Analysis of the Genome and Transcriptome of <i>Cryptococcus neoformans</i> var. <i>grubii</i> 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）是一种致病担子菌酵母，每年造成超过60万例死亡。该菌存在两种血清型（A与D），分别对应两个变种（即 grubii 变种与 neoformans 变种）。本研究对 grubii 变种的新型隐球菌进行了全基因组测序，并基于RNA测序（RNA-Seq）分析了其转录组结构。我们确定了该菌株的染色体定位，分析了着丝粒（centromeres）的序列与结构特征，并鉴定了复制起始位点（origins of replication）。本基因组采用自动化注释与人工审核相结合的方式完成注释。研究共鉴定到超过4万个内含子，覆盖99%以上的表达基因；尽管绝大多数内含子位于编码序列（CDS）中，但也在非翻译区（UTRs）中发现了超过2000个内含子。研究利用含多聚腺苷酸的测序读段，定位了超过80%基因的多聚腺苷酸化位点（polyadenylation sites），对该位点周边序列的分析发现了一种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种混杂RNA（miscRNA），这类RNA可发生剪接和/或多聚腺苷酸化，但未表现出明显的编码能力。最后，本基因组序列为实验室传代后获得的H99菌株变异体的比较分析提供了基础。本研究鉴定的突变谱为解析实验室菌株微进化的遗传基础提供了新见解，并鉴定出与应激响应、交配效率及毒力相关的突变。