Biology subjects

Martinho, R. G.

Publications and source records attributed to Martinho, R. G..

5 recordsLinked to original sources

bioRxiv2022

An SNF2 helicase-like protein links mitotic transcription termination to sister chromatid resolution

AO_SCPLOWBSTRACTC_SCPLOWMitotic chromatin is largely assumed incompatible with transcription due to changes in the transcription machinery and chromosome architecture. However, the mechanisms of mitotic transcriptional inactivation and their interplay with chromosome assembly remain largely unknown. By monitoring ongoing transcription in Drosophila early embryos, we reveal that eviction of nascent mRNAs from mitotic chromatin occurs after substantial chromosome compaction and is not promoted by condensin I. Instead, we show that the timely removal of transcripts from mitotic chromatin is driven by the SNF2 helicase-like protein Lodestar (Lds), identified here as a modulator of sister chromatid cohesion defects. In addition to transcriptional termination, we uncovered that Lds cooperates with Topoisomerase 2 to ensure efficient sister chromatid resolution and mitotic fidelity. We conclude that mitotic transcriptional termination is not a passive consequence of cell cycle progression and/or chromosome compaction but occurs via dedicated mechanisms with functional parallelisms to sister chromatid resolution.

cell biology↗

bioRxiv2022

N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity

Most eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, we reveal a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. We reveal that loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility. In BriefVarland, Silva et al. define that a major cellular role of N-terminal acetylation is shielding proteins from proteasomal degradation by specific ubiquitin ligases. The human N-terminal acetyltransferase NatC protects the neddylation regulator UBE2M from degradation, while overexpression of Drosophila UBE2M/UbcE2M rescues the longevity and motility defects of NatC deletion. HighlightsO_LIN-terminal acetylation by NatC protects proteins from degradation, including UBE2M C_LIO_LIUBR4-KCMF1 targets unacetylated N-terminal Met followed by a hydrophobic residue C_LIO_LIDrosophila NatC is required for adult longevity and motility in elderly C_LIO_LIOverexpression of UBE2M/UbcE2M suppresses Drosophila NatC deletion phenotypes C_LI

molecular biology↗

bioRxiv2020

Transcription and splicing dynamics during early Drosophila development

Widespread co-transcriptional 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 co-transcriptional 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 nucleotides 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 unravels novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo.

molecular biology↗

bioRxiv2018

Absence of the Spindle Assembly Checkpoint restores mitotic fidelity upon loss of sister chromatid cohesion

Sister chromatid cohesion is essential for faithful mitosis, as premature cohesion loss leads to random chromosome segregation and aneuploidy, resulting in abnormal development. 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 knock-down of the acetyltransferase Separation anxiety (San)/Naa50, a cohesin complex stabilizer. Several genes whose function impacts wing development upon cohesion loss were identified. Surprisingly, knockdown of key Spindle Assembly Checkpoint (SAC) proteins, Mad2 and Mps1, 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 bi-orientation at mitotic entry, coupled with slow engagement of error-correction reactions. We conclude that mitotic timing determines the severity of defects associated with cohesion deficiency. Therefore, although divisions are still error-prone, SAC inactivation enhances cell survival and tissue homeostasis upon cohesion loss.

cell biology↗

bioRxiv2018

The Trithorax group protein dMLL3/4 instructs the assembly of the zygotic genome at fertilization

The transition from fertilized oocyte to totipotent embryo relies on maternally-provided factors that are synthetized and accumulated in developing oocytes. Yet, it is still unclear how oocytes regulate the expression of these embryo fate-promoting genes within the general transcriptional program of oogenesis. Here we report that the Drosophila Trithorax group protein MLL3/4 (dMLL3/4, also known as Trr) is essential for the transition to embryo fate at fertilization. In the absence of dMLL3/4, oocytes develop normally but fail to initiate the embryo mitotic divisions after fertilization. This incapability results from defects in both paternal genome reprogramming and maternal meiotic completion. We show that, during oogenesis, dMLL3/4 promotes the expression of a functionally coherent gene subset that is later required for the correct assembly of the zygotic genome. Accordingly, we identify the evolutionarily-conserved IDGF4 glycoprotein (known as oviductin in mammals) as a new oocyte-to-embryo transition gene under dMLL3/4 transcriptional control. Based on these observations, we propose that dMLL3/4 plays an instructive role in the oocyte-to-embryo transition that is functionally uncoupled from the requirements of normal oocyte differentiation.

developmental biology↗