Biology subjects

Dietrich, F. S.

Publications and source records attributed to Dietrich, F. S..

3 recordsLinked to original sources

bioRxiv2024

Distinct evolutionary trajectories following loss of RNA interference in Cryptococcus neoformans

While increased mutation rates typically have negative consequences in multicellular organisms, hypermutation can be advantageous for microbes adapting to the environment. Previously, we identified two hypermutator Cryptococcus neoformans clinical isolates that rapidly develop drug resistance due to transposition of a retrotransposon, Cnl1. Cnl1-mediated hypermutation is caused by a nonsense mutation in the gene encoding a novel RNAi component, Znf3, combined with a tremendous transposon burden. To elucidate adaptative mechanisms following RNAi loss, two bioinformatic pipelines were developed to identify RNAi loss-of-function mutations in a collection of 387 sequenced C. neoformans isolates. Remarkably, several RNAi-loss isolates were identified that are not hypermutators and have not accumulated transposons. To test if these RNAi loss-of-function mutations can cause hypermutation, the mutations were introduced into a non-hypermutator strain with a high transposon burden, which resulted in a hypermutator phenotype. To further investigate if RNAi-loss isolates can become hypermutators, in vitro passaging was performed. Although no hypermutators were found in two C. neoformans RNAi-loss strains after short-term passage, hypermutation was observed in a passaged C. deneoformans strain with increased transposon burden. Consistent with a two-step evolution, when an RNAi-loss isolate was crossed with an isolate containing a high Cnl1 burden, F1 hypermutator progeny inheriting a high transposon burden were identified. In addition to Cnl1 transpositions, insertions of a novel gigantic DNA transposon KDZ1 ([~]11 kb), contributed to hypermutation in the progeny. Our results suggest that RNAi loss is relatively common (7/387, [~]1.8%) and enables distinct evolutionary trajectories: hypermutation following transposon accumulation or survival without hypermutation. Significance StatementThere is a dearth of antifungal drugs available to treat Cryptococcus neoformans, a human fungal pathogen of global impact. We previously identified natural hypermutators with a loss-of-function mutation in the RNAi machinery and transposon expansion. Here, we identified several novel natural isolates with RNAi defects, none of which are hypermutators or have undergone transposon expansion. Furthermore, we demonstrate that these isolates can lie on a pathway to hypermutation following introduction of a transposon burden. In addition, a novel DNA transposon class was discovered that contributes to antifungal drug resistance. These findings highlight the importance of transposons in driving rapid adaptation in the absence of RNAi and reveal distinct evolutionary trajectories following RNAi loss, a relatively common event in C. neoformans.

genetics↗

bioRxiv2023

Core gene set of the species Saccharomyces cerevisiae

Examination of the genome sequence of Saccharomyces cerevisiae strain S288c and 93 additional diverse strains allows identification of the 5885 genes that make up the core set of genes in this species and gives a better sense of the organization and plasticity of this genome. S. cerevisiae strains each contain dozens to hundreds of strain-specific genes. In addition to a variable content of retrotransposons Ty1-Ty6, some strains contain a novel transposable element, Ty7. Examination further shows that some annotated putative protein coding genes are likely artifacts. We propose altering approximately 5% of the current annotations in the widely used reference strain S288c. Potential null alleles are common and found in all 94 strains examined, with these potential null alleles typically containing a single stop codon or frameshift. There are also gene remnants, pseudogenes, and variable arrays of genes. Among the core genes there are now only 364 protein coding genes of unknown function, classified as uncharacterized in the Saccharomyces Genome Database. This work suggests that there is a role for carefully edited and annotated genome sequences in understanding the genome organization and content of a species. We propose that gene remnants be added to the repertoire of features found in the S. cerevisiae genome, and likely other fungal species.

genetics↗

bioRxiv2021

Comparative analysis of RNA enrichment methods for preparation of Cryptococcus neoformans RNA sequencing libraries

Ribosomal RNA (rRNA) is the major RNA constituent of cells, therefore most RNA sequencing (RNA-Seq) experiments involve removal of rRNA. This process, called RNA enrichment, is done primarily to reduce cost: without rRNA removal, deeper sequencing would need to be performed to balance the sequencing reads wasted on rRNA. The ideal RNA enrichment method would remove all rRNA without affecting other RNA in the sample. We have tested the performance of three RNA enrichment methods on RNA isolated from Cryptococcus neoformans, a fungal pathogen of humans. We show that the RNase H depletion method unambiguously outperforms the commonly used Poly(A) isolation method: the RNase H method more efficiently depletes rRNA while more accurately recapitulating the expression levels of other RNA observed in an unenriched "gold standard". The RNase H depletion method is also superior to the Ribo-Zero depletion method as measured by rRNA depletion efficiency and recapitulation of protein-coding gene expression levels, while the Ribo-Zero depletion method performs moderately better in preserving non-coding RNA (ncRNA). Finally, we have leveraged this dataset to identify novel long non-coding RNA (lncRNA) genes and to accurately map the C. neoformans mitochondrial rRNA genes. ARTICLE SUMMARYWe compare the efficacy of three different RNA enrichment methods for RNA-Seq in Cryptococcus neoformans: RNase H depletion, Ribo-Zero depletion, and Poly(A) isolation. We show that the RNase H depletion method, which is evaluated in C. neoformans samples for the first time here, is highly efficient and specific in removing rRNA. Additionally, using data generated through these analyses, we identify novel long non-coding RNA genes in C. neoformans. We conclude that RNase H depletion is an effective and reliable method for preparation of C. neoformans RNA-Seq libraries.

microbiology↗