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At least 235 records · Page 13Linked to original sources
PubMed2002

Novel isoform of insulin receptor substrate p53/p58 is generated by alternative splicing in the CRIB/SH3-binding region.

Insulin receptor substrate p53/p58 (IRSp53) is involved in cytoskeletal dynamics and is a candidate disease sensor in polyglutamine expansion neurodegeneration. It is widely expressed throughout the body, but its levels are dramatically elevated in forebrain regions. IRSp53 functions as a signal transducing adaptor between activated Rho family GTPases and their effectors. There are four known alternatively spliced isoforms of IRSp53 that vary by the identity of the 3'-terminal exon. We report here that there is a fifth alternatively spliced isoform, IRSp53-B, which lacks 40 amino acids abutting the CRIB/SH3 (Cdc42/Rac-interactive binding/Src homology 3)-binding site. We speculate that the novel form has an altered function related to the mechanism of autoinactivation. IRSp53-B has an odd history in the mammalian lineage, which may complicate the use of rodent models to study cytoskeletal reorganization.

Alternative Splicing↗
PubMed2001

Color bar coding the BRCA1 gene on combed DNA: a useful strategy for detecting large gene rearrangements.

Genetic linkage data have shown that alterations of the BRCA1 gene are responsible for the majority of hereditary breast and ovarian cancers. BRCA1 germline mutations, however, are found less frequently than expected. Mutation detection strategies, which are generally based on the polymerase chain reaction, therefore focus on point and small gene alterations. These approaches do not allow for the detection of large gene rearrangements, which also can be involved in BRCA1 alterations. Indeed, a few of them, spread over the entire BRCA1 gene, have been detected recently by Southern blotting or transcript analysis. We have developed an alternative strategy allowing a panoramic view of the BRCA1 gene, based on dynamic molecular combing and the design of a full four-color bar code of the BRCA1 region. The strategy was tested with the study of four large BRCA1 rearrangements previously reported. In addition, when screening a series of 10 breast and ovarian cancer families negatively tested for point mutation in BRCA1/2, we found an unreported 17-kb BRCA1 duplication encompassing exons 3 to 8. The detection of rearrangements as small as 2 to 6 kb with respect to the normal size of the studied fragment is achieved when the BRCA1 region is divided into 10 fragments. In addition, as the BRCA1 bar code is a morphologic approach, the direct observation of complex and likely underreported rearrangements, such as inversions and insertions, becomes possible.

Breast Neoplasms↗
PubMed1996

Expression patterns of two murine homologs of Drosophila single-minded suggest possible roles in embryonic patterning and in the pathogenesis of Down syndrome.

The single-minded (sim) gene encodes a transcriptional regulator that functions as a key determinant of central nervous system (CNS) midline development in Drosophila. We report here the identification of two murine homologs of sim, Sim1 and Sim2, whose products show a high degree of sequence conservation with Drosophila SIM in their amino-terminal halves, with each containing a basic helix-loop-helix domain as well as a PAS domain. Sim1 maps to the proximal region of mouse chromosome 10, whereas Sim2 maps to a portion of the distal end of chromosome 16 that is syntenic to the Down syndrome critical region of human chromosome 21. Recent exon-trapping studies have identified in the critical region several exons of a human sim homolog which appears to be the homolog of murine Sim2; this has led to the hypothesis that increased dosage of this sim homolog in cases of trisomy 21 might be a causal factor in the pathogenesis of Down syndrome. We have examined the expression patterns of the Sim genes during embryogenesis. Both genes are expressed in dynamic and selective fashion in specific neuromeric compartments of the developing forebrain, and the expression pattern of Sim2 provides evidence for early regionalization of the diencephalon prior to any overt morphological differentiation in this region. Outside the CNS, Sim1 is expressed in mesodermal and endodermal tissues, including developing somites, mesonephric duct, and foregut. Sim2 is expressed in facial and trunk cartilage, as well as trunk muscles. Both murine Sim genes are also expressed in the developing kidney. Our data suggest that the Sim genes play roles in directing the regionalization of tissues where they are expressed. Moreover, the expression pattern documented for Sim2 may provide insights into its potential roles in Down syndrome.

Amino Acid Sequence↗
PubMed2000

[The role of ergometric testing in diagnosis of latent long QT syndrome].

BACKGROUND: The long QT syndrome is a genetically determined disease based on mutations of ion membrane channel genes. The resulting prolongation of repolarization increases a risk of malignant ventricular arrhythmias and sudden death. The diagnosis is problematic in individuals with borderline or even normal corrected QT interval value. Ventricular arrhythmias in this syndrome are often provoked by exercise, therefore exercise testing is considered as a useful differential diagnostic method. METHODS: In a 24-member family an occurrence of the long QT syndrome was established clinically in 7 patients. In other 3 members borderline corrected QT interval values were found (0.44-0.46 s). In these individuals a stress testing by bicycle ergometry was performed. RESULTS: During exercise in a 28-year old man (patient III/6) the corrected QT interval prolonged to 0.53 s, in a 27-year old man (patient III/2) and in a 20-year old woman (patient III/9) physiological QT interval shortenings were observed (0.4 s during peak exercise). The diagnosis was confirmed by molecular genetic investigation. In patient III/6 (along with other symptomatic family members) a mutation in exon 7 of KCNQ1 (previously called KVLQT1) gene was found. In patients III/2 and III/9 no signs of KCNQ1 gene pathology were present. CONCLUSIONS: In families with clinically established diagnosis of long QT syndrome occurrence the exercise testing reveals previously asymptomatic individuals, at least in LQT1 type (which is the most common). In general stress testing and measurement of QT interval dynamics is a necessary part of arrhythmological investigation especially in young individuals with history of syncope.

Adult↗
PubMed1993

Protein modelling using a chimera reference protein derived from exons.

Bovine pancreatic beta-trypsin (PDB ID-code: 1TPO) which is registered in the Brookhaven Protein Data Bank (PDB) consists of four exons. The results of homology searches for each exon in the PDB showed that homologous proteins were tonin (PDB ID-code: 1TON), rat mast cell protease (PDB ID-code: 3RP2__A), kallikrein A (PDB ID-code: 2PKA__B) and kallikrein A (2PKA__B) respectively. Thus, for the three-dimensional structure prediction of 1TPO, a chimera protein was constructed from the three proteins mentioned above and the 3-D structure prediction was performed using this chimera reference protein. The modelled structure of 1TPO was energetically optimized by molecular mechanics and molecular dynamics simulation and was compared with its X-ray crystal structure registered in the PDB. The root mean square deviations (r.m.s.d.) of main chain atoms and the neighbouring active site (5 A sphere from His57, Asp102 and Ser195) between the modelled structure and the X-ray structure were 1.66 and 0.94 A respectively. Porcine pancreatic elastase (PDB ID-code: 3EST) which is registered in the PDB was used as the reference protein and the modelled structure from 3EST was also compared with the X-ray data. The r.m.s.d. of main chain atoms and that of the active site were 2.14 and 1.18 A respectively. These results clearly support the propriety of this method using the chimera reference protein.

Algorithms↗
PubMed2001

Genetic and biological variation in equine infectious anemia virus Rev correlates with variable stages of clinical disease in an experimentally infected pony.

Genetic and biological variation in the regulatory protein Rev of equine infectious anemia virus (EIAV) were examined throughout a clinically dynamic disease course of an experimentally infected pony. Following infection with the virulent EIAV(Wyo), the pony underwent a variable disease course, including an acute fever episode at 12 days postinfection (DPI), multiple recurrent fever episodes until 135 DPI, a prolonged subclinical period, and two late fever episodes. Viral RNA was isolated from the inoculum and sequential sera samples, and the rev exon 2/gp45 overlapping ORFs were amplified, cloned, and sequenced. Novel variants were found throughout infection, and genetic analyses indicated that both the Rev and gp45 ORFs were under selective pressure. The Rev variant predominant in the inoculum, R1, remained predominant during the early periods following infection (until 35 DPI); however, R1 was replaced by new predominant variants during the recurrent fever period (67-135 DPI). R1 reemerged as the predominant variant during the afebrile period, but a new predominant variant, R93, was associated with the late fever episodes. Rev variants predominant during recurrent febrile and late-febrile periods had significantly higher Rev-mediated nuclear export activity than the variants predominant during the acute and afebrile periods. Statistical correlation was found between Rev activity and different stages of clinical disease. Together, these results suggest that genetic and biological variation in rev may be a contributing factor in EIAV disease progression.

Amino Acid Sequence↗
PubMed2005

Experimental validation of the regulated expression of large numbers of non-coding RNAs from the mouse genome.

Recent large-scale analyses of mainly full-length cDNA libraries generated from a variety of mouse tissues indicated that almost half of all representative cloned sequences did not contain an apparent protein-coding sequence, and were putatively derived from non-protein-coding RNA (ncRNA) genes. However, many of these clones were singletons and the majority were unspliced, raising the possibility that they may be derived from genomic DNA or unprocessed pre-mRNA contamination during library construction, or alternatively represent nonspecific "transcriptional noise." Here we show, using reverse transcriptase-dependent PCR, microarray, and Northern blot analyses, that many of these clones were derived from genuine transcripts of unknown function whose expression appears to be regulated. The ncRNA transcripts have larger exons and fewer introns than protein-coding transcripts. Analysis of the genomic landscape around these sequences indicates that some cDNA clones were produced not from terminal poly(A) tracts but internal priming sites within longer transcripts, only a minority of which is encompassed by known genes. A significant proportion of these transcripts exhibit tissue-specific expression patterns, as well as dynamic changes in their expression in macrophages following lipopolysaccharide stimulation. Taken together, the data provide strong support for the conclusion that ncRNAs are an important, regulated component of the mammalian transcriptome.

Animals↗
PubMed2004

Dynamic regulation of estrogen receptor-beta expression by DNA methylation during prostate cancer development and metastasis.

Estrogen receptor (ER)-beta is thought to exert anti-proliferative effects in the normal prostate but supports prostate cancer (PCa) cell survival. We previously reported that the receptor's expression declined as PCa developed in the gland but reappeared in lymph node and bone metastases. To investigate whether hypermethylation was the underlying mechanism for these phenomena, we first identified two CpG islands (CGIs) encompassing 41 CpG dinucleotides, located separately in the untranslated exon 0N and the promoter region of ER-beta. Using immunostained, laser capture-microdissected samples from 56 clinical specimens, we demonstrated an inverse relationship exists between the extent of ER-beta CGI methylation and receptor expression in normal, hyperplastic, premalignant, and malignant foci of the prostate and in lymph node and bone metastases. Treatment of PCa cell lines (LNCaP and DU145), that express little ER-beta mRNA, with a demethylating agent increased levels of receptor expression thus corroborating our in vivo findings that methylation is involved in ER-beta silencing. Methylation centers in the promoter region and exon 0N were identified by hierarchical cluster analysis of bisulfite sequencing data obtained from 710 alleles. Methylation at these centers was insignificant in normal epithelium, reached 80 to 90% in grade 4/5 PCa, but declined to less than 20% in bone metastases. In addition, progressive methylation spreading from the exonic CGI to the promoter CGI, which correlated with loss of ER-beta expression, was detected in microdissected samples and in cell cultures. Using a new class of methylated oligonucleotides that mediate sequence-specific methylation in cellulo, we demonstrated that methylation of the promoter CGI, but not the exonic CGIs, led to transcriptional inactivation of ER-beta. Our results present the first evidence that epigenetic regulation of ER-beta is a reversible and tumor stage-specific process and that gene silencing via methylated oligonucleotides may have therapeutic potential in the treatment of advanced PCa.

Base Sequence↗
PubMed2006

Strength of the purifying selection against different categories of the point mutations in the coding regions of the human genome.

Using available Information on the total absolute size of the coding region of the human genome, data on codon usage and pseudogene-derived mutation rates for different single nucleotide substitutions we have estimated, for the human genome, the potential numbers of mutation events capable to produce: (1) nonsense; (2) missense (radical and conservative); (3) silent; (4) splice; and (5) protein-elongating (those changing wild-type stop codon into an amino acid encoding codon) mutations. We used the NCBI dbSNP database to retrieve data on the observed number of polymorphisms of each category. The fraction of polymorphisms in each category among all potential events in the genome depends on the strength of selection: the higher the rate of polymorphism, the weaker the selection. We used nonsense mutations as a referent group. Compared with nonsense mutations, we found that the relative selection coefficient against protein-elongating mutations was 21%, and the relative selection was 12% against missense mutations. Radical missense mutations were found to be four times more deleterious compared to conservative ones. Surprisingly, we found that silent mutations on average are not neutral; with the average harmfulness of 3% of nonsense mutations. Silent mutations may be deleterious when they affect splicing by creating cryptic donor-acceptor sites or by disturbing exonic splicing enhancers (ESESs). The average selection coefficient against splice mutations was 48% of that against nonsense mutations. Converting the relative selection coefficients into absolute ones using data on loss-of-function mutations in Saccharomyces cerevisiae and Caenorhabditis elegans, or by analysis of the expected frequency of mutations in the human genome, suggested that genetic drift could play a role in population dynamics of conservative missense and silent mutations.

Computational Biology↗
PubMed2005

Variation at two flowering time genes within and among populations of Arabidopsis thaliana: comparison with markers and traits.

Flowering Locus C (FLC) and Frigida are two interacting genes controlling flowering time variation in Arabidopsis thaliana. Variation at these genes was surveyed in 12 A. thaliana populations sampled in France. These populations were also screened for variation at molecular markers [12 microsatellites and 19 cleaved amplified polymorphic sequence (CAPS) markers] and at seven quantitative traits measured with and without vernalization. Seven populations were highly polymorphic at markers (H(S) = 0.57 at microsatellites, 0.24 at CAPS) and showed heritable variation for bolting time and some other traits. Five populations were genetically fixed or nearly fixed. Q(ST) for bolting time without vernalization was significantly higher than F(ST), suggesting local divergent selection. One of the two haplotype groups at FLC (FLC(A)) was very predominant (frequency of 99%). The first exon of Frigida showed elevated nonsynonymous variation, and nine loss-of-function mutations were found throughout the gene. The association between loss-of-function and earlier bolting was confirmed. Overall, 18 Frigida haplotypes were detected. The pattern of variation at Frigida was largely similar to that found at markers and traits, with the same populations being fixed or highly diverse. Metapopulation dynamics is thus probably the main factor shaping genetic variation in A. thaliana. However, F(ST) for functional (FRI) vs. nonfunctional (FRI(Delta)) haplotypes was significantly higher than F(ST) at markers. This suggested that loss-of-function at Frigida is under local selection for flowering time.

Arabidopsis↗
PubMed2004

Glucocorticoid down-regulation of rat glucocorticoid receptor does not involve differential promoter regulation.

The level of expression of the glucocorticoid receptor (GR) is the principal determinant of glucocorticoid sensitivity in most cells. GR levels are permanently 'set' in a tissue-specific manner in response to the perinatal environment, an effect we have previously shown to relate to differential expression of tissue-enriched alternative promoters/exons 1 of the GR gene. In adult animals, GR levels are dynamically regulated around the 'set point' by glucocorticoids themselves, with glucocorticoids down-regulating GR mRNA in most cells and tissues. Here we have examined whether autoregulation of GR mRNA by glucocorticoids involves differential promoter regulation. We show that, in contrast to tissue-specific programming of GR mRNA levels, autoregulation of GR mRNA in vivo does not involve differential regulation of variant exon 1-containing GR mRNAs in that the major variants are down-regulated to a similar extent by glucocorticoid treatment. Consistent with this, transfections of reporter constructs showed that the majority of GR promoters, which are contained within a 4.4 kb region upstream of exon 2, are similarly regulated by glucocorticoids, with two regions of the promoter redundantly required for glucocorticoid regulation. Thus transcriptional autoregulation can occur in adult tissues around the set point established by promoter selection in early life.

Animals↗
PubMed2001

[A turning point in the knowledge of the structure-function-activity relations of elastin].

In this review are presented the last new results of our research group dealing with the molecular structures (atomic level) of tropoelastin, elastin and elastin derived peptides studied by using essentially methods of bioinformatics (theoretical predictions and molecular modelling) linked to experimental circular dichroism spectroscopic studies. We already had characterized both the local secondary structure and some parts of the tertiary structure of the tropoelastin and elastin molecules (human, bovine...), by using either theoretical predictions (local secondary structure, linear epitopes...) and/or experimental data (optical spectroscopic methods: Raman scattering, infrared absorption, circular dichroism). Except the cross-linking regions which are in helical conformations, the whole tropoelastin structure displays a lot of beta-reverse turns which usually belong to irregular structures in proteins. These turns play a key role in other regularly structures orientation (alpha-helix, beta-strand), thus they are very important in the native protein 3D architecture. It is particularly true for human tropoelastin, because its sequence is rich in glycines and prolines, and these residues are frequently met in beta-turns (a beta-turn is made of four consecutive residues which are stabilized by an hydrogen bond). Several types of beta-turns can be defined with the dihedral angles values phi and psi of the two central residues. Thus, by using a very recent updated set of propensities for the amino acid residues to belong to given types of reverse beta-turns (extracted from a reference set of known 3-D structures of globular proteins), we have determined, (by using our home made software COUDES), for all possible tetrapeptides of the human tropoelastin sequence, the distribution and the characterization of the possible type of turns. Thus, it is shown that the locations and/or the types of these reverse beta-turns reveal a regularity and are not all random. This confirms our hypothesis that intra-molecular elasticity of tropoelastin could be explained by the possibility of transitions between conformations involving short beta-strands and beta-turns. This result is of great interest in the construction (by using molecular biology) of elastic biomaterials derived from the elastin sequence (particularly, the elastin derived peptides corresponding to the sequence exon 21--(exon 24--exon 24...). Our study permit also to predict the conformations of specific elastin derived peptides which could have interesting biological activity. Peptides resulting from the degradation of elastin, the insoluble polymer of tropoelastin and responsible for the elasticity of vertebrate tissues, can induce biological effects and notably the regulation of matrix metalloproteinases (MMP-s) activity. Recently, it was proposed that some elastin derived hexapeptides resulting from circular permutations of VGVAPG (a three fold repetition sequence in exon 24 of human tropoelastin) possess MMP-1 production and activation regulation properties. This effect depends on the presence of the tropoelastin specific membraneous receptor 67 KDa EBP (Elastin Binding Protein). Our results obtained by using both circular dichroism spectroscopy and linear predictions confirmed the hypothesis of a structure dependent mechanism with a possibly occurring type VIII beta-turn on the first four residues of the GXXPG sequence consensus which is only present among all active peptides. Thus, we have performed extensive molecular dynamics studies, in both implicit and explicit solvent, on these active and inactive elastin derived hexapeptides. Using our own analysis method of pattern recognition of the types of the beta-reverse-turns followed during the molecular dynamics trajectory, we found that active and inactive peptides effectively form two well distinct conformational groups in which active peptides preferentially adopt conformation close to type VIII GXXP (beta-reverse-turn. The structural role of the C terminal G residue could also be explained. Additional molecular simulations on (VGVAPG)2 and (VGVAPG)3 show the formation of two or three GXXP tetrapeptides adopting a structure close to type VIII beta-reverse-turn, suggesting a local conformational preference for this motif. This observation of a specific structural single and/or repeated motif is in agreement with the circular dichroism spectra of the involved (VGVAPG)1, (VGVAPG)2 and (VGVAPG)3 peptides and then it can be proposed that their biological activities have to be linear. The final aim of this type of work is to understand more about the sequence/structure/function/activity relationships of those structured peptides in order to propose specific sequences (corresponding to specific structures) for best biological activity results.

Amino Acid Sequence↗
PubMed1997

Mechanical stability of compact modules of barnase.

Globular proteins are composed of structural elements such as secondary structures and modules. Modules are compact segments consisting of 10-40 contiguous amino acid residues and are often encoded by exons. Therefore, the view that the modular organization of proteins is a result of exon-shuffling or -fusion is given support. Secondary structures such as alpha-helix and beta-sheet are stabilized by hydrogen bonds and are thus considered to be stable, structural elements of a globular domain. Since module boundaries are often located on alpha-helices or beta-sheets, it is not obvious whether the modules are mechanically stable. We carried out molecular dynamics simulations on modules of barnase, a bacterial RNase from Bacillus amyloliquefaciens, for 1 ns in vacuo and 150 ps in water. Five of six modules (M1, 1-24; M2, 25-52; M3, 53-73; M4, 74-88; M5, 89-98) retained native-like conformations during these simulations. Only the C-terminal module (M6, 99-110) was deformed; it is less compact than the other modules. As the modules are mechanically stable they are suitable as parts combined into proteins. Together with RNase activity of the three isolated modules of barnase, M2, M3 and M6, our study supports the view that modules were indeed original building blocks of proteins.

Bacterial Proteins↗
PubMed2002

Fast and sensitive algorithm for aligning ESTs to human genome.

There is a pressing need to align growing set of expressed sequence tags (ESTs) to newly sequenced human genome. The problem is, however, complicated by the exon/intron structure of eucaryotic genes, misread nucleotides in ESTs, and millions of repeptive sequences in genomic sequences. Indeed, to solve this, algorithms that use dynamic programming have been proposed, but in reality, these algorithms require an enormous amount of processing time. In an effort to improve the computational efficiency of these classical DP algorithms, we develop software that fully utilizes the lookup-table for allowing the efficient detection of the start- and endpoints of an EST within a given DNA sequence, and subsequently, the prompt identification of exons and introns. In addition, high sensitivity and accuracy must be achieved by calculating locations of all spliced sites correctly for more ESTs while retaining high computational efficiency. This goal is hard to accomplish in practice, owing to misread nucleotides in ESTs and repeptive sequences in the genome, but we present a couple of heuristics effective in settling this issue. Experimental results have confirmed that our technique improves the overall computation time by orders of magnitude compared with common tools such as sim4 and BLAT, and attains high sensitivity and accuracy against datasets of clean and documented genes at the same time.

Algorithms↗
PubMed2004

The von Hippel-Lindau tumor suppressor protein influences microtubule dynamics at the cell periphery.

The von Hippel-Lindau (VHL) protein protects microtubules (MTs) from destabilization by nocodazole treatment. Based on this fixed-cell assay with static end points, VHL has been reported to directly stabilize the MT cytoskeleton. To investigate the dynamic changes in MTs induced by VHL in living cells, we measured the influence of VHL on tubulin turnover using fluorescence recovery after photobleaching (FRAP). To this end, we engineered VHL-deficient renal cell carcinoma cells to constitutively incorporate fluorescently labeled tubulin and to inducibly express VHL. Induction of VHL in these cells resulted in a decrease of tubulin turnover as measured by FRAP at the cell periphery, while minimally influencing MT dynamics around the centrosome. Our data indicates that VHL changes the behavior of MTs dependent on their subcellular localization implying a role for VHL in cellular processes such as migration, polarization, and cell-cell interactions. Here we propose a complementary method to directly measure VHL-induced subcellular changes in microtubule dynamics, which may serve as a tool to study the effect of MT binding proteins such as VHL.

Carcinoma, Renal Cell↗
PubMed2004

Patterns of intron gain and loss in fungi.

Little is known about the patterns of intron gain and loss or the relative contributions of these two processes to gene evolution. To investigate the dynamics of intron evolution, we analyzed orthologous genes from four filamentous fungal genomes and determined the pattern of intron conservation. We developed a probabilistic model to estimate the most likely rates of intron gain and loss giving rise to these observed conservation patterns. Our data reveal the surprising importance of intron gain. Between about 150 and 250 gains and between 150 and 350 losses were inferred in each lineage. We discuss one gene in particular (encoding 1-phosphoribosyl-5-pyrophosphate synthetase) that displays an unusually high rate of intron gain in multiple lineages. It has been recognized that introns are biased towards the 5' ends of genes in intron-poor genomes but are evenly distributed in intron-rich genomes. Current models attribute this bias to 3' intron loss through a poly-adenosine-primed reverse transcription mechanism. Contrary to standard models, we find no increased frequency of intron loss toward the 3' ends of genes. Thus, recent intron dynamics do not support a model whereby 5' intron positional bias is generated solely by 3'-biased intron loss.

Adenosine↗
PubMed2006

Human Sec31B: a family of new mammalian orthologues of yeast Sec31p that associate with the COPII coat.

We have cloned human brain and testis Sec31B protein (also known as secretory pathway component Sec31B-1 or SEC31-like 2; GenBank accession number AF274863). Sec31B is an orthologue of Saccharomyces cerevisiae Sec31p, a component of the COPII vesicle coat that mediates vesicular traffic from the endoplasmic reticulum. Sec31B is widely expressed and enriched in cerebellum and testis. Its predicted sequence of 1180 residues (expected molecular mass 128,711 Da) shares 47.3% and 18.8% similarity to human Sec31A (also known as Sec31; GenBank accession number AF139184) and yeast Sec31p, respectively. The gene encoding Sec31B is located on chromosome 10q24 and contains 29 exons. PCR analysis of exon utilization reveals massive alternative mRNA splicing of Sec31B, with just 16 exons being constitutively utilized in all transcripts. The presence of a stop codon in exon 13 generates two families of Sec31B gene products (each displaying additional patterns of mRNA splicing): a group of full-length proteins (hereafter referred to as Sec31B-F) and also a group of truncated proteins (hereafter referred to as Sec31B-T), distinguished by their utilization of exon 13. Sec31B-F closely resembles Sec31p and Sec31A, with canonical WD repeats in an N-terminal domain that binds Sec13 and a proline-rich C-terminal region that presumably binds Sec23/24. The Sec31B-T group (molecular mass 52,983 Da) contains a preserved WD-repeat domain but lacks the C-terminal proline-rich region. When expressed as a fusion protein with eYFP in cultured cells, Sec31B-F associates with the endoplasmic reticulum and with vesicular-tubular clusters, displays restricted intracellular movement characteristic of COPII vesicle dynamics, co-distributes on organelles with Sec13, Sec31A and Sec23 (markers of the COPII coat), and concentrates with ts045-VSV-G-CFP (VSV-G) when examined early in the secretory pathway or after temperature or nocodazole inhibition. The role of the truncated form Sec31B-T appears to be distinct from that of Sec31B-F and remains unknown. We conclude that Sec31B-F contributes to the diversity of the mammalian COPII coat, and speculate that the Sec31 cage, like Sec24, might be built with isoforms tuned to specific types of cargo or to other specialized functions.

Alternative Splicing↗
PubMed2026

An endothelial RNA splicing atlas catalogs effects of IL-1β and identifies an alternative PROCR isoform with genetic links to pleiotropic vascular disease.

Alternative splicing (AS) alters the sequence and dynamics of mRNA but has not been comprehensively annotated in human endothelial cells (ECs). EC dysfunction is a hallmark of complex inflammatory diseases, including cancer and atherosclerosis. Therefore, we modeled inflammation in vitro using 53 genetically distinct human aortic EC lines exposed to interleukin-1β (IL-1β) or control media. This identified 1,224 differentially spliced transcripts (DSTs). DSTs were enriched for alternative first (AF) exon usage, including isoforms of several disease-associated and metabolic genes. To confirm that IL-1β-dependent AF exons were produced by alternative promoters, a quantitative measure of promoter activity was defined using epigenetic data. Ratios of histone 3 lysine 27 acetylation and binding of ERG and RELA between alternative promoters correlated RNA levels of AF exons, confirming our hypothesis. Finally, the effects of genetic variation on AS were investigated by mapping splicing quantitative trait loci (sQTLs). Significant sQTLs were tested for genetic colocalization with cardiovascular risk loci from genome-wide association studies. This identified 66 colocalized signals corresponding to 30 genes and 39 lead variants. The sQTLs identified here provide testable mechanisms explaining some of the genetic risk for vascular disease. For example, genetic association for a previously undescribed isoform of endothelial protein C receptor (PROCR) colocalized with genetic risk for deep vein thrombosis and coronary artery disease with opposing risk alleles. This study demonstrates the prevalence of inducible promoters upon inflammatory stimuli and shows that genetic risk for vascular disease may be in part governed through AS in ECs.

Humans↗