RNA-dependent RNA polymerases (RDR) generate double-stranded (ds)RNA triggers for RNA silencing across eukaryotes. Among the three clades, a-clade and b-clade members are key components of RNA silencing and mediators of stress responses across eukaryotes. However, c-clade members are unusual in that they are represented in phylogenetically distant plants and fungi, and their functions are unknown.Using genetic, bioinformatic and biochemical methods, we show that c-clade RDRs from Oryza sativa L. are involved in plant development as well as regulation of expression of coding and noncoding RNAs.Overexpression of c-clade RDRs in transgenic rice and tobacco plants resulted in robust growth phenotype, whereas their silencing in rice displayed strong inhibition of growth. Small (s)RNA and RNA-seq analysis of OsRDR3 mis-expression lines suggested that it is specifically involved in the regulation of repeat-rich regions in the genome. Biochemical analysis confirmed that OsRDR3 has robust polymerase activities on both single stranded (ss)RNA and ssDNA templates similar to the activities reported for a-clade RDRs such as AtRDR6.Our results provide the first evidence of the importance of c-clade RDRs in plant development, their atypical biochemical activities and their contribution to the regulation of gene expression.
tRNA-derived fragments (tRFs) have recently gained a lot of scientific interest due to their diverse regulatory roles in several cellular processes. However, their function in dynamic biological process such as development and regeneration remains unexplored. Here, we show that tRFs are dynamically expressed during planarian regeneration suggesting a possible role for these small RNAs in the regulation of regeneration. In order to characterise planarian tRFs, we first annotated 457 tRNAs in S.mediterranea combining two tRNA prediction algorithms. Annotation of tRNAs facilitated the identification of three main species of tRFs in planarians -the shorter tRF-5s and itRFs, and the abundantly expressed 5'-tsRNAs. Spatial profiling of tRFs in sequential transverse sections of planarians revealed diverse expression patterns of these small RNAs, including those that are enriched in the head and pharyngeal regions. Expression analysis of these tRF species revealed dynamic expression of these small RNAs over the course of regeneration suggesting an important role in planarian anterior and posterior regeneration. Finally, we show that 5'-tsRNA in planaria interact with all three SMEDWI proteins and an involvement of Ago1 in the processing of itRFs. In summary, our findings implicate a novel role for tRFs in planarian regeneration, highlighting their importance in regulating complex systemic processes. Our study adds to the catalogue of post-transcriptional regulatory systems in planarian, providing valuable insights on the biogenesis and the function of tRFs in neoblasts and planarian regeneration.
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