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Analysis of mammalian native elongating transcript sequencing (mNET-seq) high-throughput data
Publication . Prudêncio, Pedro; Rebelo, Kenny; Grosso, Ana Rita; Goncalo Martinho, Rui; Carmo-Fonseca, Maria
Mammalian Native Elongating Transcript sequencing (mNET-seq) is a recently developed technique that generates genome-wide profiles of nascent transcripts associated with RNA polymerase II (Pol II) elongation complexes. The ternary transcription complexes formed by Pol II, DNA template and nascent RNA are first isolated, without crosslinking, by immunoprecipitation with antibodies that specifically recognize the different phosphorylation states of the polymerase large subunit C-terminal domain (CTD). The coordinate of the 3' end of the RNA in the complexes is then identified by high-throughput sequencing. The main advantage of mNET-seq is that it provides global, bidirectional maps of Pol II CTD phosphorylation-specific nascent transcripts and coupled RNA processing at single nucleotide resolution. Here we describe the general pipeline to prepare and analyse high-throughput data from mNET-seq experiments.
The emerging role of transcriptional regulation in the oocyte-to-zygote transition
Publication . Navarro-Costa, Paulo; Martinho, Rui Goncalo
Fertilization marks the beginning of a new life by converting two terminally differentiated gametes into a single totipotent zygote. Central to this transition is a complex biological program commonly referred to as oocyte activation—an umbrella term for a series of profound changes that prepare the fertilized oocyte for totipotency. These include, among others, the completion of meiosis, the formation of the two pronuclei, and the selective translation of maternal RNAs. A remarkable aspect of oocyte activation is that it occurs in the absence of transcription. Not surprisingly, most of our knowledge of this process is centered on the posttranscriptional regulation of gene expression. Yet, a recent body of evidence has brought new focus on the fundamental importance of transcriptional regulation during oogenesis as a primer for the oocyte-to-zygote transition.
NineTeen Complex-subunit Salsa is required for efficient splicing of a subset of introns and dorsal-ventral patterning
Publication . Rathore, Om Singh; Silva, Rui D; Ascensão-Ferreira, Mariana; Matos, Ricardo; Carvalho, Célia; Marques, Bruno; Tiago, Margarida N.; Prudêncio, Pedro; Andrade, Raquel P.; Roignant, Jean-Yves; Barbosa-Morais, Nuno; Martinho, Rui Goncalo
The NineTeen Complex (NTC), also known as pre-mRNA-processing factor 19 (Prp19) complex, regulates distinct spliceosome conformational changes necessary for splicing. During Drosophila midblastula transition, splicing is particularly sensitive to mutations in NTC-subunit Fandango, which suggests differential requirements of NTC during development. We show that NTC-subunit Salsa, the Drosophila ortholog of human RNA helicase Aquarius, is rate-limiting for splicing of a subset of small first introns during oogenesis, including the first intron of gurken Germline depletion of Salsa and splice site mutations within gurken first intron impair both adult female fertility and oocyte dorsal-ventral patterning, due to an abnormal expression of Gurken. Supporting causality, the fertility and dorsal-ventral patterning defects observed after Salsa depletion could be suppressed by the expression of a gurken construct without its first intron. Altogether, our results suggest that one of the key rate-limiting functions of Salsa during oogenesis is to ensure the correct expression and efficient splicing of the first intron of gurken mRNA. Retention of gurken first intron compromises the function of this gene most likely because it undermines the correct structure and function of the transcript 5'UTR.
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Fundação para a Ciência e a Tecnologia
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3599-PPCDT
Número da atribuição
PTDC/BEX-BID/0395/2014