Altering the 3′ UTR endonucleolytic cleavage site of a Chlamydomonas chloroplast mRNA affects 3′-end maturation invitro but not invivo

Altering the 3′ UTR endonucleolytic cleavage site of a Chlamydomonas chloroplast mRNA affects... The 3′ ends of chloroplast mRNAs are produced by the processing of longer precursors. The 3′ ends of most plastid mRNAs are located at, or several nucleotides downstream of, stem-loop structures, which act as 3′-end-processing signals and RNA stability elements. In chloroplasts of the green alga Chlamydomonas reinhardtii, 3′-end maturation of atpB mRNA involves endonucleolytic cleavage of the pre-mRNA at an AU-rich site located about 10 nucleotides downstream of the stem-loop structure. This cleavage is followed by exonucleolytic resection to generate the mature 3′ end. In order to define critical nucleotides of the endonucleolytic cleavage site, we mutated its sequence. Incubation of synthetic atpB pre-RNAs containing these mutations in a chloroplast protein extract resulted in the accumulation of 3′-end-processed products. However, in two cases where the AU-rich sequence of this site was replaced with a GC-rich one, the 3′ end of the stable processing product differed from that of the wild-type product. To examine whether these mutations affected atpB mRNA processing or accumulation in vivo, the endogenous 3′ UTR was replaced with mutated sequences by biolistic transformation of Chlamydomonas chloroplasts. Analysis of the resulting strains revealed that the accumulation of atpB mRNA was approximately equal to that of wild-type cells, and that a wild-type atpB 3′ end was generated. These results imply that Chlamydomonas atpB 3′ processing parallels the situation with other endonucleases such as Escherichia coli RNAse E, where specific sequences are required for correct in vitro processing, but in vivo these mutations can be overcome. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Molecular Biology Springer Journals

Altering the 3′ UTR endonucleolytic cleavage site of a Chlamydomonas chloroplast mRNA affects 3′-end maturation invitro but not invivo

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Publisher
Springer Journals
Copyright
Copyright © 1999 by Kluwer Academic Publishers
Subject
Life Sciences; Biochemistry, general; Plant Sciences; Plant Pathology
ISSN
0167-4412
eISSN
1573-5028
D.O.I.
10.1023/A:1006252201661
Publisher site
See Article on Publisher Site

Abstract

The 3′ ends of chloroplast mRNAs are produced by the processing of longer precursors. The 3′ ends of most plastid mRNAs are located at, or several nucleotides downstream of, stem-loop structures, which act as 3′-end-processing signals and RNA stability elements. In chloroplasts of the green alga Chlamydomonas reinhardtii, 3′-end maturation of atpB mRNA involves endonucleolytic cleavage of the pre-mRNA at an AU-rich site located about 10 nucleotides downstream of the stem-loop structure. This cleavage is followed by exonucleolytic resection to generate the mature 3′ end. In order to define critical nucleotides of the endonucleolytic cleavage site, we mutated its sequence. Incubation of synthetic atpB pre-RNAs containing these mutations in a chloroplast protein extract resulted in the accumulation of 3′-end-processed products. However, in two cases where the AU-rich sequence of this site was replaced with a GC-rich one, the 3′ end of the stable processing product differed from that of the wild-type product. To examine whether these mutations affected atpB mRNA processing or accumulation in vivo, the endogenous 3′ UTR was replaced with mutated sequences by biolistic transformation of Chlamydomonas chloroplasts. Analysis of the resulting strains revealed that the accumulation of atpB mRNA was approximately equal to that of wild-type cells, and that a wild-type atpB 3′ end was generated. These results imply that Chlamydomonas atpB 3′ processing parallels the situation with other endonucleases such as Escherichia coli RNAse E, where specific sequences are required for correct in vitro processing, but in vivo these mutations can be overcome.

Journal

Plant Molecular BiologySpringer Journals

Published: Oct 19, 2004

References

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