Addition of destabilizing poly (A)-rich sequences to endonuclease cleavage sites during the degradation of chloroplast mRNA - PubMed (original) (raw)

Addition of destabilizing poly (A)-rich sequences to endonuclease cleavage sites during the degradation of chloroplast mRNA

I Lisitsky et al. Proc Natl Acad Sci U S A. 1996.

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.

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Figures

Figure 1

Posttranscriptional addition of poly(A)-rich sequences to spinach chloroplast psbA mRNA. (A) Nucleotide sequences and locations of the poly(A)-rich stretches that were PCR amplified from oligo(dT)-primed chloroplast cDNA. A schematic representation of the psbA RNA 3′ region is shown. The open box denotes the coding region. The single line represents the 3′ UTR in which the inverted repeats are symbolized by a stem–loop structure. The psbA gene is numbered according to Zurawski et al. (24), and the nucleotides where poly(A)-rich sequences had been added are numbered 1–7, underlined, and printed in boldface type. The poly(A)-rich addition sites coincident with endonucleolytic cleavage sites are circled (numbers 2, 3, and 5). The 3′ end of the mature psbA mRNA is located at nucleotides 1159A and 1160G, immediately following the inverted repeats that form the stem–loop structure (24); two of the poly(A)-rich addition sites, 6 and 7, were located at this position. The gene-specific primers are indicated by arrows. The poly(A)-rich stretches that are shown below, numbered 1–7, are those that were posttranscriptionally added to sites 1–7, respectively. The nucleotides that are not adenines are shaded. (B) Size of poly(A) tracts in chloroplast RNA. Total RNA from mature leaf cells (T) and purified chloroplast RNA (Cp) were labeled with [32P]pCp, followed by complete digestion of the RNAs with RNase T1 and RNase A. An end-labeled, 35-nt-long oligonucleotide was run in the same gel as a size marker (M).

Figure 2

Determination of the initial cleavage sites in the degradation of psbA mRNA. (A) Characterization of psbA degradation intermediates by high-resolution RNA blot analysis. Lysed chloroplasts from spinach leaves were incubated at 25°C for 180 min, and the RNA was recovered and separated in denaturing polyacrylamide gels, which were electroblotted to nylon membranes and hybridized with a32P-radiolabeled oligonucleotide complementary to the 5′ or 3′ end of psbA mRNA. (B) Determination of the initial psbA decay intermediate cleavage sites by primer extension analysis. The 5′ ends of the degradation intermediates were determined by primer extension analysis. Oligonucleotide primers complementary to positions 801–823 (Upper) or complementary to positions 1084–1105 (Lower) of the coding region of psbA mRNA were used. Lanes G, A, T, and C show the sequencing reactions of the corresponding cloned fragments. Nucleotide numbers of reverse transcriptase stops in the_psbA_ gene are indicated. Positions of the three corresponding poly(A)-rich addition sites are given in parentheses.

Figure 3

Polyadenylylation of synthetic_psbA_ transcripts in vitro using an extract of soluble chloroplast proteins. (A) Radioactive RNA corresponding to the unprocessed 3′ UTR of the chloroplast_psbA_ mRNA was incubated in the presence of 0.5 mM ATP with chloroplast extract (Cp Extract) or yeast poly(A) polymerase. Lane −, RNA that was incubated for 60 min without addition of protein. The lengths of the RNAs in nucleotides are shown at the left. (B) The synthetic RNA described in A was incubated for 1 h with the chloroplast protein extract in the presence of 0.5 mM ATP (lane A), CTP (lane C), GTP (lane G), or UTP (lane U). (C) RNA terminating with a stem–loop structure is poorly polyadenylylated in chloroplast protein extract. Synthetic RNAs corresponding to the unprocessed precursor, mature 3′ end and part of the coding region of the petD 3′ end RNA, were incubated for the times indicated in the figure with chloroplast extract in the presence of 0.5 mM ATP. A schematic representation of the RNA substrates is shown on the right. The open box denotes the amino acid coding region of the mRNA.

Figure 4

Effect of polyadenylylation on the 3′ end processing and degradation of RNAs in vitro. In vitro_-synthesized [32P]RNA corresponding to the 3′ end of the psbA precursor RNA (A) or the_petD amino acid coding region and part of the 3′ UTR (B), and the same RNAs that were first polyadenylylated_in vitro,_ were incubated in the chloroplast protein extract without the addition of ATP, either each alone (A) or as a mixture (B). Samples were taken at the times indicated in the figure and analyzed by denaturing gel electrophoresis and autoradiography.

References

1. Jackson R J, Standart N. Cell. 1990;62:15–24. - PubMed
1. Baker E J. In: Control of mRNA Stability. Belasco J G, Brawerman G, editors. New York: Academic; 1993. pp. 367–415.
1. Sachs A B. Cell. 1993;74:413–421. - PubMed
1. Wahle E, Keller W. Annu Rev Biochem. 1992;61:419–440. - PubMed
1. Manley J L. Curr Opin Genet Dev. 1995;5:222–228. - PubMed

Addition of destabilizing poly (A)-rich sequences to endonuclease cleavage sites during the degradation of chloroplast mRNA - PubMed (original) (raw)