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

Burke, J. E.; Longhurst, A. D.; Natarajan, P.; Rao, B.; Liu, S. J.; Sales-Lee, J.; Mortensen, Y.; Moresco, J.; Diedrich, J. K.; Yates, J. R.; Madhani, H. D.

doi:10.1101/577924

bioRxiv · 10.1101/577924

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

Burke, J. E.·Longhurst, A. D.·Natarajan, P.·Rao, B.·Liu, S. J.·Sales-Lee, J.·Mortensen, Y.·Moresco, J.·Diedrich, J. K.·Yates, J. R.·Madhani, H. D.

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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. 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.

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Burke, J. E., Longhurst, A. D., Natarajan, P., Rao, B., Liu, S. J., Sales-Lee, J., Mortensen, Y., Moresco, J., Diedrich, J. K., Yates, J. R., Madhani, H. D.. 2019-03-14. A non-Dicer RNase III and four other novel factors required for RNAi-mediated transposon suppression in the human pathogenic yeast C. neoformans. https://doi.org/10.1101/577924

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