Quantitative global studies reveal differential translational control by start codon context across the fungal kingdom

Abstract

Eukaryotic protein synthesis generally initiates at a start codon defined by an AUG and its surrounding Kozak sequence context, but the quantitative importance of this context in different species is unclear. We tested this concept in two pathogenic Cryptococcus yeast species by genome-wide mapping of translation and of mRNA 5′ and 3′ ends. We observed thousands of AUG-initiated upstream open reading frames (uORFs) that are a major contributor to translation repression. uORF use depends on the Kozak sequence context of its start codon, and uORFs with strong contexts promote nonsense-mediated mRNA decay. Transcript leaders in Cryptococcus and other fungi are substantially longer and more AUG-dense than in Saccharomyces. Numerous Cryptococcus mRNAs encode predicted dual-localized proteins, including many aminoacyl-tRNA synthetases, in which a leaky AUG start codon is followed by a strong Kozak context in-frame AUG, separated by mitochondrial-targeting sequence. Analysis of other fungal species shows that such dual-localization is also predicted to be common in the ascomycete mould, Neurospora crassa. Kozak-controlled regulation is correlated with insertions in translational initiation factors in fidelity-determining regions that contact the initiator tRNA. Thus, start codon context is a signal that quantitatively programs both the expression and the structures of proteins in diverse fungi.

Application of an Optimized Annotation Pipeline to theCryptococcus DeuterogattiiGenome Reveals Dynamic Primary Metabolic Gene Clusters and Genomic Impact of RNAi Loss

“…RNA-seq libraries from four growth conditions (exponential phase at 30°C, + exponential phase at 37°C, stationary phase at 30°C, and stationary phase at 37°), conducted in triplicate, of C. neoformans H99 and C. deneoformans JEC21 used in this study have been previously described (WALLACE et al 2020). The C. deuterogattii R265 strain was grown in YPD at 30°C and 37°C under agitation to exponential or early stationary phase as previously described (WALLACE et al 2020).…”

“…The evaluation of the pipeline sensitivity and precision for gene prediction was performed by comparing the predicted annotations against the H99 and JEC21 reference annotations (WALLACE et al, 2020) with the GFFCompare program (PERTEA AND PERTEA 2020).…”

“…neoformans H99 genome were input into the BRAKER1 and Cufflink-CodingQuarry pipelines (WALLACE et al 2020). For the Cufflink-CodingQuarry-based analyses, we used two alternative protocols.…”

“…There are currently eight recognized pathogenic species of Cryptococcus (HAGEN et al 2015;Farrer et al, 2019). Manual annotation of the Cryptococcus neoformans and Cryptococcus deneoformans reference genomes revealed complex and dynamic transcriptomes (JANBON et al 2014;WALLACE et al 2020). These annotations were recently completed through precise identification of the transcript leader (TL) and 3'UTR sequences through TSS-seq and 3UTR-seq analyses; these annotations are likely the most complete and detailed annotations in intron-rich fungi (WALLACE et al 2020).…”

“…Manual annotation of the Cryptococcus neoformans and Cryptococcus deneoformans reference genomes revealed complex and dynamic transcriptomes (JANBON et al 2014;WALLACE et al 2020). These annotations were recently completed through precise identification of the transcript leader (TL) and 3'UTR sequences through TSS-seq and 3UTR-seq analyses; these annotations are likely the most complete and detailed annotations in intron-rich fungi (WALLACE et al 2020). With 99.5% of 6,795 annotated coding genes containing introns, five to six introns per coding gene, and 37,832 introns in total, an automatic annotation of these genomes would be considered highly challenging even with the large sets of RNA-seq data that have been produced (WILM et al 2007;JANBON 2018;WALLACE et al 2020).…”