UCSF

ABSTRACTInvestigating the biosynthesis and function of newly identified intronic small interference RNA in Caenorhabditis elegans

Small interfering RNA (siRNA) are critical regulators of gene expression in many animals. In the model organism C. elegans, our lab found that disruption of the siRNA machinery results in an inability to sense and adapt to specific odorants. Specifically, we found that mutants defective for the RNase III nuclease DCR-1, the double-stranded RNA binding protein RDE-4, the RNA-dependent RNA polymerase RRF-3, or the nuclear RNAi Argonaute NRDE-3 are defective in olfactory adaptation to the odorant, butanone. The lab performed extensive small RNA sequencing experiments to better understand the role of siRNA in olfactory adaptation. By performing an in-depth survey of these siRNA libraries, we identified a class of small RNA (average size, 21 nucleotides) that mapped to intronic regions of more than 30% of the genes in the C. elegans genome. These small RNAs, that map to introns, were mentioned in the Craig Mello lab’s 2009 publication but were largely ignored by the field. We term these understudied small RNA–that map to intronic regions–“isiRNA”, and have performed extensive bioinformatic analysis to understand their origin and function. The major findings we reach from this work are: 1. Two separate bioinformatic pipelines confirm the presence of isiRNA in multiple independent, public-domain small RNA seq C. elegans datasets. 2. isiRNA map non-randomly to longer introns. 3. A diverse set of common siRNA factors are required for accumulation of both intronic siRNA (isiRNA) and exonic siRNA (esiRNA) in the germline. 4. isiRNA are likely produced via the WAGOs pathway. 5. isiRNA may be amplified by the mutator complex which is at the periphery of P granules. 6. isiRNA levels are dependent on many exo-RNAi factors. 7. isiRNA is independent from the Enhanced for Exogenous RNAi (ERI) endogenous RNAi biosynthesis pathway. 8. isiRNA are tertiary RNA. 9. isiRNA map to genes that have significantly more alternative splice variants. 10. isiRNA binds to the germline specific Argonaute, HRDE-1, in order to repress transcription. 11. HRDE-1 and CSR-1 may compete for isiRNA. 12. isiRNA binds to Argonaute CSR-1 to promote the production of siRNA. 13. Cold shock and exogenous RNAi promote the binding of esiRNA and isiRNA to NRDE-3. 14. Lack of ERGO-1 increased the number of genes with higher isiRNA than esiRNA reads. 15. WAGO-1 is required for biosynthesis of germline specific isiRNA. Together, these data reveal a previously uncharacterized population of small RNAs with potentially critical functions in diverse aspects of isiRNA maturation and regulation of gene expression. Future wet-lab experiments will be required to probe the biology of this intriguing class of small RNAs.