Loading...
5 results
Search Results
Now showing 1 - 5 of 5
- NineTeen Complex-subunit Salsa is required for efficient splicing of a subset of introns and dorsal-ventral patterningPublication . Rathore, Om; Silva, Rui D.; Ascensao-Ferreira, Mariana; Matos, Ricardo; Carvalho, Celia; Marques, Bruno; Tiago, Margarida N.; Prudencio, Pedro; Andrade, Raquel P.; Roignant, Jean-Yves; Barbosa-Morais, Nuno; Martinho, Rui GoncaloThe 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.
- Absence of N-terminal acetyltransferase diversification during evolution of eukaryotic organismsPublication . Rathore, Om; Faustino, Alexandra; Prudencio, Pedro; Van Damme, Petra; Cox, C. J.; Martinho, Rui GoncaloProtein N-terminal acetylation is an ancient and ubiquitous co-translational modification catalyzed by a highly conserved family of N-terminal acetyltransferases (NATs). Prokaryotes have at least 3 NATs, whereas humans have six distinct but highly conserved NATs, suggesting an increase in regulatory complexity of this modification during eukaryotic evolution. Despite this, and against our initial expectations, we determined that NAT diversification did not occur in the eukaryotes, as all six major human NATs were most likely present in the Last Eukaryotic Common Ancestor (LECA). Furthermore, we also observed that some NATs were actually secondarily lost during evolution of major eukaryotic lineages; therefore, the increased complexity of the higher eukaryotic proteome occurred without a concomitant diversification of NAT complexes.
- Regulation of pre-mRNA splicing during Drosophila developmentPublication . Rathore, Om; Martinho, Rui GonçaloThe spliceosome is a very dynamic molecular machine, whose compositional and conformational remodeling is crucial for pre-mRNA splicing. Although splicing is biochemically simple, with essentially two nucleophilic attacks, the spliceosome is nevertheless remarkably complex, as it needs to be in one hand extremely precise in splice-site recognition, but in the other hand must accommodate an array of alternative splicing events capable of generating transcript diversity. Multiple studies in yeast and human cells have shown that loss of distinct subunits of the spliceosome impaired splicing of distinct subsets of introns (Dix et al, 1999; Lygerou et al, 1999; Larson et al, 2016), suggesting a significant degree of splicing plasticity among different classes of introns. To better understand the contribution of such splicing plasticity to differential gene expression during development of multicellular organisms, we performed a screen to identify spliceosome subunits whose depletion by RNA interference (RNAi) was associated to specific phenotypes during oogenesis and/or early embryonic development. Our working hypothesis is that those subunits would be particularly rate limiting for splicing of small subsets of introns. We identified Salsa, a subunit of the spliceosome Nine Teen Complex (NTC), as being required for dorsal ventral (D/V) patterning of the Drosophila egg. Germ-line specific depletion of Salsa during oogenesis produced a highly penetrant ventralization phenotype (spindle phenotype) and dramatically reduced fertility. Since Gurken expression (a TGFα family signaling ligand) is crucial for D/V patterning of the developing oocyte, we decided to investigate if Salsa was rate limiting for Gurken expression. We observed that splicing of the first intron of gurken transcript was particularly sensitive to Salsa depletion, whereas anterior dorsal localization of gurken mRNA, and consequently Gurken protein, was highly abnormal. Since it was previously suggested that the 5´UTR of gurken is important for its anterior dorsal localization (Saunders & Cohen, 1999a), our current working model is that retention of the proximal intron, within the 5´UTR of gurken transcript, disturbs the folding of an unknown RNA motif important for its correct localization. Interestingly, analysis of the Drosophila ovaries transcriptome after depletion of Salsa further confirmed that this RNA helicase is particularly rate limiting for splicing of small proximal introns; including for example the proximal 5´UTRlocalized intron of tra2, a gene whose alternative splicing is crucial for sexdetermination.
- Naa50/San-dependent N-terminal acetylation of Scc1 is potentially important for sister chromatid cohesionPublication . Ribeiro, Ana Luisa; Silva, Rui D.; Foyn, Havard; Tiago, Margarida N.; Rathore, Om; Arnesen, Thomas; Martinho, Rui GoncaloThe gene separation anxiety (san) encodes Naa50/San, a N-terminal acetyltransferase required for chromosome segregation during mitosis. Although highly conserved among higher eukaryotes, the mitotic function of this enzyme is still poorly understood. Naa50/San was originally proposed to be required for centromeric sister chromatid cohesion in Drosophila and human cells, yet, more recently, it was also suggested to be a negative regulator of microtubule polymerization through internal acetylation of beta Tubulin. We used genetic and biochemical approaches to clarify the function of Naa50/San during development. Our work suggests that Naa50/San is required during tissue proliferation for the correct interaction between the cohesin subunits Scc1 and Smc3. Our results also suggest a working model where Naa50/San N-terminally acetylates the nascent Scc1 polypeptide, and that this co-translational modification is subsequently required for the establishment and/or maintenance of sister chromatid cohesion.
- Absence of the spindle assembly checkpoint restores mitotic fidelity upon loss of sister chromatid cohesionPublication . Silva, Rui; Mirkovic, Mihailo; Guilgur, Leonardo G.; Rathore, Om; Martinho, Rui Goncalo; Oliveira, Raquel A.The fidelity of mitosis depends on cohesive forces that keep sister chromatids together. This is mediated by cohesin that embraces sister chromatid fibers from the time of their replication until the subsequent mitosis [1-3]. Cleavage of cohesin marks anaphase onset, where single chromatids are dragged to the poles by the mitotic spindle [4-6]. Cohesin cleavage should only occur when all chromosomes are properly bio-oriented to ensure equal genome distribution and prevent random chromosome segregation. Unscheduled loss of sister chromatid cohesion is prevented by a safeguard mechanism known as the spindle assembly checkpoint (SAC) [7, 8]. To identify specific conditions capable of restoring defects associated with cohesion loss, we screened for genes whose depletion modulates Drosophila wing development when sister chromatid cohesion is impaired. Cohesion deficiency was induced by knockdown of the acetyltransferase separation anxiety (San)/Naa50, a cohesin complex stabilizer [9-12]. Several genes whose function impacts wing development upon cohesion loss were identified. Surprisingly, knockdown of key SAC proteins, Mad2 and Mpsl, suppressed developmental defects associated with San depletion. SAC impairment upon cohesin removal, triggered by San depletion or artificial removal of the cohesin complex, prevented extensive genome shuffling, reduced segregation defects, and restored cell survival. This counterintuitive phenotypic suppression was caused by an intrinsic bias for efficient chromosome biorientation at mitotic entry, coupled with slow engagement of error-correction reactions. Thus, in contrast to SAC's role as a safeguard mechanism for mitotic fidelity, removal of this checkpoint alleviates mitotic errors when sister chromatid cohesion is compromised.