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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.
Polycomb group (PcG) proteins prevent the assembly of abnormal synaptonemal complex structures during meiosis
Publication . Feijão, Tália; Marques, Bruno; Silva, Rui D.; Carvalho, Célia; Sobral, Daniel; Matos, Ricardo; Tan, Tian; Pereira, António; Morais de Sá, Eurico; Maiato, Hélder; DeLuca, Steven Z.; Martinho, Rui Goncalo
The synaptonemal complex (SC) is a proteinaceous scaffold that is assembled between paired homologous chromosomes during the onset of meiosis. Timely expression of SC coding genes is essential for SC assembly and successful meiosis. However, SC components have an intrinsic tendency to self-organize into abnormal repetitive structures, which are not assembled between the paired homologs and whose formation is potentially deleterious for meiosis and gametogenesis. This creates an interesting conundrum, where SC genes need to be robustly expressed during meiosis, but their expression must be carefully regulated to prevent the formation of anomalous SC structures. In this manuscript, we show that the Polycomb group protein Sfmbt, the Drosophila ortholog of human MBTD1 and L3MBTL2, is required to avoid excessive expression of SC genes during prophase I. Although SC assembly is normal after Sfmbt depletion, SC disassembly is abnormal with the formation of multiple synaptonemal complexes (polycomplexes) within the oocyte. Overexpression of the SC gene corona and depletion of other Polycomb group proteins are similarly associated with polycomplex formation during SC disassembly. These polycomplexes are highly dynamic and have a well-defined periodic structure. Further confirming the importance of Sfmbt, germ line depletion of this protein is associated with significant metaphase I defects and a reduction in female fertility. Since transcription of SC genes mostly occurs during early prophase I, our results suggest a role of Sfmbt and other Polycomb group proteins in downregulating the expression of these and other early prophase I genes during later stages of meiosis.
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.
Transcription and splicing dynamics during early Drosophila development
Publication . Prudêncio, Pedro; Savisaar, Rosina; Rebelo, Kenny; Martinho, Rui Goncalo; Carmo-Fonseca, Maria
Widespread cotranscriptional splicing has been demonstrated from yeast to human. However, most studies to date addressing the kinetics of splicing relative to transcription used either Saccharomyces cerevisiae or metazoan cultured cell lines. Here, we adapted native elongating transcript sequencing technology (NET-seq) to measure cotranscriptional splicing dynamics during the early developmental stages of Drosophila melanogaster embryos. Our results reveal the position of RNA polymerase II (Pol II) when both canonical and recursive splicing occur. We found heterogeneity in splicing dynamics, with some RNAs spliced immediately after intron transcription, whereas for other transcripts no splicing was observed over the first 100 nt of the downstream exon. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. We studied the splicing dynamics of both nascent pre-mRNAs transcribed in the early embryo, which have few and short introns, as well as pre-mRNAs transcribed later in embryonic development, which contain multiple long introns. As expected, we found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. We further observed that genes transcribed in the early embryo tend to be isolated in the genome whereas genes transcribed later are often overlapped by a neighboring convergent gene. In isolated genes, transcription termination occurred soon after the polyadenylation site, while in overlapped genes, Pol II persisted associated with the DNA template after cleavage and polyadenylation of the nascent transcript. Taken together, our data unravel novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo.
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Fundação para a Ciência e a Tecnologia
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9471 - RIDTI
Número da atribuição
PTDC/BIA-BID/28441/2017