Drosophila melanogaster retrotransposon and inverted repeat-derived endogenous siRNAs are differentially processed in distinct cellular locations.

ABSTRACT:

Background

Endogenous small interfering (esi)RNAs repress mRNA levels and retrotransposon mobility in Drosophila somatic cells by poorly understood mechanisms. 21 nucleotide esiRNAs are primarily generated from retrotransposons and two inverted repeat (hairpin) loci in Drosophila culture cells in a Dicer2 dependent manner. Additionally, proteins involved in 3' end processing, such as Symplekin, CPSF73 and CPSR100, have been recently implicated in the esiRNA pathway.

Results

Here we present evidence of overlap between two essential RNA metabolic pathways: esiRNA biogenesis and mRNA 3' end processing. We have identified a nucleus-specific interaction between the essential esiRNA cleavage enzyme Dicer2 (Dcr2) and Symplekin, a component of the core cleavage complex (CCC) required for 3' end processing of all eukaryotic mRNAs. This interaction is mediated by the N-terminal 271 amino acids of Symplekin; CCC factors CPSF73 and CPSF100 do not contact Dcr2. While Dcr2 binds the CCC, Dcr2 knockdown does not affect mRNA 3' end formation. RNAi-depletion of CCC components Symplekin and CPSF73 causes perturbations in esiRNA abundance that correlate with fluctuations in retrotransposon and hairpin esiRNA precursor levels. We also discovered that esiRNAs generated from retrotransposons and hairpins have distinct physical characteristics including a higher predominance of 22 nucleotide hairpin-derived esiRNAs and differences in 3' and 5' base preference. Additionally, retrotransposon precursors and derived esiRNAs are highly enriched in the nucleus while hairpins and hairpin derived esiRNAs are predominantly cytoplasmic similar to canonical mRNAs. RNAi-depletion of either CPSF73 or Symplekin results in nuclear retention of both hairpin and retrotransposon precursors suggesting that polyadenylation indirectly affects cellular localization of Dcr2 substrates.

Conclusions

Together, these observations support a novel mechanism in which differences in localization of esiRNA precursors impacts esiRNA biogenesis. Hairpin-derived esiRNAs are generated in the cytoplasm independent of Dcr2-Symplekin interactions, while retrotransposons are processed in the nucleus.

SUBMITTER: Harrington AW 

PROVIDER: S-EPMC5392987 | biostudies-literature |

REPOSITORIES: biostudies-literature