小鼠SmX5基因mRNA的选择性剪接

通过RT-PCR技术,发现小鼠SmX5基因的另外3种选择性剪接体,分别命名为SmX5a,b和c.小鼠SmX5a cDNA具有完整的内含子1,而SmX5b cDNA含有部分内含子2。SmX5c的保留片断和SmX5b在相同位置起始,但保留部分延伸经过余下的内含子2。余下的DNA序列在所有内含子-外显子边界处都符合剪接的“GT—AG”规则。RT-PCR的表达分析显示这3种异构体都是稳定的转录产物,均通过剪接体特异性引物的PCR反应在mRNA水平上得到证实。和SmX5相比较,剪接体的出现频率都很低。RT-PCR表达分析提示,XmX5和它的3种异构体存在于小鼠的脑、肾、睾丸、胸腺、肝、脾和心脏。SmX5a主要在胸腺中表达,而SmX5b和c在所有组织都可以检测到.SmX5不同选择剪接体及其组织特异的不同剪接方式的存在,表明SmX5的表达是复杂的,并且所有4种SmX5 mRNA水平的调控对于在不同细胞类型的mRNA前体的剪接机制维持具有重要作用。     

Differential expression and developmental regulation of a novel alpha-dystrobrevin isoform in muscle

Alpha-dystrobrevin is a dystrophin-related protein expressed primarily in skeletal muscle, heart, lung and brain. In skeletal muscle, alpha-dystrobrevin is a component of the dystrophin-associated glycoprotein complex and is localized to the sarcolemma, presumably through interactions with dystrophin and utrophin. Alternative splicing of the alpha-dystrobrevin gene generates multiple isoforms which have been grouped into three major classes: alpha-DB1, alpha-DB2, and alpha-DB3. Various isoforms have been shown to interact with a variety of proteins; however, the physiological function of the alpha-dystrobrevins remains unknown. In the present study, we have cloned a novel alpha-dystrobrevin cDNA encoding a protein (referred to as alpha-DB2b) with a unique 11 amino acid C-terminal tail. Using RT PCR with primers specific to the new isoform, we have characterized its expression in skeletal muscle, heart, and brain, and in differentiating C2C12 muscle cells. We show that alpha-DB2b is expressed in skeletal muscle, heart and brain, and that exons 12 and 13 are alternatively spliced in alpha-DB2b to generate at least three splice variants. The major alpha-DB2b splice variant expressed in adult skeletal muscle and heart contains exons 12 and 13, while in adult brain, two alpha-DB2b splice variants are expressed at similar levels. This is consistent with the preferential expression of exons 12 and 13 in other alpha-dystrobrevin isoforms in skeletal muscle and heart. Similarly, in alpha-DB1 the first 21 nucleotides of exon 18 are preferentially expressed in skeletal muscle and heart relative to brain. We also show that the expression of alternatively spliced alpha-DB2b is developmentally regulated in muscle; during differentiation of C2C12 cells, alpha-DB2b expression switches from an isoform lacking exons 12 and 13 to one containing them. We demonstrate similar developmental upregulation of exons 12, 13, and 18 in alpha-DB1 and of exons 12 and 13 in alpha-DB2a. Finally, we show that alpha-DB2b protein is expressed in adult skeletal muscle, suggesting that it has a functional role in adult muscle. Together, these data suggest that alternatively spliced variants of the new alpha-dystrobrevin isoform, alpha-DB2b, are differentially expressed in various tissues and developmentally regulated during muscle cell differentiation in a fashion similar to that previously described for alpha-dystrobrevin isoforms.     

Identification and characterization of nonmuscle myosin II-C, a new member of the myosin II family

A previously unrecognized nonmuscle myosin II heavy chain (NMHC II), which constitutes a distinct branch of the nonmuscle/smooth muscle myosin II family, has recently been revealed in genome data bases. We characterized the biochemical properties and expression patterns of this myosin. Using nucleotide probes and affinity-purified antibodies, we found that the distribution of NMHC II-C mRNA and protein (MYH14) is widespread in human and mouse organs but is quantitatively and qualitatively distinct from NMHC II-A and II-B. In contrast to NMHC II-A and II-B, the mRNA level in human fetal tissues is substantially lower than in adult tissues. Immunofluorescence microscopy showed distinct patterns of expression for all three NMHC isoforms. NMHC II-C contains an alternatively spliced exon of 24 nucleotides in loop I at a location analogous to where a spliced exon appears in NMHC II-B and in the smooth muscle myosin heavy chain. However, unlike neuron-specific expression of the NMHC II-B insert, the NMHC II-C inserted isoform has widespread tissue distribution. Baculovirus expression of noninserted and inserted NMHC II-C heavy meromyosin (HMM II-C/HMM II-C1) resulted in significant quantities of expressed protein (mg of protein) for HMM II-C1 but not for HMM II-C. Functional characterization of HMM II-C1 by actin-activated MgATPase activity demonstrated a V(max) of 0.55 + 0.18 s(-1), which was half-maximally activated at an actin concentration of 16.5 + 7.2 microm. HMM II-C1 translocated actin filaments at a rate of 0.05 + 0.011 microm/s in the absence of tropomyosin and at 0.072 + 0.019 microm/s in the presence of tropomyosin in an in vitro motility assay.     

The alpha- and beta-isoforms of the inhibitor protein of the 3',5'-cyclic adenosine monophosphate-dependent protein kinase: characteristics and tissue- and developmental-specific expression.

The inhibitor protein (PKI) of the cAMP-dependent protein kinase was first characterized from rabbit skeletal muscle. More recently a form of PKI was isolated and cloned from rat testis which shares relatively limited amino acid sequence with the rabbit skeletal muscle form. We have now isolated a cDNA from rat brain which encodes a protein corresponding to the rabbit skeletal muscle PKI. This establishes the presence of the "skeletal muscle" and "testis" proteins in the same species and therefore that they clearly represent distinct isoforms. We have also demonstrated that the isoform from testis, like the skeletal muscle isoform, is specific for the cAMP-dependent protein kinase and that it is able to inhibit this enzyme when expressed in cultured JEG-3 cells. Both forms contain the five specific amino acid recognition determinants which have been shown to be required for high affinity binding to the protein kinase catalytic site, although there is some noted lack of conservation of codons used for these residues. Overall, the two rat isoforms are only 41% identical at the amino acid level and 46% at the level of coding nucleotides. We propose that the rabbit skeletal muscle and rat testis forms be designated PKI alpha and PKI beta, respectively. Using Northern blot analysis, we have examined the tissue distribution of the two forms in the rat and their relative expression during development. In the adult rat, mRNA of the PKI alpha species is highest in muscle (both skeletal and cardiac) and brain (cortex and cerebellum).(ABSTRACT TRUNCATED AT 250 WORDS)     

gamma-Syntrophin scaffolding is spatially and functionally distinct from that of the alpha/beta syntrophins

The syntrophins are a family of scaffolding proteins with multiple protein interaction domains that link signaling proteins to dystrophin family members. Each of the three most characterized syntrophins (alpha, beta1, beta2) contains a PDZ domain that binds a unique set of signaling proteins including kinases, ion and water channels, and neuronal nitric oxide synthase (nNOS). The PDZ domains of the gamma-syntrophins do not bind nNOS. In vitro pull-down assays show that the gamma-syntrophins can bind dystrophin but have unique preferences for the syntrophin binding sites of dystrophin family members. Despite their ability to bind dystrophin in vitro, neither gamma-syntrophin isoform co-localizes with dystrophin in skeletal muscle. Furthermore, gamma-syntrophins do not co-purify with dystrophin isolated from mouse tissue. These data suggest that the interaction of gamma-syntrophin with dystrophin is transient and potentially subject to regulatory mechanisms. gamma1-Syntrophin is highly expressed in brain and is specifically localized in hippocampal pyramidal neurons, Purkinje neurons in cerebellum, and cortical neurons. gamma2-Syntrophin is expressed in many tissues including skeletal muscle where it is found only in the subsynaptic space beneath the neuromuscular junction. In both neurons and muscle, gamma-syntrophin isoforms localize to the endoplasmic reticulum where they may form a scaffold for signaling and trafficking.     

Phosphorylation of the cardiac isoform of calsequestrin in cultured rat myotubes and rat skeletal muscle.

Calsequestrin is a high-capacity Ca(2+)-binding protein and a major constituent of the sarcoplasmic reticulum (SR) of both skeletal and cardiac muscle. Two isoforms of calsequestrin, cardiac and skeletal muscle forms, have been described which are products of separate genes. Purified forms of the two prototypical calsequestrin isoforms, dog cardiac and rabbit fast-twitch skeletal muscle calsequestrins, serve as excellent substrates for casein kinase II and are phosphorylated on distinct sites (Cala, S.E. and Jones, L.R. (1991) J. Biol. Chem 266, 391-398). Dog cardiac calsequestrin is phosphorylated at a 50 to 100-fold greater rate than is rabbit skeletal muscle calsequestrin, and only the dog cardiac isoform contains endogenous Pi on casein kinase II phosphorylation sites. In this study, we identified and examined both calsequestrin isoforms in rat muscle cultures and homogenates to demonstrate that the cardiac isoform of calsequestrin in rat skeletal muscle was phosphorylated in vivo on sites which are phosphorylated by casein kinase II in vitro. Phosphorylation of rat skeletal muscle calsequestrin was not detected. In tissue homogenates, cardiac and skeletal muscle calsequestrin isoforms were both found to be prominent substrates for endogenous casein kinase II activity with cardiac calsequestrin the preferred substrate. In addition, these studies revealed that the cardiac isoform of calsequestrin was the predominant form expressed in skeletal muscle of fetal rats and cultured myotubes.     

Expression profiling of the gamma-subunit isoforms of AMP-activated protein kinase suggests a major role for gamma3 in white skeletal muscle

Expression patterns of the three isoforms of the regulatory gamma-subunit of AMP-activated protein kinase (AMPK) were determined in various tissues from adult humans, mice, and rats, as well as in human primary muscle cells. Real-time PCR-based quantification of mRNA showed similar expression patterns in the three species and a good correlation with protein expression in mice and rats. The gamma3-isoform appeared highly specific to skeletal muscle, whereas gamma1 and gamma2 showed broad tissue distributions. Moreover, the proportion of white, type IIb fibers in the mouse and rat muscle samples, as indicated by real-time PCR quantification of Atp1b2 mRNA, showed a strong positive correlation with the expression of gamma3. In samples of white skeletal muscle, gamma3 clearly appeared to be the most abundant gamma-isoform. Differentiation of human primary muscle cells from myoblasts into multinucleated myotubes was accompanied by upregulation of gamma3 mRNA expression, whereas levels of gamma1 and gamma2 remained largely unchanged. However, even in these cultured myotubes, gamma2 was the most highly expressed isoform, indicating a considerable difference compared with adult skeletal muscle. Immunoblot analysis of mouse gastrocnemius and quadriceps muscle extracts precipitated with a gamma3-specific antibody showed that gamma3 was exclusively associated with the alpha2- and beta2-subunit isoforms. The observation that the AMPKgamma3 isoform is expressed primarily in white skeletal muscle, in which it is the predominant gamma-isoform, strongly suggests that gamma3 has a key role in this tissue.