RNA helicase p68 (DDX5) regulates tau exon 10 splicing by modulating a stem-loop structure at the 5' splice site

Regulation of tau exon 10 splicing plays an important role in tauopathy. One of the cis elements regulating tau alternative splicing is a stem-loop structure at the 5' splice site of tau exon 10. The RNA helicase(s) modulating this stem-loop structure was unknown. We searched for splicing regulators interacting with this stem-loop region using an RNA affinity pulldown-coupled mass spectrometry approach and identified DDX5/RNA helicase p68 as an activator of tau exon 10 splicing. The activity of p68 in stimulating tau exon 10 inclusion is dependent on RBM4, an intronic splicing activator. RNase H cleavage and U1 protection assays suggest that p68 promotes conformational change of the stem-loop structure, thereby increasing the access of U1snRNP to the 5' splice site of tau exon 10. This study reports the first RNA helicase interacting with a stem-loop structure at the splice site and regulating alternative splicing in a helicase-dependent manner. Our work uncovers a previously unknown function of p68 in regulating tau exon 10 splicing. Furthermore, our experiments reveal functional interaction between two splicing activators for tau exon 10, p68 binding at the stem-loop region and RBM4 interacting with the intronic splicing enhancer region.     

Alternative splicing within the chicken c-ets-1 locus: implications for transduction within the E26 retrovirus of the c-ets proto-oncogene.

Two overlapping c-ets-1 cDNA clones were isolated which contained the alpha and beta genomic sequences homologous to the 5' end of v-ets not detected in the previously described c-ets RNA species or proteins. Nucleotide sequencing demonstrated that these cDNAs corresponded to the splicing of alpha and beta to a common set of 3' exons (a through F) already found in the p54c-ets-1 mRNA. They contained an open reading frame of 1,455 nucleotides which could encode a polypeptide of 485 amino acids with a predicted molecular mass of 53 kilodaltons. However, when expressed in COS-1 cells, the cDNAs directed the synthesis of a protein with an apparent molecular mass in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 68 kilodaltons, p68c-ets-1, comigrating with a protein expressed at low levels in normal chicken spleen cells. These two proteins were shown to be identical by partial digestion with protease V8. Northern (RNA) blot hybridization analysis with the p68c-ets-1 -specific sequence and RNase protection experiments showed that the corresponding mRNA was expressed in normal chicken spleen and not in normal chicken thymus or in various T lymphoid cell lines. Thus, two closely related proteins, having distinct amino-terminal parts, are generated within the same locus by alternative addition of different 5' exons, alpha and beta or I54, respectively, onto a common set of 3' exons (a to F). Finally, we demonstrate that an aberrant splicing event between a cryptic splice donor site in c-myb exon E6 and the normal splice acceptor site of c-ets-1 exon alpha involved in the genesis of the E26 myb-ets sequence.     

Genome-wide detection of tissue-specific alternative splicing in the human transcriptome

We have developed an automated method for discovering tissue-specific regulation of alternative splicing through a genome-wide analysis of expressed sequence tags (ESTs). Using this approach, we have identified 667 tissue-specific alternative splice forms of human genes. We validated our muscle-specific and brain-specific splice forms for known genes. A high fraction (8/10) were reported to have a matching tissue specificity by independent studies in the published literature. The number of tissue-specific alternative splice forms is highest in brain, while eye-retina, muscle, skin, testis and lymph have the greatest enrichment of tissue-specific splicing. Overall, 10-30% of human alternatively spliced genes in our data show evidence of tissue-specific splice forms. Seventy-eight percent of our tissue-specific alternative splices appear to be novel discoveries. We present bioinformatics analysis of several tissue-specific splice forms, including automated protein isoform sequence and domain prediction, showing how our data can provide valuable insights into gene function in different tissues. For example, we have discovered a novel kidney-specific alternative splice form of the WNK1 gene, which appears to specifically disrupt its N-terminal kinase domain and may play a role in PHAII hypertension. Our database greatly expands knowledge of tissue-specific alternative splicing and provides a comprehensive dataset for investigating its functional roles and regulation in different human tissues.     

Theoretical analysis of alternative splice forms using computational methods

Nowadays understanding alternative splicing is one of the greatest challenges in biology, because it is a genetic process much more important than thought at the time of its discovery. In this paper, we explain the approach of using the different available databases and software tools to start a large scale investigation of alternative splice forms. To collect information about alternative splicing we investigated known data in the databases using different computational methods. The investigations proceeded from the genomic sequence data to structural protein data. Then, we interpreted those data to find the relationship between alternative splice forms and protein function and structure. We found some interesting features of alternative splicing which are presented here. We discuss the results of one chosen example. They concern the coverage quality of the protein sequence of a known structure, an EST analysis, the validation of splice variants, the determination of the alternative splice type, and finally the link between alternative splicing and disease.     

Alternatively spliced forms in the carboxy-terminal domain of the p53 protein regulate its ability to promote annealing of complementary single strands of nucleic acids.

The carboxy-terminal domain of the p53 protein comprising amino acid residues 311 to 393 is able to promote the reassociation of single-stranded RNA or DNA into duplex hybrids. This domain is as efficient as the intact p53 protein in both the rate and the extent of the double-stranded product produced in this reaction. Both wild-type and mutant p53 proteins from cancerous cells carry out this reaction. The monoclonal antibody PAb421, which detects an epitope between residues 370 and 378, blocks the ability of p53 to reassociate single strands of RNA or DNA. Similarly, the alternative splice form of the murine p53 protein, which removes amino acid residues 364 to 390 and replaces them with 17 new amino acids, does not carry out the reassociation reaction with RNA or DNA. This is the first indication of functionally distinct properties of the alternative splice forms of p53. These results suggest that this splice alternative can regulate a p53-mediated reaction that may be related to the functions of this protein.     

Expression and intracellular localization of Pyk2 in normal and v-src transformed chicken epiphyseal chondrocytes

The expression and localization of prolin-rich tyrosine kinase 2 (Pyk2) were studied in chick embryo epiphyseal chondrocytes. Two immunoreactive bands were detected in chondrocytes, a major band with an apparent Mr of 123 kDa and a minor band with an apparent Mr of 68 kDa. The major band appears to migrate as a doublet with apparent Mr of 116/123 kDa. Increased levels of the three forms of Pyk2 were observed in v-src transformed chondrocytes as compared to control uninfected chondrocytes. Immunofluorescent staining shows that Pyk2 is clearly visible in the cytosol and in the perinuclear region of control and v-src-chondrocytes and displays a pattern very similar to the distribution of the mitochondrial marker Mito Tracker. More, immunofluorescent staining shows that Pyk2 is nuclear in most chondrocytes. By subcellular fractionation, the p116/123 Pyk2 doublet, was found to be accumulated mainly in the cytoplasm while the p68 Pyk2 form, was found to be accumulated exclusively in the nucleus. The differential nuclear/cytoplasmic distribution of the Pyk2 forms remains unchanged after v-Src-induced transformation. The p68 Pyk2 form could no longer be detected by using a N-terminus domain-specific anti-Pyk2 antibody. Consistently, Pyk2 immunoreactivity was restricted to the cytoplasm of control and v-src transformed chondrocytes. Thus it appears that the p68 Pyk2 form that accumulates in the nucleus has a deletion in the N-terminus region.     

Primary structure of the human, membrane-associated Ca2+-binding protein p68 a novel member of a protein family.

cDNA clones encoding human 'p68', a membrane-associated Ca2+-binding protein, were isolated from a lambda gt11 expression library of the human T-leukaemia cell line J6, by using a rabbit antiserum against denatured purified lymphocyte p68, and from a liver cDNA library by using 32P-labelled p68 cDNA fragments. The amino acid sequence of p68, deduced from the sequences of overlapping cDNA clones, is described. The results show that p68 is closely related to a family of proteins which includes intracellular substrates of the EGF receptor and pp60src tyrosine kinases. The p68 amino acid sequence is internally repetitive, being constructed from eight repeats of varying lengths, each of which contains a highly conserved sequence. Multiple copies of the latter sequence are also present in the related proteins p36, lipocortin I and protein II. We discuss how the common structural features of these proteins may reflect common functions and, furthermore, how the eight repeat structure of p68 may have evolved. The sequences of independent cDNAs suggest that alternatively-spliced mRNAs could encode different p68 protein species. This suggestion is consistent with the observation that purified p68 migrates as a closely-spaced doublet when analysed by SDS-PAGE.     

Noisy splicing drives mRNA isoform diversity in human cells

While the majority of multiexonic human genes show some evidence of alternative splicing, it is unclear what fraction of observed splice forms is functionally relevant. In this study, we examine the extent of alternative splicing in human cells using deep RNA sequencing and de novo identification of splice junctions. We demonstrate the existence of a large class of low abundance isoforms, encompassing approximately 150,000 previously unannotated splice junctions in our data. Newly-identified splice sites show little evidence of evolutionary conservation, suggesting that the majority are due to erroneous splice site choice. We show that sequence motifs involved in the recognition of exons are enriched in the vicinity of unconserved splice sites. We estimate that the average intron has a splicing error rate of approximately 0.7% and show that introns in highly expressed genes are spliced more accurately, likely due to their shorter length. These results implicate noisy splicing as an important property of genome evolution.     

In vitro splicing of cardiac troponin T precursors. Exon mutations disrupt splicing of the upstream intron.

A single cardiac troponin T (cTNT) gene generates two mRNAs by including or excluding the 30-nucleotide exon 5 during pre-mRNA processing. Transfection analysis of cTNT minigenes has previously demonstrated that both mRNAs are expressed from unmodified minigenes, and mutations within exon 5 can lead to complete skipping of the exon. These results suggested a role for exon sequence in splice site recognition. To investigate this potential role, an in vitro splicing system using cTNT precursors has been established. Two-exon precursors containing the alternative exon and either the upstream exon or downstream exon were spliced accurately and efficiently in vitro. The mutations within the alternative exon that resulted in exon skipping in vivo specifically blocked splicing of the upstream intron in vitro and had no effect on removal of the downstream intron. In addition, the splicing intermediates of these two precursors have been characterized, and the branch sites utilized on the introns flanking the alternative exon have been determined. Potential roles of exon sequence in splice site selection are discussed. These results establish a system that will be useful for the biochemical characterization of the role of exon sequence in splice site selection.     

A conformational switch in the Piccolo C2A domain regulated by alternative splicing

C2 domains are widespread Ca2+-binding modules. The active zone protein Piccolo (also known as Aczonin) contains an unusual C2A domain that exhibits a low affinity for Ca2+, a Ca2+-induced conformational change and Ca2+-dependent dimerization. We show here that removal of a nine-residue sequence by alternative splicing increases the Ca2+ affinity, abolishes the conformational change and abrogates dimerization of the Piccolo C2A domain. The NMR structure of the Ca2+-free long variant provides a structural basis for these different properties of the two splice forms, showing that the nine-residue sequence forms a beta-strand otherwise occupied by a nonspliced sequence. Consequently, Ca2+-binding to the long Piccolo C2A domain requires a marked rearrangement of secondary structure that cannot occur for the short variant. These results reveal a novel mechanism of action of C2 domains and uncover a structural principle that may underlie the alteration of protein function by short alternatively spliced sequences.     

水稻NBS-LRR基因选择性剪接的全基因组检测及分析

选择性剪接是促进基因组复杂性和蛋白质组多样性的一种主要机制,但是对水稻NBS-LRR序列选择性剪接的全基因组分析却未见报道。通过隐马尔柯夫模型搜索,从TIGR数据库里得到了855条编码NBS-LRR基序的序列。利用这些序列在KOME、TIGR基因索引及UniProt三个数据库中进行同源搜索,获得同源的完整cDNA序列、假设一致性序列和蛋白质序列。再利用Spidey和SIM4程序把完整cDNA序列和假设一致性序列联配到相应的BAC序列上来预测选择性剪接。蛋白质序列和基因组序列之间的联配使用tBLASTn。在这875个NBS-LRR基因中,119个基因具有选择性剪接现象,其中包括71内含子保留,20个外显子跳跃,25个选择性起始,16个选择性终止,12个5′端的选择性剪接和16个3′端选择性剪接。大多数选择性剪接都为两个和多个转录本所支持。可以通过访问http://www.bioinfor.org查询这些数据。进而通过生物信息学分析剪接边界发现外显子跳跃和内含子保留的‘GT…AG’的规则不如组成型的保守。这暗示了它们是通过不同的调控机制来指导剪接变构体的形成。通过分析内含子保留对蛋白质的影响,发现选择性剪接的蛋白更倾向于改变其C端氨基酸序列。最后对选择性剪接的组织分布和蛋白质定位进行分析,结果表明选择性剪接的最大类的组织分布是根和愈伤组织。超过1/3剪接变构体的蛋白质定位是质膜和细胞质。这些选择性剪接蛋白可能在抗病信号转导中起到重要作用。     

DNA binding specificity of proteins derived from alternatively spliced mouse p53 mRNAs.

The mouse p53 gene generates two alternative splice products encoding p53 protein and a naturally occurring protein (p53as) with changes at the C-terminus. In p53as the negative regulatory region for DNA binding and PAb421 antibody binding site are replaced, and p53as is constitutively active for sequence-specific DNA binding. Using the technique of randomized synthetic oligonucleotide in cyclic amplification and selection of targets, we have found that p53as and p53 proteins have the same DNA binding specificities but that these specificities frequently diverge from the consensus of two copies of PuPuPuCATGPyPyPy. The importance of tetranucleotide CATG was confirmed but there was a less rigorous requirement for patterns of flanking or intervening sequences. In particular, the three purines upstream and three pyrimidines downstream of CATG are not required for p53 or p53as binding, 29 or more intervening nucleotides are tolerated, and one CATG is sufficient where adjacent nucleotides contain a region of homology with certain previously reported non-consensus p53 binding sequences. These results suggested further definition of the non-consensus motifs, and database searches with these uncovered additional candidate genes for p53 protein binding. We conclude that p53as and perhaps other activated forms of p53 exert their effects on the same genes and that differential activities of p53 protein forms are not due to inherently different sequence selectivities of DNA binding.     

ASAP: the Alternative Splicing Annotation Project

Recently, genomics analyses have demonstrated that alternative splicing is widespread in mammalian genomes (30-60% of genes reported to have multiple isoforms), and may be one of their most important mechanisms of functional regulation. However, by comparison with other genomics data such as genome annotation, SNPs, or gene expression, there exists relatively little database infrastructure for the study of alternative splicing. We have constructed an online database ASAP (the Alternative Splicing Annotation Project) for biologists to access and mine the enormous wealth of alternative splicing information coming from genomics and proteomics. ASAP is based on genome-wide analyses of alternative splicing in human (30 793 alternative splice relationships found) from detailed alignment of expressed sequences onto the genomic sequence. ASAP provides precise gene exon-intron structure, alternative splicing, tissue specificity of alternative splice forms, and protein isoform sequences resulting from alternative splicing. Moreover, it can help biologists design probe sequences for distinguishing specific mRNA isoforms. ASAP is intended to be a community resource for collaborative annotation of alternative splice forms, their regulation, and biological functions. The URL for ASAP is http://www.bioinformatics.ucla.edu/ASAP.     

Two splice variants of CaMKII-anchoring protein are present in the sarcoplasmic reticulum of rabbit fast-twitch muscle

Anchoring protein alphaKAP targets calmodulin kinase II (CaMKII) to the sarcoplasmic reticulum (SR), and in the rabbit is a substrate of CaMKII itself in fast-twitch, but not in slow-twitch muscle. This work was aimed at elucidating the molecular basis for differential phosphorylation of alphaKAP. Here we show that two, immunologically related, size forms (23 and 21 kDa) of alphaKAP are present in fast-twitch muscle SR in a 3:1 stoichiometry. Phosphorylation experiments identified the shorter form as the CaMKII specific substrate. Both forms are shown to be stably integrated into the holoenzyme. Two splice variants of alphaKAP were found in rabbit fast-twitch muscle and only one in slow-twitch muscle, using RT-PCR. Mobilities on SDS-PAGE are those expected. The shorter splice variants lacks the 33-nucleotide sequence inserted by alternative splicing present in full-length alphaKAP, akin to differences between variants A and B of brain alphaCaMKII. The absence of the 11-amino acid sequence creates a novel CaMKII phosphorylation site. Taken together our results show that alternative splicing regulates alphaKAP phosphorylation in a fiber-type specific manner.