Related Experiment Videos
E. coli RpsO mRNA decay: RNase E processing at the beginning of the coding sequence stimulates poly(A)-dependent
1UPR 9073 du CNRS, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, Paris, 75005, France.
Abstract:
The rpsO mRNA of E. coli encoding ribosomal protein S15 is destabilized by poly(A) tails posttranscriptionally added by poly(A)polymerase I. We demonstrate here that polyadenylation also contributes to the rapid degradation of mRNA fragments generated by RNase E. It was already known that an RNase E cleavage occurring at the M2 site, ten nucleotides downstream of the coding sequence of rpsO, removes the 3' hairpin which protects the primary transcript from the attack of polynucleotide phosphorylase and RNase II. A second RNase E processing site, referred to as M3, is now identified at the beginning of the coding sequence of rpsO which contributes together with exonucleases to the degradation of messengers processed at M2. Cleavages at M2 and M3 give rise to mRNA fragments which are very rapidly degraded in wild-type cells. Poly(A)polymerase I contributes differently to the instability of these fragments. The M3-M2 internal fragment, generated by cleavages at M3 and M2, is much more sensitive to poly(A)-dependent degradation than the P1-M2 mRNA, which exhibits the same 3' end as M3-M2 but harbours the 5' end of the primary transcript. We conclude that 5' extremities modulate the poly(A)-dependent degradation of mRNA fragments and that the 5' cleavage by RNase E at M3 activates the chemical degradation of the rpsO mRNA.
Insights
Polyadenylation destabilizes E. coli rpsO mRNA fragments. RNase E cleavage at site M3, combined with polyadenylation, accelerates mRNA degradation, with 5' end determining degradation sensitivity.
Area of Science:
- Molecular Biology
- Bacterial Gene Regulation
Background:
- Polyadenylation by poly(A)polymerase I posttranscriptionally destabilizes E. coli rpsO mRNA.
- RNase E is a key endonuclease involved in mRNA processing and degradation in E. coli.
Purpose of the Study:
- To investigate the role of polyadenylation in the degradation of mRNA fragments generated by RNase E.
- To identify and characterize new RNase E cleavage sites in rpsO mRNA.
- To elucidate how 5' and 3' extremities influence poly(A)-dependent mRNA degradation.
Main Methods:
- Identification of RNase E cleavage sites (M2 and M3) in rpsO mRNA.
- Analysis of mRNA fragment stability in wild-type E. coli.
- Differential stability assays comparing polyadenylated and non-polyadenylated mRNA fragments with varying 5' and 3' ends.
Main Results:
- RNase E cleavage at M2 and M3 sites generates mRNA fragments that are rapidly degraded.
- Polyadenylation significantly enhances the degradation of the M3-M2 internal fragment.
- The 5' end of mRNA fragments modulates their sensitivity to poly(A)-dependent degradation, with the M3-M2 fragment being more sensitive than P1-M2 mRNA.
Conclusions:
- RNase E cleavage at the M3 site activates rpsO mRNA for rapid degradation.
- The 5' extremity of mRNA fragments plays a crucial role in modulating poly(A)-dependent degradation.
- Polyadenylation, in conjunction with specific RNase E processing, is a significant pathway for bacterial mRNA decay.