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Chembiochem : a European Journal of... Sep 2021Proteins possess unique molecular recognition capabilities and enzymatic activities, features that are usually tied to a particular tertiary structure. To make use of... (Review)
Review
Proteins possess unique molecular recognition capabilities and enzymatic activities, features that are usually tied to a particular tertiary structure. To make use of proteins for biotechnological and biomedical purposes, it is often required to enforce their tertiary structure in order to ensure sufficient stability under the conditions inherent to the application of interest. The introduction of intramolecular crosslinks has proven efficient in stabilizing native protein folds. Herein, we give an overview of methods that allow the macrocyclization of expressed proteins, discussing involved reaction mechanisms and structural implications.
Topics: Protein Splicing
PubMed: 34060202
DOI: 10.1002/cbic.202100111 -
Annual Review of Biochemistry Jun 2020Splicing of the precursor messenger RNA, involving intron removal and exon ligation, is mediated by the spliceosome. Together with biochemical and genetic investigations... (Review)
Review
Splicing of the precursor messenger RNA, involving intron removal and exon ligation, is mediated by the spliceosome. Together with biochemical and genetic investigations of the past four decades, structural studies of the intact spliceosome at atomic resolution since 2015 have led to mechanistic delineation of RNA splicing with remarkable insights. The spliceosome is proven to be a protein-orchestrated metalloribozyme. Conserved elements of small nuclear RNA (snRNA) constitute the splicing active site with two catalytic metal ions and recognize three conserved intron elements through duplex formation, which are delivered into the splicing active site for branching and exon ligation. The protein components of the spliceosome stabilize the conformation of the snRNA, drive spliceosome remodeling, orchestrate the movement of the RNA elements, and facilitate the splicing reaction. The overall organization of the spliceosome and the configuration of the splicing active site are strictly conserved between human and yeast.
Topics: Catalytic Domain; Conserved Sequence; Exons; Humans; Introns; Models, Molecular; Nucleic Acid Conformation; Protein Structure, Secondary; RNA Helicases; RNA Precursors; RNA Splicing; RNA Splicing Factors; RNA, Small Nuclear; RNA-Binding Proteins; Ribonucleoprotein, U4-U6 Small Nuclear; Ribonucleoprotein, U5 Small Nuclear; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Spliceosomes
PubMed: 31815536
DOI: 10.1146/annurev-biochem-013118-111024 -
Journal of Molecular and Cellular... Jun 2023Alternative splicing generates specialized protein isoforms that allow the heart to adapt during development and disease. The recent discovery that mutations in the...
Alternative splicing generates specialized protein isoforms that allow the heart to adapt during development and disease. The recent discovery that mutations in the splicing factor RNA-binding protein 20 (RBM20) cause a severe form of familial dilated cardiomyopathy has sparked a great interest in alternative splicing in the field of cardiology. Since then, identification of splicing factors controlling alternative splicing in the heart has grown at a rapid pace. Despite the intriguing observation that a certain overlap exists between the targets of some splicing factors, an integrated and systematic analysis of their splicing networks is missing. Here, we compared the splicing networks of individual splicing factors by re-analyzing original RNA-sequencing data from eight previously published mouse models, in which a single splicing factor has been genetically deleted (i.e. HNRNPU, MBNL1/2, QKI, RBM20, RBM24, RBPMS, SRSF3, SRSF4). We show that key splicing events in Camk2d, Ryr2, Tpm1, Tpm2 and Pdlim5 require the combined action of the majority of these splicing factors. Additionally, we identified common targets and pathways among splicing factors, with the largest overlap between the splicing networks of MBNL, QKI and RBM24. We also re-analyzed a large-scale RNA-sequencing study on hearts of 128 heart failure patients. Here, we observed that MBNL1, QKI and RBM24 expression varied greatly. This variation in expression correlated with differential splicing of their downstream targets as found in mice, suggesting that aberrant splicing by MBNL1, QKI and RBM24 might contribute to the disease mechanism in heart failure.
Topics: Mice; Animals; RNA Splicing Factors; Heart; Alternative Splicing; Heart Failure; RNA; RNA-Binding Proteins
PubMed: 37059416
DOI: 10.1016/j.yjmcc.2023.04.003 -
Methods in Enzymology 2021Conventional site-directed mutagenesis and genetic code expansion approaches have been instrumental in providing detailed functional and pharmacological insight into...
Conventional site-directed mutagenesis and genetic code expansion approaches have been instrumental in providing detailed functional and pharmacological insight into membrane proteins such as ion channels. Recently, this has increasingly been complemented by semi-synthetic strategies, in which part of the protein is generated synthetically. This means a vast range of chemical modifications, including non-canonical amino acids (ncAA), backbone modifications, chemical handles, fluorescent or spectroscopic labels and any combination of these can be incorporated. Among these approaches, protein trans-splicing (PTS) is particularly promising for protein reconstitution in live cells. It relies on one or more split inteins, which can spontaneously and covalently link flanking peptide or protein sequences. Here, we describe the use of PTS and its variant tandem PTS (tPTS) in semi-synthesis of ion channels in Xenopus laevis oocytes to incorporate ncAAs, post-translational modifications or metabolically stable mimics thereof. This strategy has the potential to expand the type and number of modifications in ion channel research.
Topics: Inteins; Ion Channels; Peptides; Protein Engineering; Protein Splicing; Trans-Splicing
PubMed: 34120713
DOI: 10.1016/bs.mie.2021.01.028 -
PLoS Genetics Sep 2023Transcription factors (TFs) play critical roles in specifying many aspects of neuronal cell fate including dendritic morphology. How TFs are accurately regulated during...
Transcription factors (TFs) play critical roles in specifying many aspects of neuronal cell fate including dendritic morphology. How TFs are accurately regulated during neuronal morphogenesis is not fully understood. Here, we show that LIM homeodomain protein MEC-3, the key TF for C. elegans PVD dendrite morphogenesis, is regulated by both alternative splicing and an E3 ubiquitin ligase. The mec-3 gene generates several transcripts by alternative splicing. We find that mbl-1, the orthologue of the muscular dystrophy disease gene muscleblind-like (MBNL), is required for PVD dendrite arbor formation. Our data suggest mbl-1 regulates the alternative splicing of mec-3 to produce its long isoform. Deleting the long isoform of mec-3(deExon2) causes reduction of dendrite complexity. Through a genetic modifier screen, we find that mutation in the E3 ubiquitin ligase EEL-1 suppresses mbl-1 phenotype. eel-1 mutants also suppress mec-3(deExon2) mutant but not the mec-3 null phenotype. Loss of EEL-1 alone leads to excessive dendrite branches. Together, these results indicate that MEC-3 is fine-tuned by alternative splicing and the ubiquitin system to produce the optimal level of dendrite branches.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Dendrites; Eels; RNA Splicing; Ubiquitin-Protein Ligases
PubMed: 37729192
DOI: 10.1371/journal.pgen.1010941 -
Frontiers in Plant Science 2023For the past two decades, the study of alternative splicing (AS) and its involvement in plant development and stress response has grown in popularity. Only recently... (Review)
Review
For the past two decades, the study of alternative splicing (AS) and its involvement in plant development and stress response has grown in popularity. Only recently however, has the focus shifted to the study of how AS regulation (or lack-thereof) affects downstream mRNA and protein landscapes and how these AS regulatory events impact plant development and stress tolerance. In humans, protein phosphorylation represents one of the predominant mechanisms by which AS is regulated and thus the protein kinases governing these phosphorylation events are of interest for further study. Large-scale phosphoproteomic studies in plants have consistently found that RNA splicing-related proteins are extensively phosphorylated, however, the signaling pathways involved in AS regulation have not been resolved. In this mini-review, we summarize our current knowledge of the three major splicing-related protein kinase families in plants that are suggested to mediate AS phospho-regulation and draw comparisons to their metazoan orthologs. We also summarize and contextualize the phosphorylation events identified as occurring on splicing-related protein families to illustrate the high degree to which splicing-related proteins are modified, placing a new focus on elucidating the impacts of AS at the protein and PTM-level.
PubMed: 37780493
DOI: 10.3389/fpls.2023.1249057 -
Trends in Cell Biology Jan 2020Cell migration is essential for normal development, neural patterning, pathogen eradication, and cancer metastasis. Pre-mRNA processing events such as alternative... (Review)
Review
Cell migration is essential for normal development, neural patterning, pathogen eradication, and cancer metastasis. Pre-mRNA processing events such as alternative splicing and alternative polyadenylation result in greater transcript and protein diversity as well as function and activity. A critical role for alternative pre-mRNA processing in cell migration has emerged in axon outgrowth during neuronal development, immune cell migration, and cancer metastasis. These findings suggest that migratory signals result in expression changes of post-translational modifications of splicing or polyadenylation factors, leading to splicing events that generate promigratory isoforms. We summarize this recent progress and suggest emerging technologies that may facilitate a deeper understanding of the role of alternative splicing and polyadenylation in cell migration.
Topics: Alternative Splicing; Animals; Cell Movement; Humans; Models, Biological; Polyadenylation; Protein Isoforms; RNA Processing, Post-Transcriptional
PubMed: 31810769
DOI: 10.1016/j.tcb.2019.10.007 -
Advanced Science (Weinheim,... Nov 2023Mesenchymal glioblastoma (GBM) is highly resistant to radio-and chemotherapy and correlates with worse survival outcomes in GBM patients; however, the underlying...
Mesenchymal glioblastoma (GBM) is highly resistant to radio-and chemotherapy and correlates with worse survival outcomes in GBM patients; however, the underlying mechanism determining the mesenchymal phenotype remains largely unclear. Herein, it is revealed that FBXO7, a substrate-recognition component of the SCF complex implicated in the pathogenesis of Parkinson's disease, confers mesenchymal properties and chemoresistance in GBM by controlling Rbfox2-mediated alternative splicing. Specifically, FBXO7 ubiquitinates Rbfox2 Lys249 through K63-linked ubiquitin chains upon arginine dimethylation at Arg341 and Arg441 by PRMT5, leading to Rbfox2 stabilization. FBXO7 controls Rbfox2-mediated splicing of mesenchymal genes, including FoxM1, Mta1, and Postn. FBXO7-induced exon Va inclusion of FoxM1 promotes FoxM1 phosphorylation by MEK1 and nuclear translocation, thereby upregulates CD44, CD9, and ID1 levels, resulting in GBM stem cell self-renewal and mesenchymal transformation. Moreover, FBXO7 is stabilized by temozolomide, and FBXO7 depletion sensitizes tumor xenografts in mice to chemotherapy. The findings demonstrate that the FBXO7-Rbfox2 axis-mediated splicing contributes to mesenchymal transformation and tumorigenesis, and targeting FBXO7 represents a potential strategy for GBM treatment.
Topics: Animals; Humans; Mice; Alternative Splicing; Drug Resistance, Neoplasm; F-Box Proteins; Glioblastoma; Protein-Arginine N-Methyltransferases; Repressor Proteins; RNA Splicing; RNA Splicing Factors; Trans-Activators
PubMed: 37822160
DOI: 10.1002/advs.202303561 -
BioEssays : News and Reviews in... Nov 2019The major transcript variants of human protein-coding genes are annotated to a certain degree of accuracy combining manual curation, transcript data, and proteomics... (Review)
Review
The major transcript variants of human protein-coding genes are annotated to a certain degree of accuracy combining manual curation, transcript data, and proteomics evidence. However, there is considerable disagreement on the annotation of about 2000 genes-they can be protein-coding, noncoding, or pseudogenes-and on the annotation of most of the predicted alternative transcripts. Pure transcriptome mapping approaches seem to be limited in discriminating functional expression from noise. These limitations have partially been overcome by dedicated algorithms to detect alternative spliced micro-exons and wobble splice variants. Recently, knowledge about splice mechanism and protein structure are incorporated into an algorithm to predict neighboring homologous exons, often spliced in a mutually exclusive manner. Predicted exons are evaluated by transcript data, structural compatibility, and evolutionary conservation, revealing hundreds of novel coding exons and splice mechanism re-assignments. The emerging human pan-genome is necessitating distinctive annotations incorporating differences between individuals and between populations.
Topics: Algorithms; Alternative Splicing; Animals; Exons; Genome, Human; Genomics; Humans; Proteins; RNA Splicing; Transcriptome
PubMed: 31544971
DOI: 10.1002/bies.201900066 -
The Journal of Clinical Investigation May 2024Splicing factor mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but how they alter cellular functions is unclear. We show that...
Splicing factor mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but how they alter cellular functions is unclear. We show that the pathogenic SRSF2P95H/+ mutation disrupts the splicing of mitochondrial mRNAs, impairs mitochondrial complex I function, and robustly increases mitophagy. We also identified a mitochondrial surveillance mechanism by which mitochondrial dysfunction modifies splicing of the mitophagy activator PINK1 to remove a poison intron, increasing the stability and abundance of PINK1 mRNA and protein. SRSF2P95H-induced mitochondrial dysfunction increased PINK1 expression through this mechanism, which is essential for survival of SRSF2P95H/+ cells. Inhibition of splicing with a glycogen synthase kinase 3 inhibitor promoted retention of the poison intron, impairing mitophagy and activating apoptosis in SRSF2P95H/+ cells. These data reveal a homeostatic mechanism for sensing mitochondrial stress through PINK1 splicing and identify increased mitophagy as a disease marker and a therapeutic vulnerability in SRSF2P95H mutant MDS and AML.
Topics: Serine-Arginine Splicing Factors; Humans; Mitochondria; Mitophagy; Leukemia, Myeloid, Acute; Protein Kinases; Myelodysplastic Syndromes; RNA Splicing; Cell Line, Tumor; Hematologic Neoplasms; Mutation, Missense; Mice; Amino Acid Substitution; Animals
PubMed: 38713535
DOI: 10.1172/JCI175619