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Rio1 downregulates centromeric RNA levels to promote the timely assembly of structurally fit kinetochores

ABSTRACT: Kinetochores assemble on centromeres (CENs) via histone H3 variant CENP-A and low levels of CEN transcripts. RNA polymerase II (RNAPII) activity is restrained by the CEN histone code, while CEN RNA concentrations are reduced by the nuclear exosome. Using S. cerevisiae, we add kinase Rio1 to this scheme as it downregulates RNAPII, and promotes CEN RNA turnover via exoribonuclease Rat1. Transcription factor Cbf1, and the assembled kinetochore further restrain CEN transcription. CEN transcripts exist as long (up to 11,000nt) and short RNAs (119±40nt), which may underlie CEN identity and kinetochore recruitment. While also curtailed by Rio1, Rat1, and the exosome, periCEN RNAs (<200nt) accumulate at levels that are one order of magnitude higher than the CEN transcripts. Depleting Rio1 causes CEN and periCEN RNA buildup, kinetochore malformation, and chromosome loss. Depleting human orthologue RioK1 leads to CEN RNA accumulation and micronuclei formation, suggesting that Rio1/RioK1 activity at centromeres is conserved.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Romina Belli

LAB HEAD: Peter De Wulf

PROVIDER: PXD030623 | Pride | 2025-02-24

REPOSITORIES: pride

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Project description:Expression of CD40 in non-hematopoietic cells has been linked to inflammation. We presented evidence that CD40, a T-cell costimulatory molecule, is expressed in human Î²-cells and the engagement of CD40 in insulinoma cells activated the NFKB and ERK1/2 pathways. CD40 activation in human islets cells induced secretion of IL-8, MCP-1 and MIP-1 Î², which is abrogated by inhibitors of NFkB and ERK1/2 inhibitors. In this study, we have studied gene expression mediated by CD40-CD40L interaction in islet cells. This approach identified 90 genes and transcripts exhibiting at least a 1.7 fold increase in their expression intensity after treatment with soluble CD40L. A significant number of genes were related to inflammation and oxidative stress. We have a strong overexpression of CXCL1 (GroÎ±), CXCL2 (Mif2) and CXCL3; chemokines belonging to CXC family structurally related to Il-8. 11 genes were selected from this group and further quantified by Real Time PCR, including CXCL1. Activation of islet cells with CD40L induced the secretion of CXCL1 in a NFKB dependent manner. Engagement of CD40 in islet cells did not induce apoptosis, neither Î²-cell death and did not enhanced TNF-Î± mediated cell death as observed in insulinoma cells. CD40 activation in insulinoma cells, results in ERK1/2 dependent phsophorylation of synapsin I, a protein associated with the exocytosis machinery in neurons and Î²-cells. However, treatment of islets with soluble CD40L did not affect glucose induced insulin secretion. It has been reported that ductal cells always present in human islet preparations express CD40 constitutively (ref). We found that CD40-CD40L interaction in ductal cells, unlike in Î²-cells, induces secretion of diabetogenic cytokines IFNÎ³ and TNF-Î±. Furthermore, incubation of islets containing ductal cells with CD40L decreased Î²-cells viability as assessed by measurement of their mitochondrial membrane potential Experiment Overall Design: We isolated islet cells from three patients. Part of islet cells from each patient has been treated with CD40L. We compared gene expression in treated cells vs untreated for each patient using dye-swap.

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Rio1 downregulates centromeric RNA levels to promote the timely assembly of structurally fit kinetochores

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