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Somatotroph adenomas secrete supraphysiological amounts of GH, causing acromegaly. We have previously hypothesized that epithelial mesenchymal transition (EMT) may play a central role in the progression of these adenomas and that epithelial splicing regulator 1 (ESRP1) may function prominently as a master regulator of the EMT process in pituitary adenomas causing acromegaly. To further elucidate the role of ESRP1 in somatotroph adenomas and in EMT progression, we used RNA sequencing (RNAseq) to sequence somatotroph adenomas characterized by high and low ESRP1 levels. Transcripts identified by RNAseq were analyzed in 65 somatotroph adenomas and in GH-producing pituitary rat cells with a specific knockdown of Esrp1. The clinical importance of the transcripts was further investigated by correlating mRNA expression levels with clinical indices of disease activity and treatment response. Many of the transcripts and isoforms identified by RNAseq and verified by quantitative PCR were involved in vesicle transport and calcium signaling and were associated with clinical outcomes. Silencing Esrp1 in GH3 cells resulted in changes of gene expression overlapping the data observed in human somatotroph adenomas and revealed a decreased granulation pattern and attenuated GH release. We observed an alternative splicing pattern for F-box and leucine-rich repeat protein 20, depending on the ESPR1 levels and on changes in circulating IGF-I levels after somatostatin analog treatment. Our study indicates that ESRP1 in somatotroph adenomas regulates transcripts that may be essential in the EMT progression and in the response to somatostatin analog treatment.
Alternative splicing is thought to be one of the major sources for functional diversity in higher eukaryotes. Interestingly, when mapping splicing events onto protein structures, about half of the events affect structured and even highly conserved regions i.e. are non-trivial on the structure level. This has led to the controversial hypothesis that such splice variants result in nonsense-mediated mRNA decay or non-functional, unstructured proteins, which do not contribute to the functional diversity of an organism. Here we show in a comprehensive study on alternative splicing that proteins appear to be much more tolerant to structural deletions, insertions and replacements than previously thought. We find literature evidence that such non-trivial splicing isoforms exhibit different functional properties compared to their native counterparts and allow for interesting regulatory patterns on the protein network level. We provide examples that splicing events may represent transitions between different folds in the protein sequence-structure space and explain these links by a common genetic mechanism. Taken together, those findings hint to a more prominent role of splicing in protein structure evolution and to a different view of phenotypic plasticity of protein structures.
Epithelial-Splicing-Regulatory-Protein 1 (Esrp1) is a cell-type specific RNA-binding protein (RBP) that is essential for mammalian development through maintenance of epithelial cell properties including barrier function. Esrp1 also regulates splicing during the epithelial to mesenchymal transition (EMT). It contains three highly conserved RNA recognition motifs (RRMs) in the absence of other clearly defined protein domains. Esrp1 itself is also alternatively spliced to produce multiple protein isoforms. Here we determined that two competing alternative 5' splice sites in exon 12 yield Esrp1 isoforms with differential nucleocytoplasmic localization. We carried out a detailed characterization of the Esrp1 peptide that is sufficient to confer nuclear localization. Furthermore, we identified splice variants encoding distinct nuclear and cytoplasmic isoforms of fusilli, the D. Melanogaster Esrp1 ortholog. Our observations demonstrate that the production of both nuclear and cytoplasmic Esrp1 isoforms through alternative splicing is phylogenetically conserved; strongly suggesting it is biologically significant. Thus, while previous studies have described extensive regulation by nuclear Esrp1 to promote epithelial specific splicing, it will be of great interest to study the contribution of cytoplasmic Esrp1 in maintenance of epithelial cell functions.
Inteins are protein splicing elements that mediate their excision from precursor proteins and the joining of the flanking protein sequences (exteins). In this study, protein splicing was controlled by splitting precursor proteins within the Psp Pol-1 intein and expressing the resultant fragments in separate hosts. Reconstitution of an active intein was achieved by in vitro assembly of precursor fragments. Both splicing and intein endonuclease activity were restored. Complementary fragments from two of the three fragmentation positions tested were able to splice in vitro. Fragments resulting in redundant overlaps of intein sequences or containing affinity tags at the fragmentation sites were able to splice. Fragment pairs resulting in a gap in the intein sequence failed to splice or cleave. However, similar deletions in unfragmented precursors also failed to splice or cleave. Single splice junction cleavage was not observed with single fragments. In vitro splicing of intein fragments under native conditions was achieved using mini exteins. Trans-splicing allows differential modification of defined regions of a protein prior to extein ligation, generating partially labeled proteins for NMR analysis or enabling the study of the effects of any type of protein modification on a limited region of a protein.
Increasing interest and awareness of protein aggregation as being implicated in neurodegenerative processes has developed in recent years. One novel mechanism for this may be transglutaminase (TGase)-mediated protein crosslinking, that is involved in a variety of natural processes ranging from the stabilization of fibrin clots to production of the epidermal cell envelope and the fluid barrier of the skin. TGases are also implicated in both function and dysfunction of the central (CNS) and peripheral (PNS) nervous systems. The most ubiquitously expressed member of the TGase family, known as tissue TGase (tTG) or TG2, which, in addition to catalyzing the production of epsilon-lysine to gamma-glutaminyl isodipeptide bonds, serves a dual function as the G-protein Galpha(h) and is both expressed and active in PNS and CNS. It differs from other members of the TGase gene family in this regard and may implicate it in 'switches' from life or trophic signaling to those associated with apoptosis. In this regard, recent data indicate that one or more TGases are involved in neurodegenerative disorders such as the Qn/CAG repeat disorders, as well as Alzheimer's and Parkinson's diseases. As do many genes, particularly those highly expressed in the CNS, tTG undergoes alternative processing. Elevated expression and alternative splicing, resulting in a short (S) isoform of tTG with more active crosslinking activity, are associated with increased neuronal loss in affected regions in the demented brain. Our recent and novel data indicate that tTG mRNA, protein, and TGase activity are elevated in certain neurodegenerative diseases, and are accompanied by transcription of this S splice variant that results in unregulated crosslinking, unique to neurodegenerative disorders.
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.
The spliceosome is assembled via sequential interactions of pre-mRNA with five small nuclear RNAs and many proteins. Recent determination of cryo-EM structures for several spliceosomal complexes has provided deep insights into interactions between spliceosomal components and structural changes of the spliceosome between steps, but information on how the proteins interact with pre-mRNA to mediate the reaction is scarce. By systematic analysis of proteins interacting with the splice sites (SSs), we have identified many previously unknown interactions of spliceosomal components with the pre-mRNA. Prp8 directly binds over the 5'SS and the branch site (BS) for the first catalytic step, and the 5'SS and 3'SS for the second step. Switching the Prp8 interaction from the BS to the 3'SS requires Slu7, which interacts dynamically with pre-mRNA first, and then interacts stably with the 3'-exon after Prp16-mediated spliceosome remodeling. Our results suggest that Prp8 plays a key role in positioning the 5'SS and 3'SS, facilitated by Slu7 through interactions with Prp8 and substrate RNA to advance exon ligation. We also provide evidence that Prp16 first docks on the intron 3' tail, then translocates in the 3' to 5' direction on remodeling the spliceosome.
The retention and splicing (RES) complex is a conserved spliceosome-associated module that was shown to enhance splicing of a subset of transcripts and promote the nuclear retention of unspliced pre-mRNAs in yeast. The heterotrimeric RES complex is organized around the Snu17p protein that binds to both the Bud13p and Pml1p subunits. Snu17p exhibits an RRM domain that resembles a U2AF homology motif (UHM) and Bud13p harbors a Trp residue reminiscent of an UHM-ligand motif (ULM). It has therefore been proposed that the interaction between Snu17p and Bud13p resembles canonical UHM-ULM complexes. Here, we have used biochemical and NMR structural analysis to characterize the structure of the yeast Snu17p-Bud13p complex. Unlike known UHMs that sequester the Trp residue of the ULM ligand in a hydrophobic pocket, Snu17p and Bud13p utilize a large interaction surface formed around the two helices of the Snu17p domain. In total 18 residues of the Bud13p ligand wrap around the Snu17p helical surface in an U-turn-like arrangement. The invariant Trp(232) in Bud13p is located in the center of the turn, and contacts surface residues of Snu17p. The structural data are supported by mutational analysis and indicate that Snu17p provides an extended binding surface with Bud13p that is notably distinct from canonical UHM-ULM interactions. Our data highlight structural diversity in RRM-protein interactions, analogous to the one seen for nucleic acid interactions.
Anecdotal evidence of the involvement of alternative splicing (AS) in the regulation of protein-protein interactions has been reported by several studies. AS events have been shown to significantly occur in regions where a protein interaction domain or a short linear motif is present. Several AS variants show partial or complete loss of interface residues, suggesting that AS can play a major role in the interaction regulation by selectively targeting the protein binding sites. In the present study we performed a statistical analysis of the alternative splicing of a non-redundant dataset of human protein-protein interfaces known at molecular level to determine the importance of this way of modulation of protein-protein interactions through AS.
The muscleblind-like (MBNL) family of proteins are key developmental regulators of alternative splicing. Sequestration of MBNL proteins by expanded CUG/CCUG repeat RNA transcripts is a major pathogenic mechanism in the neuromuscular disorder myotonic dystrophy (DM). MBNL1 contains four zinc finger (ZF) motifs that form two tandem RNA binding domains (ZF1-2 and ZF3-4) which each bind YGCY RNA motifs. In an effort to determine the differences in function between these domains, we designed and characterized synthetic MBNL proteins with duplicate ZF1-2 or ZF3-4 domains, referred to as MBNL-AA and MBNL-BB, respectively. Analysis of splicing regulation revealed that MBNL-AA had up to 5-fold increased splicing activity while MBNL-BB had 4-fold decreased activity compared to a MBNL protein with the canonical arrangement of zinc finger domains. RNA binding analysis revealed that the variations in splicing activity are due to differences in RNA binding specificities between the two ZF domains rather than binding affinity. Our findings indicate that ZF1-2 drives splicing regulation via recognition of YGCY RNA motifs while ZF3-4 acts as a general RNA binding domain. Our studies suggest that synthetic MBNL proteins with improved or altered splicing activity have the potential to be used as both tools for investigating splicing regulation and protein therapeutics for DM and other microsatellite diseases.
The lack of tools to identify causative variants from sequencing data greatly limits the promise of precision medicine. Previous studies suggest that one-third of disease-associated alleles alter splicing. We discovered that the alleles causing splicing defects cluster in disease-associated genes (for example, haploinsufficient genes). We analyzed 4,964 published disease-causing exonic mutations using a massively parallel splicing assay (MaPSy), which showed an 81% concordance rate with splicing in patient tissue. Approximately 10% of exonic mutations altered splicing, mostly by disrupting multiple stages of spliceosome assembly. We present a large-scale characterization of exonic splicing mutations using a new technology that facilitates variant classification and keeps pace with variant discovery.
Wntless (Wls) is a protein that regulates secretion of Wnt signaling molecules from Wnt-producing cells. Wnt signaling is known to be critical for several developmental and homeostatic processes. However, Wnt-independent functions of Wls are now being elucidated. Primates express an alternative splice variant of Wls (here termed WlsX). WlsX contains an alternatively spliced COOH-terminus, and does not appear to be able to sustain significant levels of WNT secretion because of its inability to undergo retrograde trafficking to the endoplasmic reticulum. The functional significance for this alternatively spliced form of Wls has not yet been elucidated. We previously identified a cohort of Wls interacting proteins using a combination of yeast 2-hybrid and candidate gene approaches.
Auxilliary splicing sequences in exons, known as enhancers (ESEs) and silencers (ESSs), have been subject to strong selection pressures at the RNA and protein level. The protein component of this splicing code is substantial, recently estimated at ∼50% of the total information within ESEs, but remains poorly understood. The ESE/ESS profiles were previously associated with the Irving-Williams (I-W) stability series for divalent metals, suggesting that the ESE/ESS evolution was shaped by metal binding sites. Here, we have examined splicing activities of exonic sequences that encode protein binding sites for Ca2+, a weak binder in the I-W affinity order. We found that predicted exon inclusion levels for the EF-hand motifs and for Ca2+-binding residues in nonEF-hand proteins were higher than for average exons. For canonical EF-hands, the increase was centred on the EF-hand chelation loop and, in particular, on Ca2+-coordinating residues, with a 1>12>3∼5>9 hierarchy in the 12-codon loop consensus and usage bias at codons 1 and 12. The same hierarchy but a lower increase was observed for noncanonical EF-hands, except for S100 proteins. EF-hand loops preferentially accumulated exon splits in two clusters, one located in their N-terminal halves and the other around codon 12. Using splicing assays and published crosslinking and immunoprecipitation data, we identify candidate trans-acting factors that preferentially bind conserved GA-rich motifs encoding negatively charged amino acids in the loops. Together, these data provide evidence for the high capacity of codons for Ca2+-coordinating residues to be retained in mature transcripts, facilitating their exon-level expansion during eukaryotic evolution.
Exonic splicing enhancers (ESEs) are pre-mRNA cis-acting elements required for splice-site recognition. We previously developed a web-based program called ESEfinder that scores any sequence for the presence of ESE motifs recognized by the human SR proteins SF2/ASF, SRp40, SRp55 and SC35 (http://rulai.cshl.edu/tools/ESE/). Using ESEfinder, we have undertaken a large-scale analysis of ESE motif distribution in human protein-coding genes. Significantly higher frequencies of ESE motifs were observed in constitutive internal protein-coding exons, compared with both their flanking intronic regions and with pseudo exons. Statistical analysis of ESE motif frequency distributions revealed a complex relationship between splice-site strength and increased or decreased frequencies of particular SR protein motifs. Comparison of constitutively and alternatively spliced exons demonstrated slightly weaker splice-site scores, as well as significantly fewer ESE motifs, in the alternatively spliced group. Our results underline the importance of ESE-mediated SR protein function in the process of exon definition, in the context of both constitutive splicing and regulated alternative splicing.
Wilm's tumor protein 1 (WT1), a protein implicated in various cancers and developmental disorders, consists of two major isoforms: WT1(-KTS), a transcription factor, and WT1(+KTS), a post-transcriptional regulator that binds to RNA and can interact with splicing components. Here we show that WT1 interacts with the novel splicing regulator RBM4. Each protein was found to colocalize in nuclear speckles and to cosediment with supraspliceosomes in glycerol gradients. RBM4 conferred dose-dependent and cell-specific regulation of alternative splicing of pre-mRNAs transcribed from several reporter genes. We found that overexpressed WT1(+KTS) abrogated this effect of RBM4 on splice-site selection, whereas WT1(-KTS) did not. We conclude that the (+KTS) form of WT1 is able to inhibit the effect of RBM4 on alternative splicing.
Endoplasmic reticulum resident protein 44 (ERp44) is a member of the protein disulfide isomerase family and functions in oxidative protein folding in the endoplasmic reticulum. A structurally flexible C-terminal tail (C-tail) of ERp44 plays critical roles in dynamically regulating ERp44's function in protein folding quality control. The structure-function dynamics of ERp44's C-tail may be studied further using fluorescence and other techniques, if methods are found to label the C-tail site-specifically with a fluorescent group or segmentally with other desired labels. Here we have developed such methods, employing split inteins capable of protein trans-splicing, and identifying atypical S1 split inteins able to function efficiently at a suitable split site in the ERp44 sequence. One method demonstrated segmental expression of ERp44 for segmental labeling of the C-tail, another method efficiently added a commercially available fluorescent group to the C-terminus of ERp44, and both methods may also be generally useful for studying other proteins.
Protein splicing is mediated by inteins that auto-catalytically join two separated protein fragments with a peptide bond. Here we engineered a genetically encoded synthetic photoactivatable intein (named LOVInC), by using the light-sensitive LOV2 domain from Avena sativa as a switch to modulate the splicing activity of the split DnaE intein from Nostoc punctiforme. Periodic blue light illumination of LOVInC induced protein splicing activity in mammalian cells. To demonstrate the broad applicability of LOVInC, synthetic protein systems were engineered for the light-induced reassembly of several target proteins such as fluorescent protein markers, a dominant positive mutant of RhoA, caspase-7, and the genetically encoded Ca2+ indicator GCaMP2. Spatial precision of LOVInC was demonstrated by targeting activity to specific mammalian cells. Thus, LOVInC can serve as a general platform for engineering light-based control for modulating the activity of many different proteins.
Densin is a member of the leucine-rich repeat (LRR) and PDZ domain (LAP) protein family that binds several signaling molecules via its C-terminal domains, including calcium/calmodulin-dependent protein kinase II (CaMKII). In this study, we identify several novel mRNA splice variants of densin that are differentially expressed during development. The novel variants share the LRR domain but are either prematurely truncated or contain internal deletions relative to mature variants of the protein (180 kDa), thus removing key protein-protein interaction domains. For example, CaMKIIalpha coimmunoprecipitates with densin splice variants containing an intact C-terminal domain from lysates of transfected HEK293 cells, but not with variants that only contain N-terminal domains. Immunoblot analyses using antibodies to peptide epitopes in the N- and C- terminal domains of densin are consistent with developmental regulation of splice variant expression in brain. Moreover, putative splice variants display different subcellular fractionation patterns in brain extracts. Expression of green fluorescent protein (GFP)-fused densin splice variants in HEK293 cells shows that the LRR domain can target densin to a plasma membrane-associated compartment, but that the splice variants are differentially localized and have potentially distinct effects on cell morphology. In combination, these data show that densin splice variants have distinct functional characteristics suggesting multiple roles during neuronal development.
Alternative RNA splicing plays a crucial role in defining protein function. However, despite its relevance, there is a lack of tools that characterize effects of splicing on protein interaction networks in a mechanistic manner (i.e. presence or absence of protein-protein interactions due to RNA splicing). To fill this gap, we present Linear Integer programming for Network reconstruction using transcriptomics and Differential splicing data Analysis (LINDA) as a method that integrates resources of protein-protein and domain-domain interactions, transcription factor targets, and differential splicing/transcript analysis to infer splicing-dependent effects on cellular pathways and regulatory networks.
Viruses use alternative splicing to produce a broad series of proteins from small genomes by utilizing the cellular splicing machinery. Since viruses use cellular RNA binding proteins for viral RNA processing, it is presumable that the splicing of cellular pre-mRNAs is affected by viral infection. Here, we showed that herpes simplex virus type 2 (HSV-2) modifies the expression of promyelocytic leukemia (PML) isoforms by altering pre-mRNA splicing. Using a newly developed virus-sensitive splicing reporter, we identified the viral protein ICP27 as an alternative splicing regulator of PML isoforms. ICP27 was found to bind preferentially to PML pre-mRNA and directly inhibit the removal of PML intron 7a in vitro. Moreover, we demonstrated that ICP27 functions as a splicing silencer at the 3' splice site of the PML intron 7a. The switching of PML isoform from PML-II to PML-V as induced by ICP27 affected HSV-2 replication, suggesting that the viral protein modulates the splicing code of cellular pre-mRNA(s) governing virus propagation.
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