Promoter choice determines splice site selection in protocadherin alpha and gamma pre-mRNA splicing

A family of mammalian protocadherin (Pcdh) proteins is encoded by three closely linked gene clusters (alpha, beta, and gamma). Multiple alpha and gamma Pcdh mRNAs are expressed in distinct patterns in the nervous system and are generated by alternative pre-mRNA splicing between different "variable" exons and three "constant" exons within each cluster. We show that each Pcdh variable exon is preceded by a promoter and that promoter choice determines which variable exon is included in a Pcdh mRNA. In addition, we provide evidence that alternative splicing of variable exons within a gene cluster occurs via a cis-splicing mechanism. However, virtually every variable exon can engage in trans-splicing with constant exons from another cluster, albeit at a far lower level.     

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.     

GLUT14, a duplicon of GLUT3, is specifically expressed in testis as alternative splice forms

We have identified and cloned GLUT14, a novel member of the glucose transporter family. GLUT14 (SLC2A14) maps to chromosome 12p13.3 (17.1M), about 10 Mb upstream of GLUT3, with which it shares remarkable identity. Until now GLUT14 was thought to be a pseudogene. It consists of 11 exons with a genomic organization similar to that of GLUT3 and likely resulted from a duplication of GLUT3. GLUT14 has two alternatively spliced forms; the shorter form of GLUT14 (GLUT14-S) consists of 10 exons and produces a 497-amino-acid protein that is 94.5% identical to GLUT3. The long form (GLUT14-L) has an additional exon and codes for a protein with 520 amino acids that differs from GLUT14-S only at the N-terminus. GLUT14-S/L contain 12 putative membrane-spanning helices along with sugar-transporter signature motifs that have previously been shown to be essential for sugar transport activity. The putative glycosylation sites of GLUT14-S/L are present in loop 1. In contrast to the expression of GLUT3 in many tissues, both isoforms of GLUT14 are specifically expressed in testis. The mRNA level of GLUT14 in testis is about four times higher than that of GLUT3. Interestingly, the ortholog of GLUT14 is not found in mice. The multiple duplications of GLUT genes suggest that the GLUT family probably emerged by gene duplications and mutations during evolution in different lineages.     

Discovery of novel splice forms and functional analysis of cancer-specific alternative splicing in human expressed sequences

We report here a genome-wide analysis of alternative splicing in 2 million human expressed sequence tags (ESTs), to identify splice forms that are up-regulated in tumors relative to normal tissues. We found strong evidence (P < 0.01) of cancer-specific splice variants in 316 human genes. In total, 78% of the cancer-specific splice forms we detected are confirmed by human-curated mRNA sequences, indicating that our results are not due to random mis-splicing in tumors; 73% of the genes showed the same cancer-specific splicing changes in tissue-matched tumor versus normal datasets, indicating that the vast majority of these changes are associated with tumorigenesis, not tissue specificity. We have confirmed our EST results in an independent set of experimental data provided by human-curated mRNAs (P-value 10^–5.7). Moreover, the majority of the genes we detected have functions associated with cancer (P-value 0.0007), suggesting that their altered splicing may play a functional role in cancer. Analysis of the types of cancer-specific splicing shifts suggests that many of these shifts act by disrupting a tumor suppressor function. Sur prisingly, our data show that for a large number (190 in this study) of cancer-associated genes cloned originally from tumors, there exists a previously uncharacterized splice form of the gene that appears to be predominant in normal tissue.     

Amount of RNA secondary structure required to induce an alternative splice.

We set up an alternative splicing system in vitro in which the relative amounts of two spliced RNAs, one containing and the other lacking a particular exon, were directly proportional to the length of an inverted repeat inserted into the flanking introns. We then used the system to measure the effect of intramolecular complementarity on alternative splicing in vivo. We found that an alternative splice was induced in vivo only when the introns contained more than approximately 50 nucleotides of perfect complementarity, that is, only when the secondary structure was much more stable than most if not all possible secondary structures in natural mRNA precursors. We showed further that intron insertions containing long complements to splice sites and a branch point inhibited splicing in vitro but not in vivo. These results raise the possibility that in cells most pre-mRNA secondary structures either are not maintained long enough to influence splicing choices, or never form at all.     

A database on alternative splice forms on the integrated genetic map service (IGMS)

The IGMS is a comprehensive information system that combines the knowledge from genomic sequence, genetic map and genetic disorders databases. This system is updated weekly and focuses on the analysis of EST data. The IGMS identifies UniGene clusters that are differentially expressed in different types of cancer with respect to different reference tissues. The results can be combined with clinical data to asses the potential relevance of specific genes for patient survival or metastatic spread. The second application maps EST with a specific expression profile. Our third application generates a database of alternative splice forms for nine organisms from EST and mRNA sequence data. The results can be used to find splicing patterns specific for certain tissues or tumour types. Availability: http://www.bioinf.mdc-berlin.de/igms/.     

Characterization and prediction of alternative splice sites

Human alternative isoform, cryptic, skipped, and constitutive splice sites from the ALTEXTRON database were analysed regarding splice site strength, composition, GC content, position and binding site strength of polypyrimidine tract and branch site. Several features were identified which distinguish alternative isoform and cryptic splice sites, but not skipped splice sites from constitutive ones. These include splice site strength, introns GC content, U2AF35 binding site score, and oligonucleotide frequencies. For the predictive classification of splice sites, pattern recognition models for different splicing factor binding sites and oligonucleotide frequency models (OFMs) were combined using backpropagation networks. 67.45% of acceptor sites and 71.23% of donor sites are correctly classified by networks trained for classification of constitutive and alternative isoform/cryptic splice sites. A web-application for the prediction of alternative splice sites is available at http://es.embnet.org/~mwang/assp.html .     

Coevolution of genomic intron number and splice sites

Spliceosomal intron numbers and boundary sequences vary dramatically in eukaryotes. We found a striking correspondence between low intron number and strong sequence conservation of 5' splice sites (5'ss) across eukaryotic genomes. The phylogenetic pattern suggests that ancestral 5'ss were relatively weakly conserved, but that some lineages independently underwent both major intron loss and 5'ss strengthening. It seems that eukaryotic ancestors had relatively large intron numbers and 'weak' 5'ss, a pattern associated with frequent alternative splicing in modern organisms.     

Chromosomal assignment and genomic structure of Il15

Interleukin-15 (IL-15) is a novel cytokine whose effects on T-cell activation and proliferation are similar to those of interleukin-2 (IL-2), presumably because IL-15 utilizes the β and γ chains of the IL-2 receptor. Murine IL-15 cDNA and genomic clones were isolated and characterized. The murine Il15 gene was found to consist of eight exons spanning at least 34 kb and was localized to the central region of mouse chromosome 8 by interspecific backcross analysis. Intron positions in a partial human IL15 genomic clone were identical with positions of corresponding introns in the murine gene. The human IL15 gene was mapped to human chromosome 4q31 by fluorescence in situ hybridization.