Estructura y función de la Ribonucleasa R3B2 implicada en el mecanismo de silenciamiento génico de "Mucor lusitanicus"

Abstract

Post-transcriptional gene silencing (PTGS) or RNA interference (RNAi) was one of the most outstanding discoveries of the 90s decade that impacted biology fields. Nowadays multiple researches are still being conducted to understand the diverse RNAi pathways widespread among every eukaryotic lineage. These mechanisms utilize small interfering RNAs (siRNAs) to target and either degrade the complementary transcripts or block their translation into proteins. Since the initial discovery of the RNAi in fungi, a continuously expanding world of small RNAs (sRNAs) and RNAi mechanisms have demonstrated to participate in the control of fundamental processes of the lifestyle of these organisms such as growth, reproduction, stress adaptation, and pathogenesis. Here in this work, we carry out a detailed characterization of the atypical RNase R3B2 of Mucor lusitanicus in order to shed light over the complex interaction of the main RNAi pathways of this fungus, which are a canonical RNAi mechanism, where the Dicer RNases produce the siRNA, and a non-canonical RNAi pathway (NCRIP), driven by R3B2 that degrades specific endogenous transcripts without the production of typical siRNA. We determined that R3B2 binds in vitro both single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA), although it is only capable to cut ssRNA. The dimerization of the protein results in a narrowed RNA channel regarding other well described RNase III proteins that suggests a restricted access of dsRNA to the catalytic centers of the RNase. In fact, the relative position of a lysine (K84) to the catalytic centers and the charged nature of the lateral chain of the amino acid could explain the preference of U in the penultimate position of the sRNAs generated by the NCRIP, allowing us to propose a mechanistic model for ssRNA processing by R3B2. The in vivo characterization of R3B2 revealed its interaction with Dicer proteins of M. lusitanicus establishing a direct link between the canonical RNAi and the NCRIP. The yeast-two hybrid assay showed that the contact between R3B2 and Dicer-1 was stronger that the detected with Dicer-2, involving two clearly different regions of the proteins. These interactions suggest an inhibitory role of R3B2 over Dicer activity by affecting the PAZ-mediated binding of dsRNA and resembled the characterized in animals between Dicer proteins and their multi-dsRBD domain-containing partners. Our in vivo mutational analysis of the interacting regions of R3B2 revealed that both regions are essential for R3B2 in the canonical RNAi and suggest an explanation to the positive and negative role of R3B2 on the processes mediated by this pathway. The substrate specificity and these in vivo results suggest that R3B2 is necessary to cleave the ssRNA stretches of the dsRNA in order to produce adequate dsRNA termini for Dicer processing. We explored this possibility by analyzing the effect of R3B2 absence over the sRNA length and strand polarity distribution during active growth and the stationary phase. The results obtained revealed two clearly different situations in regard of the growth state and the type of genes regulated by the RNAi mechanisms of M. lusitanicus. While R3B2 is necessary to produce exonic-siRNAs (ex-siRNA) during exponential growth, it was completely dispensable or even a negative regulator of the production of these siRNAs during the stationary phase. In addition, it was dispensable for the silencing of Genomic Retrotransposable Elements of Mucoromycotina LINE1-like (Grem-LINE1) during exponential growth, but negatively regulates them during the stationary phase, as previously described.