Suresh, A. P. | Search bioRxiv

bioRxiv2022

Bub1 kinase acts as a signalling hub for the entire Cryptococcus neoformans spindle assembly checkpoint pathway

Cryptococcus neoformans (Cn) is an important human pathogen and a model system for basidiomycetes. Here we carry out a dissection of its spindle assembly checkpoint (SAC), focusing on Bub1 and Bub3. In many eukaryotes, including humans, Saccharomyces cerevisiae and Schizosaccharomyces pombe, Bub1 underwent gene duplication, generating paralogues referred to as Bub1 and BubR1 (or Mad3). Bub1 has upstream signalling functions at kinetochores, whilst BubR1/Mad3 is a component of the downstream mitotic checkpoint complex (MCC) that delays anaphase onset until all chromosomes are correctly attached. Here we demonstrate that the single CnBub1 protein carries out all the checkpoint roles of both Bub1 kinase and Mad3/BubR1. Proteomic analysis reveals kinetochore targeting via Spc105KNL1 and interactions with all downstream SAC components and effectors (Cdc20 and the anaphase promoting complex/cyclosome). We demonstrate that CnBub1 kinase activity is required to maintain prolonged checkpoint arrest. Thus CnBub1 acts as a SAC signalling hub and is a future target for anti-mitotic drugs.

cell biology↗

bioRxiv2021

Suppression of Global Protein Translation in SARS-CoV-2 Infection

Viruses employ distinct strategies to ensure efficient translation of their mRNAs over the host transcripts. SARS-CoV-2 targets host mRNAs and ribosomes to favor its own protein synthesis. However, the modulation of the key signal pathways that control the host protein translation machinery during SARS-CoV-2 infection has not been sufficiently addressed. Here, by employing an early variant and a Delta variant isolate that evolved later in the pandemic demonstrates that SARS-CoV-2 infection results in massive polysome collapse starting from 24 hpi, a hallmark of global translation inhibition. Unexpectedly, eIF2 phosphorylation, commonly targeted by viruses to induce translation arrest, was not involved in the translation arrest, suggesting that SARS-CoV-2 countermeasures by the virus to suppress ISR. eIF4E phosphorylation remained unaltered during the infection, ruling out its involvement in the preferential translation of SARS-CoV-2 transcripts. We find that SARS-CoV-2 infection consistently causes mTORC1 inhibition in a comparable manner across both variants indicating that the virus likely targets mTORC1 pathway to suppress host translation. Interestingly, mTORC1 inhibition by SARS-CoV-2 did not impact the polysomal loading of ribosomal protein transcripts rpS3 and rpL26, suggesting that 5TOP mRNAs are spared from the translation suppression and that ribosomal protein synthesis remains active during the infection. Pharmacological activation of mTORC1 did not significantly impact viral replication, suggesting that mTORC1 inhibition might be selectively restricting the host mRNAs from accessing the translation machinery, facilitating a more robust translation of viral transcripts. This study provides new insights into the molecular interactions by which SARS-CoV-2 variants, despite their different clinical outcomes, converge on a conserved mechanism to manipulate host translation regulatory pathways.

microbiology↗