A Non-Dicer RNase III and Four Other Novel Factors Required for RNAi-Mediated Transposon Suppression in the Human Pathogenic Yeast Cryptococcus neoformans

• Published in: G3 : genes - genomes - genetics 2019-07, Vol.9 (7), p.2235-2244
• Main Authors: Burke, Jordan E, Longhurst, Adam D, Natarajan, Prashanthi, Rao, Beiduo, Liu, John, Sales-Lee, Jade, Mortensen, Yasaman, Moresco, James J, Diedrich, Jolene K, Yates, John R, Madhani, Hiten D
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Cryptococcosis - microbiology; Cryptococcus neoformans - genetics; DNA Transposable Elements; Gene Dosage; Gene Expression Regulation, Bacterial; Humans; Investigations; Mutagenesis, Insertional; Mutation; Ribonuclease III - genetics; RNA Interference; RNA surveillance; RNA, Small Interfering - genetics; RNAi; transposon
• ISSN: 2160-1836; 2160-1836
• DOI: 10.1534/g3.119.400330

Abstract 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 endo-siRNA biogenesis and transposon mobilization suppression in C. neoformans.