Regulation Of C. Elegans Gene By Endogenous E. Coli Noncoding RNA DsrA

Objective:Regulation of C.elegans gene by E. coli noncoding RNA was investigated byobserving longevity of C. elegans feeding on DsrA-expressing E.coli. The regulatingpathway of this noncoding RNA and its role in species were also studied.Methods:Longevity of eleven types of C. elgans (N2, dcr-1, rde-1, rde-2, rde-4, alg-1, alg-2,rrf-3, eri-3, tm3026,tm2908) feeding on wild type E. coli K12or DsrA deletion strains(NM6003or DDS719) was examined by life span assays respectively.Semi-quantitative RT-PCR and quantitative real-time PCR were used to analyze C.elegans mRNA level influenced by DsrA, and the impacts of RNAi correlative geneon the DsrA effects. The changes of GFP signal in transgenic C. elegans, which hadbeen injected with DsrA RNA, were observed by confocal microscope. Function ofDsrA on both E. coli and C. elegans was also examined through long term mutualimpact experiment between worms and E. coli.Result:1. N2worms，feeding on DsrA mutants (NM6003or DDS719)， lived longer thanon wild-type E. coli K12(P＜0.01).2. There was no significant difference in longevity between different groups ofF42G9.6mutants (tm2908) feeding on E. coli K12or DsrA deletion mutant. However,tm2908worms on the E. coli K12lived shorter than N2worms on the same bacteria(P＜0.05).3. Semi-quantitative RT-PCR and quantitative real-time PCR results showed thatthe mRNA level of F42G9.6was lower in N2worms feeding on E. coli K12than on DsrA deficient mutants (P＜0.01).4. GFP imaging showed that injection of DsrA RNA into Pdpy-30::F42G9.6::gfptransgenic worms reduced GFP signal. Quantification of GFP intensity also showedthat DsrA decreased F42G9.6::GFP signals significantly (P＜0.01). No obviouschange was observed when PF42G9.6::gfp transgenic C. elegans had been injectedwith DsrA RNA.5. The life span assays of RNAi correlative gene mutants showed that thelongevity-decreasing effect of DsrA in rde-4mutants was not as prominent as that inN2worms(P＜0.05), while the life span of other seven types of mutant C. eleganswas significantly different between E. coli K12and DsrA mutant bacteria (P＜0.01).rde-2mutants lived much longer than N2worms on either of the two types ofbacteria.6. Real-time PCR results showed that rde-4mutants on DsrA mutant bacteriadisplayed much less increase in F42G9.6mRNA level in contrast to N2worms (P＜0.05), while rde-2mutant showed much greater increase in F42G9.6mRNA level onDsrA deletion mutant(P＜0.01).7. The results of long term mutual impact experiment between worms and E. colishowed that there were more worms on NM6003bacteria than those on K12foodafter two weeks (P＜0.05). The ratios of K12to NM6003bacteria on C. elegansplates were significantly higher than those control plates without worm (P＜0.05).Conclusion:1. DsrA-expressing E. coli decreases the longevity of C. elegans.2. E. coli noncoding RNA DsrA decreases C. elegans F42G9.6mRNA level.3. DsrA-expressing E. coli inhibits C. elegans gene expression through a pathwaydifferent from currently known feeding RNAi.4. DsrA has protective effect to E. coli.