Blocking polyadenylation of mRNA in the chloroplast inhibits its degradation

Lisitsky, Irena; Klaff, Petra; Schuster, Gadi

doi:10.1046/j.1365-313x.1997.12051173.x

Cited by 31 publications

(25 citation statements)

References 24 publications

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“…The polyadenylated molecules are then rapidly degraded by the 39/59 exonuclease polynucleotide phosphorylase (PNPase) and possibly other enzymes (Lisitsky et al, 1997a(Lisitsky et al, , 1997bWalter et al, 2002). Based on these and other results (Klaff, 1995), RNA degradation in chloroplasts is currently hypothesized to begin with endonucleolytic cleavage, for example, by the IR-recognizing enzyme CSP41 Stern, 2003a, 2003b), followed by the addition of a poly(A) or poly(A)-rich tail and exonucleolytic degradation.…”

Section: Introductionmentioning

confidence: 99%

Antisense Transcript and RNA Processing Alterations Suppress Instability of Polyadenylated mRNA in Chlamydomonas Chloroplasts

et al. 2004

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In chloroplasts, the control of mRNA stability is of critical importance for proper regulation of gene expression. The Chlamydomonas reinhardtii strain D26pAtE is engineered such that the atpB mRNA terminates with an mRNA destabilizing polyadenylate tract, resulting in this strain being unable to conduct photosynthesis. A collection of photosynthetic revertants was obtained from D26pAtE, and gel blot hybridizations revealed RNA processing alterations in the majority of these suppressor of polyadenylation (spa) strains, resulting in a failure to expose the atpB mRNA 39 poly(A) tail. Two exceptions were spa19 and spa23, which maintained unusual heteroplasmic chloroplast genomes. One genome type, termed PSþ, conferred photosynthetic competence by contributing to the stability of atpB mRNA; the other, termed PSÿ, was required for viability but could not produce stable atpB transcripts. Based on strand-specific RT-PCR, S1 nuclease protection, and RNA gel blots, evidence was obtained that the PSþ genome stabilizes atpB mRNA by generating an atpB antisense transcript, which attenuates the degradation of the polyadenylated form. The accumulation of double-stranded RNA was confirmed by insensitivity of atpB mRNA from PSþ genome-containing cells to S1 nuclease digestion. To obtain additional evidence for antisense RNA function in chloroplasts, we used strain D26, in which atpB mRNA is unstable because of the lack of a 39 stem-loop structure. In this context, when a 121-nucleotide segment of atpB antisense RNA was expressed from an ectopic site, an elevated accumulation of atpB mRNA resulted. Finally, when spa19 was placed in a genetic background in which expression of the chloroplast exoribonuclease polynucleotide phosphorylase was diminished, the PSþ genome and the antisense transcript were no longer required for photosynthesis. Taken together, our results suggest that antisense RNA in chloroplasts can protect otherwise unstable transcripts from 39!59 exonuclease activity, a phenomenon that may occur naturally in the symmetrically transcribed and densely packed chloroplast genome.

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Section: Introductionmentioning

confidence: 99%

Antisense Transcript and RNA Processing Alterations Suppress Instability of Polyadenylated mRNA in Chlamydomonas Chloroplasts

et al. 2004

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“…The chloroplast translation apparatus in many respects resembles the prokaryotic system but also has eukaryotic characteristics (45). If an equivalent to the poly(A) tail-poly(A)-binding protein-mediated translation activation mechanism exists in chloroplasts, it must involve elements other than the poly(A) tail, which actually destabilizes chloroplast transcripts (27,(30)(31)(32). One candidate element would be the 3Ј-end stemloop structure and/or proteins which bind in this region.…”

Section: Discussionmentioning

confidence: 99%

“…A model invoking mRNA circularization has been proposed, in which the mRNA 5Ј and 3Ј ends can interact via this association, which in turn is required for the initiation of translation (10,16,18,40,50). Although polyadenylation of mRNA has recently been described for spinach chloroplasts, it occurs primarily on degradation products and not at the 3Ј end of the intact transcript (27,(30)(31)(32). As in Escherichia coli (41), chloroplast polyadenylation is transient and seems to target mRNA for rapid degradation.…”

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confidence: 99%

3′-Processed mRNA Is Preferentially Translated in Chlamydomonas reinhardtii Chloroplasts

Rott

Levy

Drager

et al. 1998

Molecular and Cellular Biology

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3-end processing of nucleus-encoded mRNAs includes the addition of a poly(A) tail that is important for translation initiation. Since the vast majority of chloroplast mRNAs acquire their 3 termini by processing yet are not polyadenylated, we asked whether 3 end maturation plays a role in chloroplast translation. A general characteristic of the 3 untranslated regions of chloroplast mRNAs is an inverted repeat (IR) sequence that can fold into a stem-loop structure. These stem-loops and their flanking sequences serve as RNA 3-end formation signals. Deletion of the Chlamydomonas chloroplast atpB 3 IR in strain ⌬26 results in reduced accumulation of atpB transcripts and the chloroplast ATPase ␤-subunit, leading to weakly photosynthetic growth. Of the residual atpB mRNA in ⌬26, approximately 1% accumulates as a discrete RNA of wild-type size, while the remainder is heterogeneous in length due to the lack of normal 3 end maturation. In this work, we have analyzed whether these unprocessed atpB transcripts are actively translated in vivo. We found that only the minority population of discrete transcripts of wild-type size is associated with polysomes and thus accounts for the ATPase ␤-subunit which accumulates in ⌬26. Analysis of chloroplast rbcL mRNA revealed that transcripts extending beyond the mature 3 end were not polysome associated. These results suggest that 3-end processing of chloroplast mRNA is required for or strongly stimulates its translation.Chloroplast genes are often organized into operons and gene clusters, which are transcribed into precursor transcripts that undergo complex processing events including splicing and intercistronic cleavages (reviewed in references 35 and 49). While intercistronic cleavages form some mRNA 5Ј and 3Ј termini, these can also be formed by other types of events. For example, 5Ј ends are often formed by endonucleolytic processing of primary transcripts, and this may be the exclusive mode of 5Ј end formation in chloroplasts in the green alga Chlamydomonas reinhardtii (reviewed in reference 12). Most plastid mRNAs contain inverted-repeat (IR) sequences in their 3Ј untranslated regions, which are believed to fold into stem-loop structures. These IR sequences do not function as efficient transcription terminators but instead are thought to stabilize upstream sequences and mediate correct 3Ј-end processing (36,37,44,47,48). In most cases, the 3Ј termini of mature transcripts lie immediately downstream of the IR.Plastid 3Ј IR sequences act to stabilize upstream mRNA segments in vitro and in vivo. When RNA molecules containing the IR sequences were incubated in spinach chloroplast protein extracts, they were correctly processed at their 3Ј ends and the products were stable for several hours. However, when the IR sequences were deleted from the same RNA molecules and incubated in an identical protein extract, the RNA molecules were rapidly degraded (17,37,44,46). The ability to introduce altered genes into the chloroplast of the green alga C. reinhardtii presented the opportunity to ...

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“…Stabilize Chloroplast RNAs-To study chloroplast mRNA degradation we recently established an in vitro degradation system that faithfully reflects mRNA degradation in terms of cleavages and processing of degradation fragments after cleavage (8,9,10). This system consists of isolated, lysed chloroplasts and allows the observation of the internal mRNA that is complexed with proteins as in the native state as well as the analysis of additionally added transcripts.…”

Section: Magnesium Ionsmentioning

confidence: 99%

“…In higher plants plastid mRNA degradation is initiated by endonucleolytic cleavages (8). The resulting proximal fragments are polyadenylated as a tag for rapid exonucleolytic decay (9,10). This mechanism implies that initiation of mRNA decay is the crucial process by which the stability of a certain message is regulated.…”

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Gene-specific trans-Regulatory Functions of Magnesium for Chloroplast mRNA Stability in Higher Plants

Horlitz

Klaff

2000

Journal of Biological Chemistry

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In higher plant chloroplasts the accumulation of plastid-encoded mRNAs during leaf maturation is regulated via gene-specific mRNA stabilization. The half-lives of chloroplast RNAs are specifically affected by magnesium ions. psbA mRNA (D1 protein of photosystem II), rbcL mRNA (large subunit of ribulose-1,5-bisphosphate carboxylase), 16 S rRNA, and tRNA His gain stability at specific magnesium concentrations in an in vitro degradation system from spinach chloroplasts. Each RNA exhibits a typical magnesium concentration-dependent stabilization profile. It shows a cooperative response of the stability-regulated psbA mRNA and a saturation curve for the other RNAs. The concentration of free Mg 2؉ rises during chloroplast development within a range sufficient to mediate gene-specific mRNA stabilization in vivo as observed in vitro. We suggest that magnesium ions are a trans-acting factor mediating differential mRNA stability.

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Blocking polyadenylation of mRNA in the chloroplast inhibits its degradation

Cited by 31 publications

References 24 publications

Antisense Transcript and RNA Processing Alterations Suppress Instability of Polyadenylated mRNA in Chlamydomonas Chloroplasts

Antisense Transcript and RNA Processing Alterations Suppress Instability of Polyadenylated mRNA in Chlamydomonas Chloroplasts

3′-Processed mRNA Is Preferentially Translated in Chlamydomonas reinhardtii Chloroplasts

Gene-specific trans-Regulatory Functions of Magnesium for Chloroplast mRNA Stability in Higher Plants

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