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The Journal of Biological Chemistry May 2014Inteins are mobile genetic elements capable of self-splicing post-translationally. They exist in all three domains of life including in viruses and bacteriophage, where... (Review)
Review
Inteins are mobile genetic elements capable of self-splicing post-translationally. They exist in all three domains of life including in viruses and bacteriophage, where they have a sporadic distribution even among very closely related species. In this review, we address this anomalous distribution from the point of view of the evolution of the host species as well as the intrinsic features of the inteins that contribute to their genetic mobility. We also discuss the incidence of inteins in functionally important sites of their host proteins. Finally, we describe instances of conditional protein splicing. These latter observations lead us to the hypothesis that some inteins have adapted to become sensors that play regulatory roles within their host protein, to the advantage of the organism in which they reside.
Topics: Amino Acid Sequence; Archaea; Bacteria; Eukaryota; Evolution, Molecular; Genome; Inteins; Models, Genetic; Protein Splicing; Proteins
PubMed: 24695741
DOI: 10.1074/jbc.R114.548255 -
The Journal of Biological Chemistry Oct 2023Alternative splicing in the 3'UTR of mammalian genes plays a crucial role in diverse biological processes, including cell differentiation and development. SAM68 is a key...
Alternative splicing in the 3'UTR of mammalian genes plays a crucial role in diverse biological processes, including cell differentiation and development. SAM68 is a key splicing regulator that controls the diversity of 3'UTR isoforms through alternative last exon (ALE) selection. However, the tissue/cell type-specific mechanisms underlying the splicing control at the 3' end and its functional significance remain unclear. Here, we show that SAM68 regulates ALE splicing in a dose-dependent manner and the neuronal splicing is differentially regulated depending on the characteristics of the target transcript. Specifically, we found that SAM68 regulates interleukin-1 receptor-associated protein splicing through the interaction with U1 small nuclear ribonucleoprotein. In contrast, the ALE splicing of protocadherin-15 (Pcdh15), a gene implicated in several neuropsychiatric disorders, is independent of U1 small nuclear ribonucleoprotein but modulated by the calcium/calmodulin-dependent protein kinase signaling pathway. We found that the aberrant ALE selection of Pcdh15 led to a conversion from a membrane-bound to a soluble isoform and consequently disrupted its localization into excitatory and inhibitory synapses. Notably, the neuronal expression of the soluble form of PCDH15 preferentially affected the number of inhibitory synapses. Moreover, the soluble form of PCDH15 interacted physically with α-neurexins and further disrupted neuroligin-2-induced inhibitory synapses in artificial synapse formation assays. Our findings provide novel insights into the role of neuron-specific alternative 3'UTR isoform selections in synapse development.
PubMed: 37595869
DOI: 10.1016/j.jbc.2023.105168 -
Bio-protocol Aug 2021Inteins garner significant interest from both basic and applied researchers due to their unique catalytic abilities. Herein, we describe a protocol for accurately...
Inteins garner significant interest from both basic and applied researchers due to their unique catalytic abilities. Herein, we describe a protocol for accurately monitoring protein splicing without purification using in-gel fluorescence immediately following Tris-Glycine SDS-PAGE. Following expression in , cells are lysed by sonication, cell supernatants are separated using Tris-Glycine SDS-PAGE, and superfolder GFP (sfGFP) fluorescence is directly visualized within gels. This method is rapid, with sfGFP immediately imaged following SDS-PAGE without staining. Further, sfGFP can be specifically detected in complex samples such as cell supernatants, proteins run at expected masses, and all steps can be performed at ambient temperature. This strategy is broadly applicable beyond the study of protein splicing and can be used for sensitive and specific visualization of superfolder sfGFP-tagged proteins in-gel.
PubMed: 34541040
DOI: 10.21769/BioProtoc.4121 -
Biopolymers Sep 2013Here we review the different biochemical approaches available for the expression of backbone-cyclized polypeptides, including peptides and proteins. These methods allow... (Review)
Review
Here we review the different biochemical approaches available for the expression of backbone-cyclized polypeptides, including peptides and proteins. These methods allow for the production of circular polypeptides either in vitro or in vivo using standard recombinant DNA expression techniques. Polypeptide circularization provides a valuable tool to study the effects of topology on protein stability and folding kinetics. Furthermore, having biosynthetic access to backbone-cyclized polypeptides makes the production of genetically encoded libraries of cyclic polypeptides possible. The production of such libraries, which was previously restricted to the domain of synthetic chemistry, now offers biologists access to highly diverse and stable molecular libraries that can be screened using high-throughput methods for the rapid selection of novel cyclic polypeptide sequences with new biological activities.
Topics: Amino Acid Sequence; Cyclization; Kinetics; Peptides; Peptides, Cyclic; Proteins
PubMed: 23893781
DOI: 10.1002/bip.22306 -
Microbiology Spectrum Oct 2021tervening pro, or inteins, are mobile genetic elements that are translated within host polypeptides and removed at the protein level by splicing. In protein splicing, a...
tervening pro, or inteins, are mobile genetic elements that are translated within host polypeptides and removed at the protein level by splicing. In protein splicing, a self-mediated reaction removes the intein, leaving a peptide bond in place. While protein splicing can proceed in the absence of external cofactors, several examples of conditional protein splicing (CPS) have emerged. In CPS, the rate and accuracy of splicing are highly dependent on environmental conditions. Because the activity of the intein-containing host protein is compromised prior to splicing and inteins are highly abundant in the microbial world, CPS represents an emerging form of posttranslational regulation that is potentially widespread in microbes. Reactive chlorine species (RCS) are highly potent oxidants encountered by bacteria in a variety of natural environments, including within cells of the mammalian innate immune system. Here, we demonstrate that two naturally occurring RCS, namely, hypochlorous acid (the active compound in bleach) and -chlorotaurine, can reversibly block splicing of DnaB inteins from Mycobacterium leprae and Mycobacterium smegmatis . Further, using a reporter that monitors DnaB intein activity within M. smegmatis, we show that DnaB protein splicing is inhibited by RCS in the native host. DnaB, an essential replicative helicase, is the most common intein-housing protein in bacteria. These results add to the growing list of environmental conditions that are relevant to the survival of the intein-containing host and influence protein splicing, as well as suggesting a novel mycobacterial response to RCS. We propose a model in which DnaB splicing, and therefore replication, is paused when these mycobacteria encounter RCS. Inteins are both widespread and abundant in microbes, including within several bacterial and fungal pathogens. Inteins are domains translated within host proteins and removed at the protein level by splicing. Traditionally considered molecular parasites, some inteins have emerged in recent years as adaptive posttranslational regulatory elements. Several studies have demonstrated CPS, in which the rate and accuracy of protein splicing, and thus host protein functions, are responsive to environmental conditions relevant to the intein-containing organism. In this work, we demonstrate that two naturally occurring RCS, including the active compound in household bleach, reversibly inhibit protein splicing of Mycobacterium leprae and Mycobacterium smegmatis DnaB inteins. In addition to describing a new physiologically relevant condition that can temporarily inhibit protein splicing, this study suggests a novel stress response in Mycobacterium, a bacterial genus of tremendous importance to humans.
Topics: Chloramines; Chlorine; DNA Replication; DnaB Helicases; Gene Expression Regulation, Bacterial; Hypochlorous Acid; Inteins; Mycobacterium leprae; Mycobacterium smegmatis; Oxidants; Oxidation-Reduction; Protein Splicing; Reactive Oxygen Species; Taurine
PubMed: 34549994
DOI: 10.1128/Spectrum.00301-21 -
Biomolecules Apr 2019Although inhibition of the androgen⁻androgen receptor (AR) axis effectively represses the growth of prostate cancer, most of all cases eventually become... (Review)
Review
Although inhibition of the androgen⁻androgen receptor (AR) axis effectively represses the growth of prostate cancer, most of all cases eventually become castration-resistant prostate cancers (CRPCs). Enhancement of the expression of AR and its variants along with the downstream signals is important for disease progression. AR-V7, a constitutive active form of AR, is generated as a result of RNA splicing. RNA splicing creates multiple transcript variants from one pre-messenger RNA (mRNA) by removing introns/exons to allow mRNA translation. The molecular mechanisms leading to marked increases of AR and generation of AR-V7 have been unclear. However, recent papers highlighted the roles of RNA splicing factors which promote AR expression and production of variants. Notably, a broad range of splicing components were aberrantly regulated in CRPC tissues. Interestingly, expression of various spliceosome genes is enhanced by RNA-binding protein splicing factor proline- and glutamine-rich (PSF/SFPQ), leading to changes in the expression of AR transcript variants. Moreover, inhibition of several splicing factors repressed tumor growth in vivo. Altered expression of splicing factors is correlated to biochemical recurrence in prostate cancer patients. Thus, these findings suggest that splicing factors would be a potential therapeutic target. This review focuses on the emerging roles of splicing factors in prostate cancer progression and AR signaling.
Topics: Animals; Humans; Male; Prostatic Neoplasms; RNA Splicing Factors; Receptors, Androgen; Signal Transduction
PubMed: 30939845
DOI: 10.3390/biom9040131 -
PLoS Biology Oct 2019The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNAs. At its functional core lies the essential pre-mRNA processing factor 8 (Prp8)...
The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNAs. At its functional core lies the essential pre-mRNA processing factor 8 (Prp8) protein. Across diverse eukaryotes, this protein cofactor of RNA catalysis harbors a self-splicing element called an intein. Inteins in Prp8 are extremely pervasive and are found at 7 different sites in various species. Here, we focus on the Prp8 intein from Cryptococcus neoformans (Cne), a human fungal pathogen. We solved the crystal structure of this intein, revealing structural homology among protein splicing sequences in eukaryotes, including the Hedgehog C terminus. Working with the Cne Prp8 intein in a reporter assay, we find that the biologically relevant divalent metals copper and zinc inhibit intein splicing, albeit by 2 different mechanisms. Copper likely stimulates reversible modifications on a catalytically important cysteine, whereas zinc binds at the terminal asparagine and the same critical cysteine. Importantly, we also show that copper treatment inhibits Prp8 protein splicing in Cne. Lastly, an intein-containing Prp8 precursor model is presented, suggesting that metal-induced protein splicing inhibition would disturb function of both Prp8 and the spliceosome. These results indicate that Prp8 protein splicing can be modulated, with potential functional implications for the spliceosome.
Topics: Asparagine; Binding Sites; Cloning, Molecular; Copper; Cryptococcus neoformans; Crystallography, X-Ray; Cysteine; Escherichia coli; Fungal Proteins; Gene Expression; Genes, Reporter; Genetic Vectors; Green Fluorescent Proteins; Inteins; Maltose-Binding Proteins; Models, Molecular; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; RNA Splicing; RNA-Binding Proteins; Recombinant Fusion Proteins; Sequence Alignment; Spliceosomes; Structural Homology, Protein; Zinc
PubMed: 31600193
DOI: 10.1371/journal.pbio.3000104 -
American Journal of Physiology. Cell... May 2016A wide variety of cellular function depends on the dynamics of intracellular Ca(2+) signals. Especially for relatively slow and lasting processes such as gene... (Review)
Review
A wide variety of cellular function depends on the dynamics of intracellular Ca(2+) signals. Especially for relatively slow and lasting processes such as gene expression, cell proliferation, and often migration, cells rely on the store-operated Ca(2+) entry (SOCE) pathway, which is particularly prominent in immune cells. SOCE is initiated by the sensor proteins (STIM1, STIM2) located within the endoplasmic reticulum (ER) registering the Ca(2+) concentration within the ER, and upon its depletion, cluster and trap Orai (Orai1-3) proteins located in the plasma membrane (PM) into ER-PM junctions. These regions become sites of highly selective Ca(2+) entry predominantly through Orai1-assembled channels, which, among other effector functions, is necessary for triggering NFAT translocation into the nucleus. What is less clear is how the spatial and temporal spread of intracellular Ca(2+) is shaped and regulated by differential expression of the individual SOCE genes and their splice variants, their heteromeric combinations and pre- and posttranslational modifications. This review focuses on principle mechanisms regulating expression, splicing, and targeting of Ca(2+) release-activated Ca(2+) (CRAC) channels.
Topics: Animals; Calcium; Calcium Release Activated Calcium Channels; Gene Expression Regulation; Humans; Protein Processing, Post-Translational; Protein Splicing; Structure-Activity Relationship
PubMed: 26911279
DOI: 10.1152/ajpcell.00034.2016 -
European Journal of Biochemistry Feb 2004The introduction of noncanonical amino acids and biophysical probes into peptides and proteins, and total or segmental isotopic labelling has the potential to greatly... (Review)
Review
The introduction of noncanonical amino acids and biophysical probes into peptides and proteins, and total or segmental isotopic labelling has the potential to greatly aid the determination of protein structure, function and protein-protein interactions. To obtain a peptide as large as possible by solid-phase peptide synthesis, native chemical ligation was introduced to enable synthesis of proteins of up to 120 amino acids in length. After the discovery of inteins, with their self-splicing properties and their application in protein synthesis, the semisynthetic methodology, expressed protein ligation, was developed to circumvent size limitation problems. Today, diverse expression vectors are available that allow the production of N- and C-terminal fragments that are needed for ligation to produce large amounts and high purity protein(s) (protein alpha-thioesters and peptides or proteins with N-terminal Cys). Unfortunately, expressed protein ligation is still limited mainly by the requirement of a Cys residue. Of course, additional Cys residues can be introduced into the sequence by site directed mutagenesis or synthesis, however, those mutations may disturb protein structure and function. Recently, alternative ligation approaches have been developed that do not require Cys residues. Accordingly, it is theoretically possible to obtain each modified protein using ligation strategies.
Topics: Animals; Cysteine; Endopeptidases; Esters; Humans; Isotope Labeling; Models, Molecular; Mutagenesis, Site-Directed; Peptide Fragments; Protein Splicing; Proteins; Sulfhydryl Compounds
PubMed: 14764082
DOI: 10.1111/j.1432-1033.2004.03978.x -
Communications Biology Jul 2021Cytosine or adenine base editors (CBEs or ABEs) hold great promise in therapeutic applications because they enable the precise conversion of targeted base changes...
Cytosine or adenine base editors (CBEs or ABEs) hold great promise in therapeutic applications because they enable the precise conversion of targeted base changes without generating of double-strand breaks. However, both CBEs and ABEs induce substantial off-target DNA editing, and extensive off-target RNA single nucleotide variations in transfected cells. Therefore, the potential effects of deaminases induced by DNA base editors are of great importance for their clinical applicability. Here, the transcriptome-wide deaminase effects on gene expression and splicing is examined. Differentially expressed genes (DEGs) and differential alternative splicing (DAS) events, induced by base editors, are identified. Both CBEs and ABEs generated thousands of DEGs and hundreds of DAS events. For engineered CBEs or ABEs, base editor-induced variants had little effect on the elimination of DEGs and DAS events. Interestingly, more DEGs and DAS events are observed as a result of over expressions of cytosine and adenine deaminases. This study reveals a previously overlooked aspect of deaminase effects in transcriptome-wide gene expression and splicing, and underscores the need to fully characterize such effects of deaminase enzymes in base editor platforms.
Topics: Aminohydrolases; Cytosine; Gene Editing; Gene Expression; Humans; Protein Splicing
PubMed: 34272468
DOI: 10.1038/s42003-021-02406-5