Developmental mRNA clearance by PIWI-bound endo-siRNAs in Paramecium

The clearance of untranslated mRNAs by Argonaute proteins is essential for embryonic development in metazoans. However, it is currently unknown whether a similar process exists in unicellular eukaryotes. The ciliate Paramecium tetraurelia harbors a vast array of Argonaute proteins of the Piwi-clade involved in various small RNA pathways, many of which have not yet been investigated. In this work, we investigated the function of a Piwi protein, Ptiwi08, whose expression is limited to a narrow time window during development, concomitant with the start of zygotic transcription. We show that Ptiwi08 acts in an endo-siRNA pathway involved in the clearance of untranslated mRNAs at the post-transcriptional level. These endo-siRNAs are found in clusters that are strictly antisense to their target mRNAs and are a subset of siRNA producing clusters (SRCs). Furthermore, we demonstrate that endo-siRNAs are 2’-O methylated by Hen1 and require Dcr1 for their biogenesis. Our findings suggests that small RNA-mediated developmental mRNA clearance extends beyond metazoans and may be a more widespread mechanism than previously anticipated.