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

Abstract

The human pathogenic yeast <i>Cryptococcus neoformans</i> 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 <i>C. neoformans HARBINGER</i> family DNA transposon <i>HAR1</i> Validation experiments uncovered five novel genes (<i>RDE1-5</i>) required for <i>HAR1</i> suppression and global production of suppressive endo-siRNAs. The <i>RDE</i> genes do not impact transcript levels, suggesting the endo-siRNAs do not act by impacting target transcript synthesis or turnover. <i>RDE3</i> encodes a non-Dicer RNase III related to <i>S. cerevisiae</i> Rnt1, <i>RDE4</i> encodes a predicted terminal nucleotidyltransferase, while <i>RDE5</i> has no strongly predicted encoded domains. Affinity purification-mass spectrometry studies suggest that Rde3 and Rde5 are physically associated. <i>RDE1</i> encodes a G-patch protein homologous to the <i>S. cerevisiae</i> 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 <i>PRP43</i>, 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 <i>C. neoformans</i>.