide chemical probe EV96 and its analogues lead to a selective T cell state-dependent loss of interleukin 2-inducible T cell kinase (ITK) by targeting one of the core splicing factors SF3B1. Mechanistic investigations suggest that the state-dependency stems from a combination of differential protein turnover rates and availability of functional mRNA pools that can be depleted due to extensive alternative splicing. We further introduce a comprehensive list of proteins involved in splicing and leverage both cysteine- and protein-directed activity-based protein profiling (ABPP) data with electrophilic scout fragments to demonstrate covalent ligandability for many classes of splicing factors and splicing regulators in primary human T cells. Taken together, our findings show how chemical perturbation of splicing can lead to immune state-dependent changes in protein expression and provide evidence for the broad potential to target splicing factors with covalent chemistry.
PubMed: 38187674
DOI: 10.1101/2023.12.18.572199 -
Biosensors Apr 2022Cell-based sensors provide a flexible platform for screening biologically active targets and for monitoring their interactions in live cells. Their applicability extends... (Review)
Review
Cell-based sensors provide a flexible platform for screening biologically active targets and for monitoring their interactions in live cells. Their applicability extends across a vast array of biological research and clinical applications. Particularly, cell-based sensors are becoming a potent tool in drug discovery and cell-signaling studies by allowing function-based screening of targets in biologically relevant environments and enabling the in vivo visualization of cellular signals in real-time with an outstanding spatiotemporal resolution. In this review, we aim to provide a clear view of current cell-based sensor technologies, their limitations, and how the recent improvements were using intein-mediated protein engineering. We first discuss the characteristics of cell-based sensors and present several representative examples with a focus on their design strategies, which differentiate cell-based sensors from in vitro analytical biosensors. We then describe the application of intein-mediated protein engineering technology for cell-based sensor fabrication. Finally, we explain the characteristics of intein-mediated reactions and present examples of how the intein-mediated reactions are used to improve existing methods and develop new approaches in sensor cell fabrication to address the limitations of current technologies.
Topics: Biosensing Techniques; Inteins; Protein Engineering; Signal Transduction
PubMed: 35624584
DOI: 10.3390/bios12050283