Alternatively spliced products of the human kinesin light chain 1 (KNS2) gene

Conventional kinesin is a microtubule-based molecular motor involved in the transport of membranous and non-membranous cargoes. The kinesin holoenzyme exists as a heterotetramer, consisting of two heavy chain and two light chain subunits. It is thought that one function of the light chains is to interact with the cargo. Alternative splicing of kinesin light chain pre-mRNA has been observed in lower organisms, although evidence for alternative splicing of the human gene has not been reported. We have identified 19 variants of the human KNS2 gene ( KLC1 ) that are generated by alternative splicing of downstream exons, but calculate that KNS2 has the potential to produce 285 919 spliceforms. Corresponding spliceforms of the mouse KLC1 gene were also identified. The alternative exons are all located 3' of exon 12 and the novel spliceforms produce both alternative carboxy termini and alternative 3' untranslated regions. The observation of multiple light chain isoforms is consistent with their proposed role in specific cargo attachment.     

ASDB: database of alternatively spliced genes

Version 2.1 of ASDB (Alternative Splicing Data Base) contains 1922 protein and 2486 DNA sequences. The protein entries from SWISS-PROT are joined into clusters corresponding to alternatively spliced variants of one gene. The DNA division consists of complete genes with alternative splicing mentioned or annotated in GenBank. The search engine allows one to search over SWISS-PROT and GenBank fields and then follow the links to all variants. The database can be assessed at the URL http://cbcg.nersc.gov/asdb     

Alternatively spliced exon B of myosin Va is essential for binding the tail-associated light chain shared by dynein

A 10 kDa dynein light chain (DLC), previously identified as a tail light chain of myosin Va, may function as a cargo-binding and/or regulatory subunit of both myosin and dynein. Here, we identify and characterize the binding site of DLC on myosin Va. Fragments of the human myosin Va tail and the DLC2 isoform were expressed, and their complex formation was analyzed by pull-down assays, gel filtration, and spectroscopic methods. DLC2 was found to bind as a homodimer to a approximately 15 residue segment (Ile1280-Ile1294) localized between the medial and distal coiled-coil domains of the tail. The binding region contains the three residues coded by the alternatively spliced exon B (Asp1284-Lys1286). Removal of exon B eliminates DLC2 binding. Co-localization experiments in a transfected mammalian cell line confirm our finding that exon B is essential for DLC2 binding. Using circular dichroism, we demonstrate that binding of DLC2 to a approximately 85 residue disordered domain (Pro1235-Arg1320) induces some helical structure and stabilizes both flanking coiled-coil domains (melting temperature increases by approximately 7 degrees C). This result shows that DLC2 promotes the assembly of the coiled-coil domains of myosin Va. Nuclear magnetic resonance spectroscopy and docking simulations show that a 15 residue peptide (Ile1280-Ile1294) binds to the surface grooves on DLC2 similarly to other known binding partners of DLCs. When our data are taken together, they suggest that exon B and its associated DLC2 have a significant effect on the structure of parts of the coiled-coil tail domains and such a way could influence the regulation and cargo-binding function of myosin Va.     

Alternatively spliced human tissue factor: a circulating,soluble, thrombogenic protein

Tissue factor (TF) is an essential enzyme activator that forms a catalytic complex with FVII(a) and initiates coagulation by activating FIX and FX, ultimately resulting in thrombin formation. TF is found in adventitia of blood vessels and the lipid core of atherosclerotic plaques. In unstable coronary syndromes, plaque rupture initiates coagulation by exposing TF to blood. Biologically active TF has been detected in vessel walls and circulating blood. Elevated intravascular TF has been reported in diverse pro-thrombotic syndromes such as myocardial infarction, sepsis, anti-phospholipid syndrome and sickle-cell disease. It is unclear how TF circulates, although it may be present in pro-coagulant microparticles. We now report identification of a form of human TF generated by alternative splicing. Our studies indicate that alternatively spliced human tissue factor (asHTF) contains most of the extracellular domain of TF but lacks a transmembrane domain and terminates with a unique peptide sequence. asHTF is soluble, circulates in blood, exhibits pro-coagulant activity when exposed to phospholipids, and is incorporated into thrombi. We propose that binding of asHTF to the edge of thrombi contributes to thrombus growth by creating a surface that both initiates and propagates coagulation.     

Alternatively spliced CD44 isoforms containing exon v10 promote cellular adhesion through the recognition of chondroitin sulfate-modified CD44

Correlations have been noted between the expression of certain alternatively spliced CD44 isoforms and the metastatic propensity of various histologically distinct tumor cell types. The precise mechanism by which particular CD44 isoforms contribute to the metastatic process is, however, unclear. In the present study we demonstrate that CD44R2, a CD44 isoform highly expressed on activated and transformed hemopoietic cells, can recognize and bind a common determinant present on CD44H and CD44R1. Importantly, CD44H lacked this activity. Pretreatment of TIL1 cells expressing CD44H or CD44R1 with chondroitinase ABC inhibited adhesion to CD44R2, suggesting that the unique inserted region present within the CD44R2 molecule, encoded by exon v10, mediates cell adhesion by potentiating the recognition of chondroitin sulfate moieties presented in association with other CD44 molecules. These data help explain the differential involvement of v10-containing CD44 isoforms in tumor metastasis.     

EDAS, databases of alternatively spliced human genes

EDAS, a database of alternatively spliced human genes, contains data on the alignment of proteins, mRNAs, and EST. It contains information on all exons and introns observed, as well as elementary alternatives formed from them. The database makes it possible to filter the output data by changing the cut-off threshold by the significance level. The database is accessible at http://www.gene-bee.msu.ru/edas/.     

Positive selection in alternatively spliced exons of human genes

Alternative splicing is a well-recognized mechanism of accelerated genome evolution. We have studied single-nucleotide polymorphisms and human-chimpanzee divergence in the exons of 6672 alternatively spliced human genes, with the aim of understanding the forces driving the evolution of alternatively spliced sequences. Here, we show that alternatively spliced exons and exon fragments (alternative exons) from minor isoforms experience lower selective pressure at the amino acid level, accompanied by selection against synonymous sequence variation. The results of the McDonald-Kreitman test suggest that alternatively spliced exons, unlike exons constitutively included in the mRNA, are also subject to positive selection, with up to 27% of amino acids fixed by positive selection.     

Alternatively and constitutively spliced exons are subject to different evolutionary forces

There has been a controversy on whether alternatively spliced exons (ASEs) evolve faster than constitutively spliced exons (CSEs). Although it has been noted that ASEs are subject to weaker selective constraints than CSEs, so they evolve faster, there have also been studies that indicated slower evolution in ASEs than in CSEs. In this study, we retrieve more than 5,000 human-mouse orthologous exons and calculate the synonymous (KS) and nonsynonymous (KA) substitution rates in these exons. Our results show that ASEs have higher KA values and higher KA/KS ratios than CSEs, indicating faster amino acid-level evolution in ASEs. The faster evolution may be in part due to weaker selective constraints. It is also possible that the faster rate is in part due to faster functional evolution in ASEs. On the other hand, the majority of ASEs have lower KS values than CSEs. With reference to the substitution rate in introns, we show that the KS values in ASEs are close to the neutral substitution rate, whereas the synonymous substitution rate in CSEs has likely been accelerated. The elevated synonymous rate in CSEs is not related to CpG dinucleotides or low-complexity regions of protein but may be weakly related to codon usage bias. The overall trends of higher KA and lower KS in ASEs than in CSEs are also observed in human-rat and mouse-rat comparisons. Therefore, our observations hold for mammals of different molecular clocks.     

Alternatively spliced mRNA of the pX region of human T lymphotropic virus type I proviral genome.

The pX region of human T lymphotropic virus type I (HTLV-I) is believed to be expressed as a consequence of a 2 step splicing. It is conceivable, however, that a donor site of the 1st splicing and an acceptor site of the 2nd splicing results in the production of an alternatively spliced mRNA which is capable of coding p21X-III. This possibility was examined by amplifying cDNA derived from HTLV-I+ cells between the 5' LTR and pX region. Bands of 2 different sizes were consistently observed. Sequencing of the longer band corresponded to a cDNA derived from a double-spliced pX mRNA as previously reported. The shorter band was derived from a single-spliced mRNA. HTLV-I+ cell lines had both mRNAs to a varying degree. Expression of p40tax and p21X-III seem to be well correlated with a double-spliced and a single-spliced mRNA, respectively.