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Polyadenylation of three classes of chloroplast RNA in Chlamydomonas reinhardtii

Published online by Cambridge University Press:  01 April 2000

YUTAKA KOMINE
Affiliation:
Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, New York 14853, USA
LINDA KWONG
Affiliation:
Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, New York 14853, USA
MONTSERRAT C. ANGUERA
Affiliation:
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA
GADI SCHUSTER
Affiliation:
Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel
DAVID B. STERN
Affiliation:
Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, New York 14853, USA
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Abstract

Three classes of RNA, represented by atpB and petD mRNAs, Arg and Glu tRNAs, and 5S rRNA, were found to exist in polyadenylated form in Chlamydomonas reinhardtii chloroplasts. Sequence analysis of cDNA clones derived from reverse transcriptase-polymerase chain reaction protocols used to select polyadenylated RNAs revealed that, at least for the mRNAs and tRNAs, there are three apparent types of polyadenylation. In the first case, the poly(A) tail is added at or near the mature 3′ end, even when this follows a strong secondary structure. In the second case, the tail is added to pre-mRNA or pre-tRNA, suggesting a possible competition between polyadenylation and RNA-processing pathways. Finally, in all cases, the poly(A) tail can be added internally, possibly as a part of an RNA-decay pathway. The tails found in Chlamydomonas chloroplasts differ from those of spinach chloroplasts in adenine content, being nearly homopolymeric (>98% adenine) versus 70% in spinach, and are similar in length to those of Escherichia coli, being mostly between 20 and 50 nt. In vitro assays using a Chlamydomonas chloroplast protein extract showed that a 3′ end A25 tail was sufficient to stimulate rapid degradation of atpB RNA in vitro, with a lesser effect for petD, and only minor effects on trnE. We therefore propose that polyadenylation contributes to mRNA degradation in Chlamydomonas chloroplasts, but that its effect may vary.

Type
Research Article
Copyright
© 2000 RNA Society

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