Zein-Sabatto, H. | Search bioRxiv

bioRxiv2024

Centrocortin potentiates co-translational localization of its mRNA to the centrosome via dynein

Centrosomes rely upon proteins within the pericentriolar material to nucleate and organize microtubules. Several mRNAs also reside at centrosomes, although less is known about how and why they accumulate there. We previously showed that local Centrocortin (Cen) mRNA supports centrosome separation, microtubule organization, and viability in Drosophila embryos. Here, using Cen mRNA as a model, we examine mechanisms of centrosomal mRNA localization. We find that while the Cen N-terminus is sufficient for protein enrichment at centrosomes, multiple domains cooperate to concentrate Cen mRNA at this location. We further identify an N-terminal motif within Cen that is conserved among dynein cargo adaptor proteins and test its contribution to RNA localization. Our results support a model whereby Cen protein enables the accumulation of its own mRNA to centrosomes through a mechanism requiring active translation, microtubules, and the dynein motor complex. Taken together, our data uncover the basis of translation-dependent localization of a centrosomal RNA required for mitotic integrity. SummaryEnrichment of Centrocortin (Cen) mRNA at centrosomes is required for mitotic fidelity. This study describes a mechanism underlying co-translational Cen mRNA targeting involving microtubules, the dynein motor, and a highly conserved dynein binding motif within the Cen coding sequence.

cell biology↗

bioRxiv2021

Drosophila centrocortin is dispensable for centriole duplication but contributes to centrosome separation

Centrosomes are microtubule-organizing centers that duplicate exactly once to organize the bipolar mitotic spindle required for error-free mitosis. Prior work indicated that Drosophila centrocortin (cen) is required for normal centrosome separation, although a role in centriole duplication was not closely examined. Through time-lapse recordings of rapid syncytial divisions, we monitored centriole duplication and the kinetics of centrosome separation in control versus cen null embryos. Our data suggest that although cen is dispensable for centriole duplication, it contributes to centrosome separation.

cell biology↗

bioRxiv2021

An RNAi screen in C. elegans for genes that play a role in secretion and cleavage of VAPB MSP domain.

VAPB (VPR-1 in C. elegans) is a type-II ER transmembrane protein whose N-terminal Major Sperm Protein domain (MSPd) is cleaved and secreted. Mutations in the MSPd of human VAPB cause impaired secretion and are associated with Amyotrophic Lateral Sclerosis (ALS). In C. elegans, the secreted MSPd signals non-cell-autonomously to regulate striated muscle mitochondrial morphology and gonad development. As VAPB/VPR-1 does not have a signal peptide and its MSPd extends into the cytosol, it is unclear how the protein is proteolytically cleaved and secreted. To identify genes that are involved in VPR-1 cleavage and unconventional secretion, we performed an RNA interference (RNAi) screen in C. elegans. Worms null for vpr-1 are sterile and have striated muscle mitochondrial abnormalities. These defects can be rescued by vpr-1 expression in the neurons, germline, or intestine, implying that these three tissues share a common machinery to cleave and secrete the MSPd. Examination of shared gene expression in these tissues revealed a list of 422 genes, which we targeted with RNAi. vpr-1 null worms expressing vpr-1 from intestine were used in the screen, and the brood size of these worms after RNAi knockdown was scored. Disruption of factors involved in VPR-1 MSPd processing and/or secretion should revert fertility phenotypes in these worms. We identified many genes that induce compromised fertility when knocked down in these but not wild type worms, including a V-SNARE, several proteasome components, stress response molecules, and mitochondrial genes. Our screen thus identified many potential players involved in MSPd processing and/or secretion. SummaryThe MSP domain (MSPd) of a type-II ER transmembrane protein called VAPB is cleaved and secreted to function as a non-cell-autonomous signal. The topology of VAPB positions MSPd in the cytosol. It is thus unclear how MSPd is cleaved from VAPB and released extracellularly. Using C. elegans, we screened 422 genes by RNAi to identify potential candidates regulating MSPd cleaving and secretion. We identified the Golgi v-SNARE YKT-6 and several components of the 20S and 19S proteasome that may mediate MSPd trafficking and cleaving, respectively. These results have promising implications in advancing our understanding of MSPd signaling.

cell biology↗