Biology subjects

Robert, M.-C.

Publications and source records attributed to Robert, M.-C..

3 recordsLinked to original sources

bioRxiv2020

A conserved choreography of mRNAs at centrosomes reveals a localization mechanism involving active polysome transport

Local translation allows for a spatial control of gene expression. Here, we used high-throughput smFISH to screen centrosomal protein-coding genes, and we describe 8 human mRNAs accumulating at centrosomes. These mRNAs localize at different stages during cell cycle with a remarkable choreography, indicating a finely regulated translational program at centrosomes. Interestingly, drug treatments and reporter analyses revealed a common translation-dependent localization mechanism requiring the nascent protein. Using ASPM and NUMA1 as models, single mRNA and polysome imaging revealed active movements of endogenous polysomes towards the centrosome at the onset of mitosis, when these mRNAs start localizing. ASPM polysomes associate with microtubules and localize by either motor-driven transport or microtubule pulling. Remarkably, the Drosophila orthologs of the human centrosomal mRNAs also localize to centrosomes and also require translation. These data identify a conserved family of centrosomal mRNAs that localize by active polysomes transport mediated by nascent proteins.

cell biology↗

bioRxiv2020

Stochastic pausing at latent HIV-1 promoters generates transcriptional bursting

Promoter-proximal polymerase pausing is a key process regulating gene expression. In latent HIV-1 cells, it prevents viral transcription and is essential for latency maintenance, while in acutely infected cells the viral factor Tat releases paused polymerase to induce viral expression. Pausing is fundamental for HIV-1, but how it contributes to bursting and stochastic viral reactivation is unclear. Here, we performed single molecule imaging of HIV-1 transcription, and we developed a quantitative analysis method that manages multiple time scales from seconds to days, and that rapidly fits many models of promoter dynamics. We found that RNA polymerases enter a long-lived pause at latent HIV-1 promoters (>20 minutes), thereby effectively limiting viral transcription. Surprisingly and in contrast to current models, pausing appears stochastic and not obligatory, with only a small fraction of the polymerases undergoing long-lived pausing in absence of Tat. One consequence of stochastic pausing is that HIV-1 transcription occurs in bursts in latent cells, thereby facilitating latency exit and providing a rationale for the stochasticity of viral rebounds.

systems biology↗

bioRxiv2020

A localization screen reveals translation factories and widespread co-translational RNA targeting

Local translation allows a spatial control of gene expression. Here, we performed a dual protein/mRNA localization screen, using smFISH on 523 human cell lines expressing GFP-tagged genes. A total of 32 mRNAs displayed specific cytoplasmic localizations, and we observed local translation at unexpected locations, including cytoplasmic protrusions, cell edges, endosomes, Golgi, the nuclear envelope and centrosomes, the latter being cell cycle dependent. Quantitation of mRNA distribution and automatic pattern classification revealed a high degree of localization heterogeneity between cells. Surprisingly, mRNA localization frequently required ongoing translation, indicating widespread co-translational RNA targeting. Interestingly, while P-body accumulation was frequent (15 mRNAs), four mRNAs accumulated in foci that were distinct structures. These foci lacked the mature protein, but nascent polypeptide imaging showed that they were specialized translation factories. For {beta}-catenin, foci formation was regulated by Wnt, relied on APC-dependent polysome aggregation, and led to nascent protein degradation. Thus, translation factories uniquely regulate nascent protein metabolism and create a fine granular compartmentalization of translation.

cell biology↗