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. Overall design: 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