Secondary Structures Common to Chloroplast mRNA 3′-Untranslated Regions Direct Cleavage by CSP41, an Endoribonuclease Belonging to the Short Chain Dehydrogenase/Reductase Superfamily

Thomas J Bollenbach,David B Stern

doi:10.1074/jbc.m303559200

Abstract

CSP41 (chloroplast stem-loop-binding protein of 41 kDa), a chloroplast endonuclease belonging to the SDR superfamily, preferentially cleaves stem-loop-containing RNAs in vitro. This potentially directs it to the 3'-ends of mature chloroplast mRNAs, which generally possess such structures. To understand the basis for this discrimination, the RNA elements directing CSP41 cleavage of petD RNA in vitro were dissected. Substrates containing fully base-paired stem-loops were optimal substrates, whereas deletion of part of the stem-loop decreased activity by 100-fold, and deletion of the distal arm of the stem-loop abolished cleavage, even in substrates containing the primary CSP41 cleavage site. Competition assays showed that the decrease in activity resulted from decreased affinity for the RNA by CSP41. Mutations of the residues at the scissile bond and mutations and deletions at the terminal loop of the stem had a moderate effect on activity but no effect on cleavage site specificity, suggesting that CSP41 has no sequence specificity. Titration of ethidium bromide into the assay decreased activity to a basal level of approximately 18%, and introduction of a single base bulge into either arm of the stem-loop decreased cleavage at the primary cleavage site by up to 70%. This suggests that changing the structure of the helical stem has a mild effect on activity. Deletion analysis of CSP41 suggests that the specificity domain lies in the first 73 amino acids of the protein, a domain that also contains a putative dehydrogenaselike mononucleotide binding motif. These results are consistent with a broad role for CSP41 in the degradation of stem-loop-containing mRNAs.

Highlights

In the chloroplasts of higher plants and green algae, gene expression is regulated primarily at the post-transcriptional level
Mutations of the residues at the scissile bond and mutations and deletions at the terminal loop of the stem had a moderate effect on activity but no effect on cleavage site specificity, suggesting that CSP41 has no sequence specificity
We first used a series of deletion mutants of the preferred petD RNA substrate, as shown in Fig. 1A. petD⌬18 corresponds to the mature form of the petD mRNA 3Ј-untranslated regions (UTRs), whereas petD⌬63 and petD⌬24 remove part or all of the stemloop structure but still retain the primary cleavage site for CSP41 at position 136

Summary

Introduction

In the chloroplasts of higher plants and green algae, gene expression is regulated primarily at the post-transcriptional level. Whereas CSP41 was shown to cleave double-stranded RNA substrates, its activity was optimal with stem-loop-containing RNAs [15].

Results

Conclusion