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Addition of destabilizing poly (A)-rich sequences to endonuclease cleavage sites during the degradation of
I Lisitsky1, P Klaff, G Schuster
1Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
Abstract:
In this work, we report the posttranscriptional addition of poly(A)-rich sequences to mRNA in chloroplasts of higher plants. Several sites in the coding region and the mature end of spinach chloroplast psbA mRNA, which encodes the D1 protein of photosystem II, are detected as polyadenylylated sites. In eukaryotic cells, the addition of multiple adenosine residues to the 3' end of nuclear RNA plays a key role in generating functional mRNAs and in regulating mRNA degradation. In bacteria, the adenylation of several RNAs greatly accelerates their decay. The poly(A) moiety in the chloroplast, in contrast to that in eukaryotic nuclear encoded and bacterial RNAs, is not a ribohomopolymer of adenosine residues, but clusters of adenosines bounded mostly by guanosines and rarely by cytidines and uridines; it may be as long as several hundred nucleotides. Further analysis of the initial steps of chloroplast psbA mRNA decay revealed specific endonuclease cleavage sites that perfectly matched the sites where poly(A)-rich sequences were added. Our results suggest a mechanism for the degradation of psbA mRNA in which endonucleolytic cleavages are followed by the addition of poly(A)-rich sequences to the upstream cleavage products, which target these RNAs for rapid decay.
Insights
Chloroplasts in plants add unique poly(A)-rich sequences to messenger RNA (mRNA), influencing its degradation. This process differs from nuclear and bacterial RNA modification, suggesting a novel decay pathway for chloroplast psbA mRNA.
Area of Science:
- Plant molecular biology
- Chloroplast gene expression
- RNA metabolism
Background:
- Posttranscriptional modification of RNA is crucial for gene expression in eukaryotes and prokaryotes.
- Nuclear mRNA receives a poly(A) tail for stability and translation, while bacterial RNA adenylation often signals decay.
- Chloroplasts, the sites of photosynthesis in plants, possess their own genetic material and regulatory mechanisms.
Purpose of the Study:
- To investigate the phenomenon of polyadenylation in chloroplasts of higher plants.
- To characterize the nature and location of poly(A)-rich sequences on chloroplast mRNA.
- To elucidate the role of these sequences in chloroplast mRNA degradation.
Main Methods:
- Analysis of spinach chloroplast psbA mRNA.
- Identification of polyadenylylated sites using molecular techniques.
- Investigation of mRNA decay intermediates and cleavage sites.
- Characterization of the composition of chloroplast poly(A)-rich sequences.
Main Results:
- Poly(A)-rich sequences were found to be posttranscriptionally added to chloroplast mRNA, including spinach psbA mRNA.
- These sequences are not simple poly(A) homopolymers but contain other nucleotides and can be extensive.
- Specific endonuclease cleavage sites on psbA mRNA correlated with the addition sites of poly(A)-rich sequences.
- The addition of these sequences appears to target upstream cleavage products for rapid decay.
Conclusions:
- Chloroplasts employ a unique mechanism for mRNA degradation involving poly(A)-rich sequence addition.
- This process differs significantly from polyadenylation in eukaryotic nuclei and bacteria.
- The findings suggest a novel pathway for regulating chloroplast gene expression through targeted mRNA decay.