A myosin phosphatase targeting subunit isoform transition defines a smooth muscle developmental phenotypic switch

Smooth muscle myosin phosphatasedephosphorylates the regulatory myosin light chain and thus mediatessmooth muscle relaxation. The activity of this myosin phosphatase isdependent upon its myosin-targeting subunit (MYPT1). Isoforms of MYPT1have been identified, but how they are generated and their relationship to smooth muscle phenotypes is not clear. Cloning of the middle sectionof chicken and rat MYPT1 genes revealed that each gene gave rise toisoforms by cassette-type alternative splicing of exons. In chicken, a123-nucleotide exon was included or excluded from the mature mRNA,whereas in rat two exons immediately downstream were alternative. MYPT1isoforms lacking the alternative exon were only detected in maturechicken smooth muscle tissues that display phasic contractileproperties, but the isoform ratios were variable. The patterns ofexpression of rat MYPT1 mRNA isoforms were more complex, with threemajor and two minor isoforms present in all smooth muscle tissues atvarying stoichiometries. Isoform switching was identified in thedeveloping chicken gizzard, in which the exon-skipped isoform replacedthe exon-included isoform around the time of hatching. This isoformswitch occurred after transitions in myosin heavy chain and myosinlight chain (MLC₁₇) isoforms and correlated with aseveralfold increase in the rate of relaxation. The developmentalswitch of MYPT1 isoforms is a good model for determining the mechanismsand significance of alternative splicing in smooth muscle.

     

Localization of a new prostate-specific antigen-related serine protease gene, KLK4, is evidence for an expanded human kallikrein gene family cluster on chromosome 19q13.3-13.4.

The human tissue kallikrein (KLK) family of serine proteases, which is important in post-translational processing events, currently consists of just three genes-tissue kallikrein (KLK1), KLK2, and prostate-specific antigen (PSA) (KLK3)-clustered at chromosome 19q13. 3-13.4. We identified an expressed sequence tag from an endometrial carcinoma cDNA library with 50% identity to the three known KLK genes. Primers designed to putative exon 2 and exon 3 regions from this novel kallikrein-related sequence were used to polymerase chain reaction-screen five cosmids spanning 130 kb around the KLK locus on chromosome 19. This new gene, which we have named KLK4, is 25 kb downstream of the KLK2 gene and follows a region that includes two other putative KLK-like gene fragments. KLK4 spans 5.2 kb, has an identical genomic structure-five exons and four introns-to the other KLK genes and is transcribed on the reverse strand, in the same direction as KLK1 but opposite to that of KLK2 and KLK3. It encodes a 254-amino acid prepro-serine protease that is most similar (78% identical) to pig enamel matrix serine protease but is also 37% identical to PSA. These data suggest that the human kallikrein gene family locus on chromosome 19 is larger than previously thought and also indicate a greater sequence divergence within this family compared with the highly conserved rodent kallikrein genes.     

Human cystathionine β-synthase: gene organization and expression of different 5′ alternative splicing

The human cystathionine β-synthase (CBS) gene spans in excess of 30 kb and consists of 19 exons, with three different 5′ untranslated regions including three different exons 1 (exons 1 a, b, and c). Exon la and 1b are 390 bp apart from each other and are linked to exon 2 in cDNA « a » and cDNA « b ». Exon 1c, which linked to exon 5 in cDNA « c », is 7 kb apart from exon 1b. All splice sites conform to the GT/AG rule, including those from exon la or 1b to exon 2 and from exon 1c to exon 5. Upstream of exons la and 1b, we found two putative promoter sequences with high C + G nucleotide content, one CAAT box at —70 nucleotides (for exon lb), no TATA box, several Sp1 binding regulatory consensus sequences, and some other regulatory sequences. Human adult and fetal Northern blots hybridized with total cDNA containing exon 1b, or specific probes from exons 1 (b and c) showed mRNAs of 2.5 kb, 2.7 kb, and 3.7 kb. These results suggest that the mRNAs containing the different exons 1 are under the control of different promoters.     

Distinct targeting and recycling properties of two isoforms of the iron transporter DMT1 (NRAMP2, Slc11A2)

The metal transporter DMT1 (Slc11a2) plays a vital role in iron metabolism. Alternative splicing of the 3' exon generates two DMT1 isoforms with different C-terminal protein sequences and a 3' untranslated region harboring (isoform I, +IRE) or not (isoform II, -IRE), an iron-responsive element. Isoform I is expressed at the plasma membrane of certain epithelial cells including the duodenum brush border, where it is essential for the absorption of nutritional iron. Isoform II is expressed in many cells and is essential for the acquisiton of transferrin iron from acidified endosomes. The targeting and trafficking properties of DMT1 isoforms I and II were studied in transfected LLC-PK(1) kidney cells, with respect to isoform-specific differences in function, subcellular localization, endocytosis kinetics, and fate upon internalization. Isoform I showed higher surface expression and was internalized from the plasma membrane with slower kinetics than that of isoform II. As opposed to isoform II, which is efficiently sorted to recycling endosomes upon internalization, isoform I was not efficiently recycled and was targeted to lysosomes. Thus, alternative splicing of DMT1 critically regulates the subcellular localization and site of Fe(2+) transport.     

Expression pattern of an evolutionarily conserved splice variant in the rat Tacc2 gene

The transforming acidic coiled‐coil containing protein 2 (Tacc2) gene and its paralogs, Tacc1 and Tacc3 encode proteins that are associated with the centrosome and involved in microtubule assembly during the cell cycle. Tacc2 produces several splice variants, which are poorly characterized, especially in the rat. Characterization of the temporal/spatial expression patterns of these isoforms would be useful in understanding their distinct and overlapping functions. By comparative sequence analyses of Tacc2 in multiple species, we identified a third splice variant in rat, which is much shorter in size (1,021 aa) than the longest isoform (2,834 aa). This newly identified Tacc2 splice variant (isoform 3) uses a distinct first exon and generates a different open reading frame. Although Isoform 3 is expressed predominantly during developmental stages, the long Tacc2 isoform (isoform 1) is distributed mainly in adult tissues. Multiple protein sequence analyses revealed that Tacc2 Isoform 3 could be the ancient form, as it is conserved in mammals, birds, and amphibians; whereas the long Tacc2 isoforms may have evolved in the mammalian lineage by adding exons toward the 5′ region of the ancient isoform. genesis 52:378–386, 2014. © 2014 Wiley Periodicals, Inc.     

Kallikreins are associated with secondary progressive multiple sclerosis and promote neurodegeneration

Tissue kallikrein KLK1 and the kallikrein-related peptidases KLK2-15 are a subfamily of serine proteases that have defined or proposed roles in a range of central nervous system (CNS) and non-CNS pathologies. To further understand their potential activity in multiple sclerosis (MS), serum levels of KLK1, 6, 7, 8 and 10 were determined in 35 MS patients and 62 controls by quantitative fluorometric ELISA. Serum levels were then correlated with Expanded Disability Status Scale (EDSS) scores determined at the time of serological sampling or at last clinical follow-up. Serum levels of KLK1 and KLK6 were elevated in MS patients (p相似文献   

Complete exon-intron organization of the human leukocyte common antigen (CD45) gene

Ten genomic DNA clones encoding the human leukocyte common Ag (LCA, CD45) gene were isolated by screening human genomic DNA libraries with LCA cDNA probes. One genomic DNA clone contains the promoter region and the first two exons, as determined by primer extension analyses and S1 nuclease protection studies as well as nucleotide sequence determination. The first exon does not encode a peptide, while the second exon contains the initiation ATG codon and encodes the signal peptide. The other nine genomic DNA clones, which are separated from the first genomic clone by an unknown distance, are connected and span a total of 73 kb. The nine connected genomic clones encode a total of 31 exons. The 33 exons encoded by these 10 genomic clones account for the entire cDNA sequences including the 5' and 3' untranslated sequences. Exon 3 and exons 7 through 15 encode the extracellular domain sequences that are common to all LCA isoforms. Differential usage of exons 4, 5, and 6, generates at least five distinct LCA isoforms. Exon 16 encodes the transmembrane peptide. The cytoplasmic region of the leukocyte common antigens is composed of two homologous domains. Exons 17 through 24 encode the first domain, and exons 25 through 32 encode the second domain. The comparison of these exons indicated that the homologous domains were generated by duplication of several exons. The most 3' exon (exon 33) encodes the carboxy terminus of the LCA molecules and includes the entire 3' untranslated sequence.     

Multiple isoforms of beta-TrCP display differential activities in the regulation of Wnt signaling

The F-box proteins beta-TrCP1 and 2 (beta-transducin repeat containing protein) have 2 and 3 isoforms, respectively, due to alternative splicing of exons encoding the N-terminal region. We identified an extra exon in between the previously known exons 1 and 2 of beta-TrCP1 and beta-TrCP2. Interestingly, sequence analysis suggested that many more isoforms are produced than previously identified, via the alternative splicing of all possible combination of exons II to V of beta-TrCP1 and exons II to IV of beta-TrCP2. Different mouse tissues show specific expression patterns of the isoforms, and the level of expression of the isoform that has been used in most published papers was very low. Yeast two-hybrid assays show that beta-TrCP1 isoforms containing exon III, which are the most highly expressed isoforms in most tissues, do not interact with Skp1. Indirect immunofluorescence analysis of transiently expressed beta-TrCP1 isoforms suggests that the presence of exon III causes beta-TrCP1 to localize in nuclei. Consistent with the above findings, isoforms including exon III showed a reduced ability to block ectopic embryonic axes induced via injection of Wnt8 or beta-catenin in Xenopus embryos. Overall, our data suggest that isoforms of beta-TrCPs generated by alternative splicing may have different biological roles.     

Differential regulation of B-raf isoforms by phosphorylation and autoinhibitory mechanisms

The B-Raf proto-oncogene encodes several isoforms resulting from alternative splicing in the hinge region upstream of the kinase domain. The presence of exon 8b in the B2-Raf(8b) isoform and exon 9b in the B3-Raf(9b) isoform differentially regulates B-Raf by decreasing and increasing MEK activating and oncogenic activities, respectively. Using different cell systems, we investigated here the molecular basis of this regulation. We show that exons 8b and 9b interfere with the ability of the B-Raf N-terminal region to interact with and inhibit the C-terminal kinase domain, thus modulating the autoinhibition mechanism in an opposite manner. Exons 8b and 9b are flanked by two residues reported to down-regulate B-Raf activity upon phosphorylation. The S365A mutation increased the activity of all B-Raf isoforms, but the effect on B2-Raf(8b) was more pronounced. This was correlated to the high level of S365 phosphorylation in this isoform, whereas the B3-Raf(9b) isoform was poorly phosphorylated on this residue. In contrast, S429 was equally phosphorylated in all B-Raf isoforms, but the S429A mutation activated B2-Raf(8b), whereas it inhibited B3-Raf(9b). These results indicate that phosphorylation on both S365 and S429 participate in the differential regulation of B-Raf isoforms through distinct mechanisms. Finally, we show that autoinhibition and phosphorylation represent independent but convergent mechanisms accounting for B-Raf regulation by alternative splicing.