Restraint of the G2/M Transition by the SR/RRM Family mRNA Shuttling Binding Protein SNXAHRB1 in Aspergillus nidulans

Abstract

Control of the eukaryotic G2/M transition by CDC2/CYCLINB is tightly regulated by protein-protein interactions, protein phosphorylations, and nuclear localization of CDC2/CYCLINB. We previously reported a screen, in Aspergillus nidulans, for extragenic suppressors of nimX2 cdc2 that resulted in the identification of the cold-sensitive snxA1 mutation. We demonstrate here that snxA1 suppresses defects in regulators of the CDK1 mitotic induction pathway, including nimX2 cdc2 , nimE6 cyclinB , and nimT23 cdc25 , but does not suppress G2-arresting nimA1/nimA5 mutations, the S-arresting nimE10 cyclinB mutation, or three other G1/S phase mutations. snxA encodes the A. nidulans homolog of Saccharomyces cerevisiae Hrb1/Gbp2; nonessential shuttling messenger RNA (mRNA)-binding proteins belonging to the serine-arginine-rich (SR) and RNA recognition motif (RRM) protein family; and human heterogeneous ribonucleoprotein-M, a spliceosomal component involved in pre-mRNA processing and alternative splicing. snxA Hrb1 is nonessential, its deletion phenocopies the snxA1 mutation, and its overexpression rescues snxA1 and DsnxA mutant phenotypes. snxA1 and a second allele isolated in this study, snxA2, are hypomorphic mutations that result from decreased transcript and protein levels, suggesting that snxA acts normally to restrain cell cycle progression. SNXA HRB1 is predominantly nuclear, but is not retained in the nucleus during the partially closed mitosis of A. nidulans. We show that the snxA1 mutation does not suppress nimX2 by altering NIMX2 CDC2 /NIME CYCLINB kinase activity and that snxA1 or DsnxA alter localization patterns of NIME CYCLINB at the restrictive temperatures for snxA1 and nimX2. Together, these findings suggest a novel and previously unreported role of an SR/RRM family protein in cell cycle regulation, specifically in control of the CDK1 mitotic induction pathway. CONTROL of the eukaryotic G2/M transition by protein kinases has been widely studied and is highly conserved among all eukaryotes from the budding and fission yeasts and filamentous fungi to metazoans (for review, see Ma and Poon 2011). The CDK1/CYCLINB protein kinase complex is a major regulator of this transition in all eukaryotes and is responsible for the phosphorylations of numerous proteins, leading to massive nuclear and cytoplasmic reorganizations that regulate mitosis (for review, see Lindqvist et al. 2009). The complex itself is tightly regulated, both temporally and spatially, to allow mitotic entry.Although CDK1/CYCLINB activity is essential for mitotic entry in all eukaryotes, structural differences in the nucleus in various organisms result in "open" mitosis (more complex eukaryotes) or "closed" mitosis (budding yeasts); these differences likely affect the temporo-spatial functioning of CDK1/ CYCLINB. The partially closed mitosis of the filamentous fungus Aspergillus nidulans is an evolutionary intermediate between open and closed mitoses and provides a system for studying mitotic entry in organisms intermediate between budding ...

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“…Moreover, single and double deletion mutant strains grew on different media and temperature conditions without showing noticeable phenotypes (Supplementary Figure S20) apart from a minor (but reproducible) increase on cell density for late cultures in the Δhrb1Δgbp2 double mutant (Supplementary Figure S20). This result suggests that these proteins have a subtle and long-term negative effect on cell growth consistent with results recently obtained for the Gbp2/Hrb1 homolog in Aspergillus nidulans ( 39 ).…”

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