Project description:mRNAs associated with microtubules during interphase, metaphase and the midbody stage of cytokinesis were sequenced. Selective midbody-localized RNAs were identified and their translational characteristics were studied.
Project description:Midbody is a transient structure assembled in the last step of mitosis and is currently conceptualized as a structural remnant subject to degradation following cytokinesis. However, our findings indicate that midbody regulation is more complicated than original thought. Knockdown spliceosome proteins in cells showed a multinuclear phenotype and abnormal midbody. Spliceosome and ribosome components are enriched in midbody. We further found that mRNA splicing and protein translation occurs in the midbody during last step of mitosis, which are necessary events for proper abscission of nascent daughter cells. Midbody spliced and translated PRC1 and NINL are essential for proper midbody function. Either PRC1 or NINL knockdown also showed a multinuclear phenotype and abnormal midbody. Treating cancer cells with both chemotherapy and spliceosome inhibitors dramatically lowers the concentration of both drugs, avoiding more severe side effects. Our study discovers a novel regulating mechanism for mitosis and provides a new strategy for cancer therapy.
Project description:The midbody is an organelle assembled at the intercellular bridge connecting the two daughter cells at the end of mitosis. It is essential for the final separation of the daughter cells and involved in other important processes, including cell fate, pluripotency, apical-basal polarity, and cilium/lumen formation. The midbody is composed of numerous proteins with diverse functions distributed in a precise pattern. Here we report the first characterization of the midbody protein-protein interaction network (interactome), which provides an extremely valuable resource for understanding its multiple roles. Analysis of this midbody interactome led to the discovery that PP1/MYPT1 phosphatase regulates microtubule dynamics in late cytokinesis through de-phosphorylation of the kinesin MKLP1/KIF23, a finding that unexpectedly expands the temporal window of activity of this phosphatase during mitosis.
Project description:Translational regulation at the stage of initiation impacts the number of ribosomes translating each mRNA molecule. For example, multiple ribosomes can engage on a single mRNA forming a polysome, resulting in highly efficient protein synthesis. However, the translational activity of single 80S ribosomes on mRNA (monosomes) is less well understood, even though these 80S monosomes represent the dominant ribosomal complexes in many tissues. Here, we used cryo-EM to determine the translational activity of 80S monosomes across different tissues in Drosophila melanogaster. We discovered that while head and embryo 80S monosomes are highly translationally active, testis and ovary 80S monosomes are translationally inactive. RNA-Seq analysis of head monosome- and polysome-translated mRNAs, revealed that head 80S monosomes preferentially translate mRNAs with TOP motifs, short 5’-UTRs, short ORFs and are enriched for uORFs. Overall, these findings highlight that regulation of translation initiation, and therefore the number of ribosomes bound per mRNA, varies substantially across tissues.
Project description:The recruitment and activation of abscission machinery following mitosis is tightly regulated in time and space, yet the mechanisms controlling this process are poorly understood. We find that RNA localization and local translation at the midbody regulates when and where abscission-regulating proteins are expressed. The 3′UTR of NET1 mRNA contains an element that is necessary and sufficient for RNA targeting to the midbody. Mislocalization of NET1 mRNA results in a loss of NET1 protein, a Rho family GEF, throughout the intercellular bridge as well as slower cell proliferation and delayed abscission. This coincides with a loss of Arp2/3 at the abscission site and is dependent upon NET1 binding to Rho family GTPases. These findings establish midbody RNA localization and local translation as a key layer of regulation over abscission timing and identify a role for NET1 as a regulator of Arp2/3-mediated branched actin accumulation at the abscission site.
Project description:Midbody is a transient structure assembled in the last step of mitosis and is currently conceptualized as a structural remnant subject to degradation following cytokinesis. However, our findings indicate that midbody regulation is more complicated than original thought. Knockdown spliceosome proteins in cells showed a multinuclear phenotype and abnormal midbody. Spliceosome and ribosome components are enriched in midbody. We further found that mRNA splicing and protein translation occurs in the midbody during last step of mitosis, which are necessary events for proper abscission of nascent daughter cells. Midbody spliced and translated PRC1 and NINL are essential for proper midbody function. Either PRC1 or NINL knockdown also showed a multinuclear phenotype and abnormal midbody. Treating cancer cells with both chemotherapy and spliceosome inhibitors dramatically lowers the concentration of both drugs, avoiding more severe side effects. Our study discovers a novel regulating mechanism for mitosis and provides a new strategy for cancer therapy.
Project description:Initiation of bacterial DNA replication takes place at the origin of replication (oriC), a region characterized by the presence of multiple DnaA boxes that serve as the binding sites for the master initiator protein DnaA. The absence or failure of DNA replication can result in bacterial cell growth arrest or death. Here, we aimed to uncover the physiological and molecular consequences of stopping replication in the model bacterium Bacillus subtilis. For this purpose, DNA replication was blocked using a CRISPRi approach specifically targeting DnaA boxes 6 and 7, which are essential for replication initiation. We characterized the phenotype of these cells and analyzed the overall changes in the proteome using quantitative mass spectrometry. Cells with arrested replication were elongating and not dividing but showed no evidence of DNA damage response (DDR). Moreover, these cells did not cease translation over time. This study sets the ground for future research on non-replicating but translationally active B. subtilis, which might be valuable for biotechnological applications.