Small RNAs (sRNAs) are key factors in the regulation of gene expression. Recently, a new class of regulatory sRNAs derived from tRNAs was described, the tRNA-derived RNA fragments (tRFs). Such RNAs range in length from 14 to 30 nucleotides and are produced from both mature and primary tRNA transcripts, with very specific cleavage sites along the tRNA sequence. Although several mechanisms have been proposed for how tRFs mediate regulation of gene expression, the exact mechanism of tRF biogenesis and its dependency upon the RNAi pathway remain unclear. Cryptococcus gattii and Cryptococcus neoformans are basidiomycetous yeasts and important human pathogens. While C. neoformans is RNAi proficient, C. gattii VGII has lost essential RNAi genes. Here, we sought to identify the tRF production profile in C. gattii VGII and C. neoformans in order to assess the RNAi-dependency of tRF production in these fungal species. We developed a RNA-sequencing-based tRF prediction workflow designed to improve the currently available prediction tools. Using this methodology, we were able to identify tRFs in both organisms. Despite the loss of the RNAi pathway, C. gattii VGII displayed a number of identified tRFs that did not differ significantly from those observed in C. neoformans. The analysis of predicted tRF targets revealed that a higher number of targets was found for C. gattii VGII tRFs compared to C. neoformans tRFs. These results support the idea that tRFs are at least partially independent of the canonical RNAi machinery, raising questions about possible compensatory roles of alternative regulatory RNAs in the absence of a functional RNAi pathway.

小 RNA (sRNA) 是基因表达调控的关键因素。最近，描述了一类新的来自 tRNA 的调节 sRNA，即 tRNA 衍生的 RNA 片段 (tRFs)。此类 RNA 的长度为 14 到 30 个核苷酸，由成熟和初级 tRNA 转录物产生，沿 tRNA 序列具有非常特定的切割位点。尽管已经提出了几种关于 tRF 如何介导基因表达调节的机制，但 tRF 生物发生的确切机制及其对 RNAi 途径的依赖仍不清楚。加蒂隐球菌和新型隐球菌是担子菌酵母和重要的人类病原体。虽然C. neoformans精通 RNAi，但C. gattiiVGII 失去了必要的 RNAi 基因。在这里，我们试图确定C. gattii VGII 和C. neoformans中的 tRF 生产概况，以评估这些真菌物种中 tRF 生产的 RNAi 依赖性。我们开发了一种基于 RNA 测序的 tRF 预测工作流程，旨在改进当前可用的预测工具。使用这种方法，我们能够识别两种生物中的 tRF。尽管失去了 RNAi 途径，C. gattii VGII 显示了许多已识别的 tRF，这些 tRF 与在新型 C. 中观察到的 tRF 没有显着差异。对预测的 tRF 目标的分析表明，与C. gattii VGII tRF 相比，发现更多的目标C. 新型隐球菌tRF。这些结果支持 tRFs 至少部分独立于经典 RNAi 机制的观点，这引发了关于在缺乏功能性 RNAi 途径的情况下替代调节 RNA 可能的补偿作用的问题。