Analysis of the Genome and Transcriptome of Cryptococcus neoformans var. grubii Reveals Complex RNA Expression and Microevolution Leading to Virulence Attenuation

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.新型隐球菌（Cryptococcus neoformans）是一种致病性担子菌酵母，每年造成超过60万例死亡。其存在两种血清型（A型和D型），分别对应两个变种，即格特隐球菌变种（C. neoformans var. grubii）和新型隐球菌变种（C. neoformans var. neoformans）。本研究对格特隐球菌变种的基因组进行了测序，并基于RNA测序（RNA-Seq）分析了其转录组结构。我们确定了其染色体定位，解析了着丝粒的序列与结构特征，并鉴定了复制起始位点。该基因组通过自动化注释与人工手动校正完成了注释工作。本研究共鉴定出40000余个内含子，覆盖99%以上的表达基因。尽管多数内含子位于编码DNA序列（CDS）中，但也在非翻译区（UTRs）内发现了2000余个内含子。研究利用携带poly(A)的测序读段，定位了80%以上基因的多聚腺苷酸化位点。对这些位点上下游序列的分析发现了一个全新的与多聚腺苷酸化位点相关的基序（AUGHAH）。此外，本研究还鉴定出1197个杂合小RNA（miscRNAs），这类RNA可进行剪接和/或多聚腺苷酸化，但未表现出明显的编码能力。最后，该基因组序列为实验室传代获得的H99菌株变异体的比较分析提供了支撑。所鉴定的突变谱为解析实验室菌株微观进化的遗传学基础提供了新视角，并鉴定出了与应激响应、交配效率及毒力相关的突变。