Biology subjects

Burke, J. E.

Publications and source records attributed to Burke, J. E..

2 recordsLinked to original sources

bioRxiv2019

Characterisation of the c10orf76-PI4KB complex, and its necessity for Golgi PI4P levels and enterovirus replication

The lipid kinase PI4KB, which generates phosphatidylinositol 4-phosphate (PI4P), is a key enzyme in regulating membrane transport and is also hijacked by multiple picornaviruses to mediate viral replication. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB-associated protein that increases PI4P levels at the Golgi, and is essential for the viral replication of specific enteroviruses. We used hydrogen deuterium exchange mass spectrometry to characterize the c10orf76-PI4KB complex and reveal that binding is mediated by the kinase linker of PI4KB, with formation of the heterodimeric complex modulated by PKA-dependent phosphorylation. Complex-disrupting mutations demonstrate that PI4KB is required for membrane recruitment of c10orf76 to the Golgi, and that an intact c10orf76-PI4KB complex is required for the replication of c10orf76-dependent enteroviruses. Intriguingly, c10orf76 was also required for proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76-dependent increase in PI4P levels at the Golgi.\n\nHighlightsO_LIc10orf76 forms a direct complex with PI4KB, with the interface formed by a disorder-to-order transition in the kinase linker of PI4KB\nC_LIO_LIThe c10orf76 binding site of PI4KB can be phosphorylated by PKA, with phosphorylation leading to decreased affinity for c10orf76\nC_LIO_LIComplex-disrupting mutants of PI4KB and c10orf76 reveal that PI4KB recruits c10orf76 to the Golgi/TGN\nC_LIO_LIDepletion of c10orf76 leads to decreases in both active Arf1 and Golgi PI4P levels\nC_LIO_LIEnteroviruses that rely on c10orf76 for replication depend on formation of the c10orf76-PI4KB complex\nC_LI

biochemistry↗

bioRxiv2019

A non-Dicer RNase III and four other novel factors required for RNAi-mediated transposon suppression in the human pathogenic yeast C. neoformans

The human pathogenic yeast Cryptococcus neoformans silences transposable elements using endo-siRNAs and an Argonaute, Ago1. Endo-siRNAs production requires the RNA-dependent RNA polymerase, Rdp1, and two partially redundant Dicer enzymes, Dcr1 and Dcr2, but is independent of histone H3 lysine 9 methylation. We describe here an insertional mutagenesis screen for factors required to suppress the mobilization of the C. neoformans HARBINGER family DNA transposon HAR1. Validation experiments uncovered five novel genes (RDE1-5) required for HAR1 suppression and global production of suppressive endo-siRNAs. Loss of the RDE genes does not impact transcript levels, suggesting the endo-siRNAs do not act by impacting target transcript synthesis or turnover. RDE3 encodes a non-Dicer RNase III related to S. cerevisiae Rnt1, RDE4 encodes a predicted terminal nucleotidyltransferase, while RDE5 has no strongly predicted encoded domains. Affinity purification-mass spectrometry studies reveal that Rde3 and Rde5 are physically associated. RDE1 encodes a G-patch protein homologous to the S. cerevisiae Sqs1/Pfa1, a nucleolar protein that directly activates the essential helicase Prp43 during rRNA biogenesis. Rde1 copurifies Rde2, another novel protein obtained in the screen, as well as Ago1, a homolog of Prp43, and numerous predicted nucleolar proteins. We also describe the isolation of conditional alleles of PRP43, which are defective in RNAi. This work reveals unanticipated requirements for a non-Dicer RNase III and presumptive nucleolar factors for endo-siRNA biogenesis and transposon mobilization suppression in C. neoformans.

molecular biology↗