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

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 .

Coordinate genomic association of transcription factors controlled by an imported quorum sensing peptide in Cryptococcus neoformans

“…In line with this idea, one of the promoters that exhibited a very dramatic dependence on Qsp1 for binding of all three transcription factors was the LAC1 gene (CNAG_03465) ( S3 Fig), which encodes the melanization factor laccase [40][41][42]. Melanization is known to be a key virulence trait for C. neoformans infection [40,43]. In our previous work, we found that cells lacking Qsp1 display altered capacities to produce melanin when plated on media containing the substrate for melanin synthesis [4].…”

“…Total RNA was isolated from 50 OD 600 's of cells as previously described [42] and libraries prepared as previously described [43]. In brief, cell pellets were lyophilized overnight and then RNA was isolated using TRIzol (Invitrogen) as previously described [42] and DNase treated as previously described [44].…”

The interplay of phenotype and genotype inCryptococcus neoformansdisease

“…As TEs can negatively disrupt the genome, most eukaryotes have evolved methods to defend against TE-induced mutagenesis. C. neoformans uses endogenously produced small RNAs (endo-siRNA) to silence TEs [107][108][109][110]. While limiting transposon mobilization to prevent detrimental mutagenesis is important under normal conditions, when adapting to a new, high-stress environment, transposons may prove beneficial.…”

Advances in Molecular Tools and In Vivo Models for the Study of Human Fungal Pathogenesis

“…In conclusion, although many studies employing transposon mutagenesis are performed in S. cerevisiae and C. albicans, there is a growing body of work using transposons in other pathogenic fungi including Cryptococcus, C. glabrata and Histoplasma [111,[135][136][137][138][139]. Since transposon mutagenesis allows genome-scale DNA modification, this strategy serves a powerful tool to generate insertional mutations.…”

“…For example, transposable element Tf1 preferentially targets promoters of genes induced by environmental stresses. However, over the past few years, some studies have emerged that contribute to our knowledge of TEs and their mechanisms [ 111 , 112 ]. Despite limitations, transposon-mediated mutagenesis has been used successfully to study protein localization, morphological transitions, phenotypic analysis and to determine genes essential for growth and virulence [ 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 ].…”