TY - JOUR
T1 - A non-dicer RNase III and four other novel factors required for RNAi-Mediated transposon suppression in the human pathogenic yeast cryptococcus neoformans
AU - Burke, Jordan E.
AU - Longhurst, Adam D.
AU - Natarajan, Prashanthi
AU - Rao, Beiduo
AU - Liu, John
AU - Sales-Lee, Jade
AU - Mortensen, Yasaman
AU - Moresco, James J.
AU - Diedrich, Jolene K.
AU - Yates, John R.
AU - Madhani, Hiten D.
N1 - Publisher Copyright:
© 2019 Burke et al.
PY - 2019/7/1
Y1 - 2019/7/1
N2 - 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. The RDE genes do 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 suggest 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 endosiRNA biogenesis and transposon mobilization suppression in C. neoformans.
AB - 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. The RDE genes do 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 suggest 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 endosiRNA biogenesis and transposon mobilization suppression in C. neoformans.
KW - RNA surveillance
KW - RNAi transposon
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U2 - 10.1534/g3.119.400330
DO - 10.1534/g3.119.400330
M3 - Article
C2 - 31092606
AN - SCOPUS:85069329942
SN - 2160-1836
VL - 9
SP - 2235
EP - 2244
JO - G3: Genes, Genomes, Genetics
JF - G3: Genes, Genomes, Genetics
IS - 7
ER -