Browsing by Author "Carmo-Fonseca, Maria"
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- Dynamic transitions in RNA polymerase II density profiles during transcription terminationPublication . Grosso, Ana Rita; Almeida, Sérgio Fernandes de; Braga, José; Carmo-Fonseca, MariaEukaryotic protein-coding genes are transcribed by RNA polymerase II (RNAPII) through a cycle composed of three main phases: initiation, elongation, and termination. Recent studies using chromatin immunoprecipitation coupled to high-throughput sequencing suggest that the density of RNAPII molecules is higher at the 3′-end relative to the gene body. Here we show that this view is biased due to averaging density profiles for "metagene" analysis. Indeed, the majority of genes exhibit little, if any, detectable accumulation of polymerases during transcription termination. Compared with genes with no enrichment, genes that accumulate RNAPII at the 3′-end are shorter, more frequently contain the canonical polyadenylation [poly(A)] signal AATAAA and G-rich motifs in the downstream sequence element, and have higher levels of expression. In 1% to 4% of actively transcribing genes, the RNAPII enriched at the 3′-end is phosphorylated on Ser5, and we provide evidence suggesting that these genes have their promoter and terminator regions juxtaposed. We also found a striking correlation between RNAPII accumulation and nucleosome organization, suggesting that the presence of nucleosomes after the poly(A) site induces pausing of polymerases, leading to their accumulation. Yet we further observe that nucleosome occupancy at the 3′-end of genes is dynamic and correlates with RNAPII density. Taken together, our results provide novel insight to transcription termination, a fundamental process that remains one of the least understood stages of the transcription cycle.
- Unconstrained mining of transcript data reveals increased alternative splicing complexity in the human transcriptomePublication . Mollet, I. G.; Ben-Dov, Claudia; Felício-Silva, Daniel; Grosso, A. R.; Eleutério, Pedro; Alves, Ruben; Staller, Ray; Silva, Tito Santos; Carmo-Fonseca, MariaMining massive amounts of transcript data for alternative splicing information is paramount to help understand how the maturation of RNA regulates gene expression. We developed an algorithm to cluster transcript data to annotated genes to detect unannotated splice variants. A higher number of alternatively spliced genes and isoforms were found compared to other alternative splicing databases. Comparison of human and mouse data revealed a marked increase, in human, of splice variants incorporating novel exons and retained introns. Previously unannotated exons were validated by tiling array expression data and shown to correspond preferentially to novel first exons. Retained introns were validated by tiling array and deep sequencing data. The majority of retained introns were shorter than 500 nt and had weak polypyrimidine tracts. A subset of retained introns matching small RNAs and displaying a high GC content suggests a possible coordination between splicing regulation and production of noncoding RNAs. Conservation of unannotated exons and retained introns was higher in horse, dog and cow than in rodents, and 64% of exon sequences were only found in primates. This analysis highlights previously bypassed alternative splice variants, which may be crucial to deciphering more complex pathways of gene regulation in human.