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J. Biol. Chem., Vol. 277, Issue 5, 3325-3333, February 1, 2002

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An mRNA 3' Processing Site Targets Downstream Sequences for Rapid Degradation in Chlamydomonas Chloroplasts^*

Amanda Hicks, Robert G. Drager, David C. Higgs^§, and David B. Stern^¶

From the  Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, New York 14853 and the ^§ Department of Biological Sciences, University of Wisconsin-Parkside, Kenosha, Wisconsin 53141

In Chlamydomonas chloroplasts, atpB pre-mRNA matures through a two-step process. Initially, endonuclease cleavage occurs 8-10 nt downstream of the mature 3' end, which itself lies at the end of a stem-loop-forming inverted repeat (IR) sequence. This intermediate product is then trimmed by a 3'  5' exonuclease activity. Although the initial endonucleolytic cleavage by definition generates two products, the downstream product of atpB pre-mRNA endonucleolytic processing cannot be detected, even transiently. This product thus appears to be highly unstable, and it can be hypothesized that specific mechanisms exist to prevent its accumulation. In experiments described here, the atpB 3' maturation site was placed upstream of reporter genes in vivo. Constructs containing both the IR and endonuclease cleavage site (ECS) did not accumulate the reporter gene mRNA, whereas constructs containing only the IR did accumulate the reporter mRNA. The ECS alone gave an intermediate result, suggesting that the IR and ECS act synergistically. Additional secondary structures were used to test whether 5'  3' and/or 3'  5' exonuclease activities mediated degradation. Because these structures did not prevent degradation, rapid endonucleolytic cleavages most likely trigger RNA destruction after ECS cleavage. On the other hand, fragments resulting from cleavage within the endogenous atpB mRNA could occasionally be detected as antisense transcripts of the adjacent reporter genes. Because endonuclease cleavages are also involved in the 5' maturation of chloroplast mRNAs, where only the downstream cleavage product accumulates, it appears that chloroplast endoribonuclease activities have evolved mechanisms to selectively stabilize different ECS products.

This manuscript is dedicated to the memory of Rob Drager.

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