Alternative splicing in colon, bladder, and prostate cancer identified by exon array analysis

Alternative splicing enhances proteome diversity and modulates cancer-associated proteins. To identify tissue- and tumor-specific alternative splicing, we used the GeneChip Human Exon 1.0 ST Array to measure whole-genome exon expression in 102 normal and cancer tissue samples of different stages from colon, urinary bladder, and prostate. We identified 2069 candidate alternative splicing events between normal tissue samples from colon, bladder, and prostate and selected 15 splicing events for RT-PCR validation, 10 of which were successfully validated by RT-PCR and sequencing. Furthermore 23, 19, and 18 candidate tumor-specific splicing alterations in colon, bladder, and prostate, respectively, were selected for RT-PCR validation on an independent set of 81 normal and tumor tissue samples. In total, seven genes with tumor-specific splice variants were identified (ACTN1, CALD1, COL6A3, LRRFIP2, PIK4CB, TPM1, and VCL). The validated tumor-specific splicing alterations were highly consistent, enabling clear separation of normal and cancer samples and in some cases even of different tumor stages. A subset of the tumor-specific splicing alterations (ACTN1, CALD1, and VCL) was found in all three organs and may represent general cancer-related splicing events. In silico protein predictions suggest that the identified cancer-specific splice variants encode proteins with potentially altered functions, indicating that they may be involved in pathogenesis and hence represent novel therapeutic targets. In conclusion, we identified and validated alternative splicing between normal tissue samples from colon, bladder, and prostate in addition to cancer-specific splicing events in colon, bladder, and prostate cancer that may have diagnostic and prognostic implications.     

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.     

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。     

Precise and parallel characterization of coding polymorphisms, alternative splicing, and modifications in human proteins by mass spectrometry

The human proteome is a highly complex extension of the genome wherein a single gene often produces distinct protein forms due to alternative splicing, RNA editing, polymorphisms, and posttranslational modifications. Such biological variation compounded by the high sequence identity within gene families currently overwhelms the complete and routine characterization of mammalian proteins by MS. A new data base of human proteins (and their possible variants) was created and searched using tandem mass spectrometric data from intact proteins. This first application of top down MS/MS to wild-type human proteins demonstrates both gene-specific identification and the unambiguous characterization of multifaceted mass shifts (Deltam values). Such Deltam values found from the precise identification of 45 protein forms from HeLa cells reveal 34 coding single nucleotide polymorphisms, two protein forms from alternative splicing, and 12 diverse modifications (not including simple N-terminal processing), including a previously unknown phosphorylation at 10% occupancy. Automated protein identification was achieved with a median expectation value of 10(-13) and often occurred simultaneously with dissection of diverse sources of protein variability as they occur in combination. Top down MS therefore has a bright future for enabling precise annotation of gene products expressed from the human genome by non-mass spectrometrists.     

CD72几种新的剪切形式的发现及它们在红斑狼疮模型鼠中的差异表达

CD72是一个重要的B细胞特异性受体,它以多种选择性剪切形式存在.在小鼠脾细胞中发现并鉴定了8种新的CD72选择性剪切形式,这些剪切形式中包含有2种独特的插入片段,一种选择性剪切保留了一个内含子(intron1),而这个内含子被翻译成氨基酸序列后并没有改变前后外显子的读码框,另一种使用了一个位于内含子之内的3′剪切位点,从而产生移码,提前终止了蛋白质的开放读码框,称为3′AS(3′alternative splicingsite).比较了CD72所有剪切形式在BALB/C小鼠和NZB/W小鼠中的差异表达,发现:a.含有3′AS的剪切形式的表达都很少;b.WT, In1, In1-Ex3和-Ex3的表达在BLAB/C小鼠中比在NZB/W小鼠中高;c.没有ITIM2的-Ex2-Ex3剪切形式在NZB/W小鼠中有特异性高表达.这些结果提示,CD72的多种选择性剪切形式在调控B细胞受体信号转导过程中可能发挥着不同的作用,并与系统性红斑狼疮的发病密切相关.     

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.     

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.     

Osteogenic role of endosomal chloride channels in MC3T3-E1 cells

PHR protein family consists of C. elegan Rpm-1/Drosophila Highwire/Zebrafish Esrom/Mouse Phr-1/Human Pam. Esrom is required for correct neurites exiting the paused state at intermediate targets as well as pteridine synthesis. This study reports the identification and characterization of two novel Esrom splice variants, named splice variants 2 (splicing out 5′ 24 bp of exon 17) and 3 (splicing out 5′ 24 bp of exons 17 and 18). Polypeptides encoded by 5′ 24 bp of exons 17 and 18 are part of basic amino-acid-rich region inside Esrom RCC1-like domain (RLD). These two splice variants maintain the whole protein reading frame and alternative exons usage patterns are conserved with mammal. At different developmental stages and adult zebrafish tissues, abundances of these splice variants are different. Importantly, by yeast two-hybrid screen and confocal colocalization analysis, it was found that alternative splicing of exon 18 regulates Esrom RLD interaction with kinesin family member 22 and G protein beta-subunit 1. Taken together, these results suggest that Esrom RLD functions are regulated by alternative splicing at temporal and spatial-specific manner.