A network of conserved co-occurring motifs for the regulation of alternative splicing.

Cis-acting short sequence motifs play important roles in alternative splicing. It is now possible to identify such sequence motifs as conserved sequence patterns in genome sequence alignments. Here, we report the systematic search for motifs in the neighboring introns of alternatively spliced exons by using comparative analysis of mammalian genome alignments. We identified 11 conserved sequence motifs that might be involved in the regulation of alternative splicing. These motifs are not only significantly overrepresented near alternatively spliced exons, but they also co-occur with each other, thus, forming a network of cis-elements, likely to be the basis for context-dependent regulation. Based on this finding, we applied the motif co-occurrence to predict alternatively skipped exons. We verified exon skipping in 29 cases out of 118 predictions (25%) by EST and mRNA sequences in the databases. For the predictions not verified by the database sequences, we confirmed exon skipping in 10 additional cases by using both RT-PCR experiments and the publicly available RNA-Seq data. These results indicate that even more alternative splicing events will be found with the progress of large-scale and high-throughput analyses for various tissue samples and developmental stages.

Methylation and silencing of the retinoic acid receptor-beta 2 gene in cervical cancer.

BACKGROUND: Expression of the retinoic acid receptor beta2 (RAR-beta2), a putative tumor suppressor gene, is reduced in various human cancers, including squamous cell carcinomas (SCC) of the uterine cervix. The mechanism of the inhibition of RAR-beta2 expression remains obscure. We examined whether methylation of RAR-beta2 gene could be responsible for this silencing in cervical SCC. METHODS: Expression of RAR-beta2 mRNA and methylation status of the 5' region of RAR-beta2 gene were examined in 20 matched specimens from patients with cervical SCC and in three cervical cancer cell lines by Northern blot analysis and methylation-specific PCR (MSP) assay or Southern blot analysis respectively. RESULTS: In 8 out 20 cervical SCC (40%) the levels of RAR-beta2 mRNA were decreased or undetectable in comparison with non-neoplastic cervix tissues. All 8 tumors with reduced levels of RAR-beta2 mRNA expression showed methylation of the promoter and the first exon expressed in the RAR-beta2 transcript. The RAR-beta2 gene from non-neoplastic cervical tissues was mostly unmethylated and expressed, but methylated alleles of the gene were found in three samples of the morphologically normal tissues adjacent to the tumors. Three cervical cancer cell lines with extremely low level of RAR-beta2 mRNA expression, SiHA, HeLA and CaSki, also showed methylation of this region of the RAR-beta2 gene. CONCLUSIONS: These findings suggest that methylation of the 5' region of RAR-beta2 gene may contribute to gene silencing and that methylation of this region may be an important and early event in cervical carcinogenesis. These findings may be useful to make retinoids more effective as preventive and therapeutic agents in combination with inhibitors of DNA methylation.

Human papilloma viruses and cervical tumours: mapping of integration sites and analysis of adjacent cellular sequences.

BACKGROUND: In cervical tumours the integration of human papilloma viruses (HPV) transcripts often results in the generation of transcripts that consist of hybrids of viral and cellular sequences. Mapping data using a variety of techniques has demonstrated that HPV integration occurred without obvious specificity into human genome. However, these techniques could not demonstrate whether integration resulted in the generation of transcripts encoding viral or viral-cellular sequences. The aim of this work was to map the integration sites of HPV DNA and to analyse the adjacent cellular sequences. METHODS: Amplification of the INTs was done by the APOT technique. The APOT products were sequenced according to standard protocols. The analysis of the sequences was performed using BLASTN program and public databases. To localise the INTs PCR-based screening of GeneBridge4-RH-panel was used. RESULTS: Twelve cellular sequences adjacent to integrated HPV16 (INT markers) expressed in squamous cell cervical carcinomas were isolated. For 11 INT markers homologous human genomic sequences were readily identified and 9 of these showed significant homologies to known genes/ESTs. Using the known locations of homologous cDNAs and the RH-mapping techniques, mapping studies showed that the INTs are distributed among different human chromosomes for each tumour sample and are located in regions with the high levels of expression. CONCLUSIONS: Integration of HPV genomes occurs into the different human chromosomes but into regions that contain highly transcribed genes. One interpretation of these studies is that integration of HPV occurs into decondensed regions, which are more accessible for integration of foreign DNA.