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.     

Global regulation of alternative splicing during myogenic differentiation

Recent genome-wide analyses have elucidated the extent of alternative splicing (AS) in mammals, often focusing on comparisons of splice isoforms between differentiated tissues. However, regulated splicing changes are likely to be important in biological transitions such as cellular differentiation, or response to environmental stimuli. To assess the extent and significance of AS in myogenesis, we used splicing-sensitive microarray analysis of differentiating C2C12 myoblasts. We identified 95 AS events that undergo robust splicing transitions during C2C12 differentiation. More than half of the splicing transitions are conserved during differentiation of avian myoblasts, suggesting the products and timing of transitions are functionally significant. The majority of splicing transitions during C2C12 differentiation fall into four temporal patterns and were dependent on the myogenic program, suggesting that they are integral components of myogenic differentiation. Computational analyses revealed enrichment of many sequence motifs within the upstream and downstream intronic regions near the alternatively spliced regions corresponding to binding sites of splicing regulators. Western analyses demonstrated that several splicing regulators undergo dynamic changes in nuclear abundance during differentiation. These findings show that within a developmental context, AS is a highly regulated and conserved process, suggesting a major role for AS regulation in myogenic differentiation.     

Detection and measurement of alternative splicing using splicing-sensitive microarrays

Splicing and alternative splicing are major processes in the interpretation and expression of genetic information for metazoan organisms. The study of splicing is moving from focused attention on the regulatory mechanisms of a selected set of paradigmatic alternative splicing events to questions of global integration of splicing regulation with genome and cell function. For this reason, parallel methods for detecting and measuring alternative splicing are necessary. We have adapted the splicing-sensitive oligonucleotide microarrays used to estimate splicing efficiency in yeast to the study of alternative splicing in vertebrate cells and tissues. We use gene models incorporating knowledge about splicing to design oligonucleotides specific for discriminating alternatively spliced mRNAs from each other. Here we present the main strategies for design, application, and analysis of spotted oligonucleotide arrays for detection and measurement of alternative splicing. We demonstrate these strategies using a two-intron yeast gene that has been altered to produce different amounts of alternatively spliced RNAs, as well as by profiling alternative splicing in NCI 60 cancer cell lines.     

Human immunodeficiency virus type 1 hnRNP A/B-dependent exonic splicing silencer ESSV antagonizes binding of U2AF65 to viral polypyrimidine tracts

Human immunodeficiency virus type 1 (HIV-1) exonic splicing silencers (ESSs) inhibit production of certain spliced viral RNAs by repressing alternative splicing of the viral precursor RNA. Several HIV-1 ESSs interfere with spliceosome assembly by binding cellular hnRNP A/B proteins. Here, we have further characterized the mechanism of splicing repression using a representative HIV-1 hnRNP A/B-dependent ESS, ESSV, which regulates splicing at the vpr 3' splice site. We show that hnRNP A/B proteins bound to ESSV are necessary to inhibit E complex assembly by competing with the binding of U2AF65 to the polypyrimidine tracts of repressed 3' splice sites. We further show evidence suggesting that U1 snRNP binds the 5' splice site despite an almost complete block of splicing by ESSV. Possible splicing-independent functions of U1 snRNP-5' splice site interactions during virus replication are discussed.     

Multiple variants and a differential splicing pattern of kinectin in human hepatocellular carcinoma.

To extend the search for hepatocellular carcinoma (HCC) associated antigens with immunogenicity for clinical applications, we constructed a cDNA expression library using resected human HCC tissue sample and screened it by serological analysis of recombinant cDNA expression library (SEREX) with autologous and allogeneic sera. A total of 24 distinct antigens were isolated and kinectin was the antigen most frequently identified. We found that kinectin was alternatively spliced at four sites and obtained all eight theoretical forms of variant, six by SEREX and two by RT-PCR, from the different splicing combinations of the last three sites. In addition, the splicing patterns of four sites were analyzed. Variant containing D2 was overexpressed in cancerous tissues and this alteration may be tumor associated. The four splicing sites, the variants generated by alternative splicing, and the humoral immune response in HCC patients, may help to analyze the role of kinectin in human HCC cell biology.     

Evolution of Nova-dependent splicing regulation in the brain

A large number of alternative exons are spliced with tissue-specific patterns, but little is known about how such patterns have evolved. Here, we study the conservation of the neuron-specific splicing factors Nova1 and Nova2 and of the alternatively spliced exons they regulate in mouse brain. Whereas Nova RNA binding domains are 94% identical across vertebrate species, Nova-dependent splicing silencer and enhancer elements (YCAY clusters) show much greater divergence, as less than 50% of mouse YCAY clusters are conserved at orthologous positions in the zebrafish genome. To study the relation between the evolution of tissue-specific splicing and YCAY clusters, we compared the brain-specific splicing of Nova-regulated exons in zebrafish, chicken, and mouse. The presence of YCAY clusters in lower vertebrates invariably predicted conservation of brain-specific splicing across species, whereas their absence in lower vertebrates correlated with a loss of alternative splicing. We hypothesize that evolution of Nova-regulated splicing in higher vertebrates proceeds mainly through changes in cis-acting elements, that tissue-specific splicing might in some cases evolve in a single step corresponding to evolution of a YCAY cluster, and that the conservation level of YCAY clusters relates to the functions encoded by the regulated RNAs.     

Genomic organization, physical mapping, and expression analysis of the human protein arginine methyltransferase 1 gene

Protein arginine methyltransferases (PRMTs) regulate mRNA processing and maturation by modulating the activity of RNA-binding proteins through methylation. The cDNA for human PRMT1 (HRMT1L2) was recently identified. In this paper, we describe the complete genomic organization of the human PRMT1 gene (GenBank Accession No. AF222689), together with its precise chromosomal localization in relation to other neighboring genes. We have also examined its expression in a total RNA panel of 26 human tissues, the BT-474 breast carcinoma cell line, and 16 breast tumors. PRMT1, which spans 11.2 kb of genomic sequence on chromosome 19q13.3, is located in close proximity to the IRF3 and RRAS genes and is transcribed in the opposite direction. It is formed of 12 coding exons and 11 intervening introns, and shows structural similarity to other PRMT genes. Three PRMT1 isoforms exist as a result of alternative mRNA splicing. Amino acid sequence comparison of the splicing variants indicates that they are all enzymatically active methyl transferases, but with different N-terminal hydrophobic regions. PRMT1 expression was detected in a variety of tissues. We have shown that the relative prevalence of alternatively spliced forms of PRMT1 is different between normal and cancerous breast tissues. Although PRMT1 was not found to be hormonally regulated by steroid hormones in breast cancer cells, our results suggest that two variants of PRMT1 are down regulated in breast cancer.     

Alternative splicing is associated with decreased expression of the redox proto-oncogene thioredoxin-1 in human cancers

Human thioredoxin-1 (Trx-1) is a small redox protein that is overexpressed in a number of human primary tumors, where it is associated with rapid cell proliferation and inhibited apoptosis. Mutation scanning denaturing high-performance liquid chromatography of Trx-1 mRNA in 58 human tumor cell lines found no evidence for changes in the base sequence of human Trx-1 mRNA. An alternatively spliced form of Trx-1 mRNA lacking exons 2 and 3 was found in 7 of the cell lines but it was not translated. The cell lines having the alternatively spliced Trx-1 mRNA had 73% lower total Trx-1 mRNA than the other cell lines, suggesting that alternative splicing may control the level of Trx-1 mRNA in some cancer cells.     

Alternative splicing,gene duplication and connectivity in the genetic interaction network of the nematode worm <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

We examined the relationship between gene duplication, alternative splicing, and connectedness in a predicted genetic interaction network using published data from the nematode worm Caenorhabditis elegans. Similar to previous results from mammals, genes belonging to families with only one member ("singletons") were significantly more likely to lack alternative splicing than were members of large multi-gene families. Genes belonging to multi-gene families lacking alternative splicing tended to have higher connectedness in the genetic interaction network than did genes in families that included one or more alternatively spliced members. Moreover, alternatively spliced genes were significantly more likely to interact with other alternatively spliced genes. These results support the hypothesis that certain key proteins with high degrees of network connectedness are subject to selection opposing the occurrence of alternatively spliced forms.