Direct targeted degradation of transposon RNAs by the non-canonical RNAi pathway of the fungus Mucor lusitanicus

Mucor lusitanicus has emerged as a model organism for studying RNAi in early-diverging fungi. This fungus exhibits intricate RNAi pathways that play crucial roles in regulating gene expression, de-stroying invasive exogenous genetic material, and controlling the movement of transposable ele-ments (TEs) to ensure genome stability. One of the most fascinating RNAi pathways of this fungus is the non-canonical RNAi pathway (NCRIP), which is independent of Dicer and Argonaute proteins and uses the atypical RNase III R3B2 to degrade specific target mRNAs, playing an essential role in genome stability and virulence. Despite accumulating data suggesting that this pathway is a deg-radation mechanism, there has been no conclusive evidence. Here, we conducted a comparative transcriptomic analysis of mRNA and small RNAs regulated by r3b2, identifying 35 direct NCRIP targets. Most of these direct NCRIP targets correspond to TEs, highlighting the significant role of this RNAi pathway in TE control. Detailed functional analysis of the NCRIP targets confirms the crucial role on r3b2 in regulating gene expression of protein-coding genes and controlling TEs other than centromeric GremLINE1 transposons, emphasizing the important role of r3b2 in genome stability. Interestingly, the RNAs of the NCRIP targets harbor a unique motif consisting of CAG repeats which are known to form hairpin structures which are targeted by RNA interference. Additionally, the generation of transformants expressing mRNAs containing the luciferase reporter gene along direct NCRIP targets reveals that this RNAi pathway is a true degradation mechanism for specific mRNAs. These results are expected to contribute to the understanding of the regulation of the NCRIP pathway through the analysis of its direct targets identified here. RNA-seq experiment to analyze the differences in gene expression at the sRNA and mRNA levels caused by R3B2 KO in M. lusitanicus. Total RNA was extracted from MU636 and MU412 (r3b2 KO) for 24 hours on YPG media (solid) at 26 ºC. For each strain, we performed three biological replicates

葡萄牙毛霉（Mucor lusitanicus）已成为研究早期分歧真菌RNA干扰（RNAi）的模式生物。该真菌拥有复杂的RNAi通路，在调控基因表达、降解入侵的外源遗传物质以及控制转座因子（transposable elements, TEs）的移动以维持基因组稳定性方面发挥关键作用。该真菌最引人关注的RNAi通路之一是非经典RNA干扰通路（non-canonical RNAi pathway, NCRIP），其不依赖Dicer与Argonaute蛋白，通过非典型核糖核酸酶III（RNase III）家族蛋白R3B2降解特定靶mRNA，在基因组稳定性与致病性中发挥核心作用。尽管已有越来越多的数据表明该通路属于降解机制，但迄今尚无确凿证据。本研究对r3b2调控的mRNA与小RNA开展了比较转录组学分析，共鉴定出35个NCRIP直接靶标。其中绝大多数直接靶标均为转座因子，凸显了该RNAi通路在转座因子调控中的重要意义。对NCRIP靶标的详细功能分析证实，R3B2在调控蛋白编码基因的表达以及控制着丝粒外GremLINE1转座子之外的转座因子方面发挥关键作用，进一步强调了R3B2在维持基因组稳定性中的重要价值。有趣的是，NCRIP靶标的RNA携带一段独特的CAG重复序列基序，该序列已知可形成发夹结构并成为RNA干扰的靶向对象。此外，通过构建共表达荧光素酶报告基因mRNA与直接NCRIP靶标的转化子，证实该RNAi通路确实是特异性mRNA的降解机制。本研究结果有望通过解析本次鉴定的直接靶标，推动学界对NCRIP通路调控机制的理解。本研究开展了RNA测序（RNA-seq）实验，以分析葡萄牙毛霉中R3B2基因敲除（knockout, KO）导致的小RNA与mRNA水平的基因表达差异。实验材料为在YPG固体培养基、26℃条件下培养24小时的MU636野生株与MU412（r3b2敲除株）。每个菌株设置3次生物学重复。