Abstract
We identified the dsRNA binding protein RbdB as an essential component in miRNA processing in Dictyostelium discoideum. RbdB is a nuclear protein that accumulates, together with Dicer B, in nucleolar foci reminiscent of plant dicing bodies. Disruption of rbdB results in loss of miRNAs and accumulation of primary miRNAs. The phenotype can be rescued by ectopic expression of RbdB thus allowing for a detailed analysis of domain function. The lack of cytoplasmic dsRBD proteins involved in miRNA processing, suggests that both processing steps take place in the nucleus thus resembling the plant pathway. However, we also find features e.g. in the domain structure of Dicer which suggest similarities to animals. Reduction of miRNAs in the rbdB- strain and their increase in the Argonaute A knock out allowed the definition of new miRNAs one of which appears to belong to a new non-canonical class.
Highlights
MicroRNAs are an abundant class of regulatory RNAs that are encoded in the genome of eukaryotes
By knock-out and complementation experiments, we identify RNA binding domain protein B (RbdB) in the amoebozoa Dictyostelium as the accessory double-strand RNA binding proteins (dsRBPs) processing component for both steps
Of special evolutionary interest is the supergroup of amoebozoa with the model organism Dictyostelium discoideum which branched off the tree of life after the plants but before the animal/fungi split [5]
Summary
MicroRNAs (miRNAs) are an abundant class of regulatory RNAs that are encoded in the genome of eukaryotes. They are involved in many biological processes, e.g. in development, differentiation and metabolism [summarized in 1,2]. At least one Argonaute-like protein, one Piwi-like protein, one Dicer and one RNA dependent RNA polymerase (RdRP) was suggested to have constituted the basic RNAi machinery in the last common ancestor of eukaryotes [3]. The D. discoideum genome encodes five Argonaute proteins of the Piwi clade (AgnA to AgnE), two Dicer homologues (DrnA and DrnB) and three RdRPs (RrpA to RrpC). Previous experiments have shown that AgnA [6] and RrpC [7] are required for the production of siRNAs while DrnB is essential for miRNA processing [8,9]. DrnA is probably the nuclease that generates siRNAs but apparently, a knockout is lethal
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