Control of mRNA stability in chloroplasts by 3′ inverted repeats: effects of stem and loop mutations on degradation of<i>psb</i>A mRNA<i>in vitro</i>

Adams, Cynthia C.; Stern, David B.

doi:10.1093/nar/18.20.6003

Cited by 85 publications

(52 citation statements)

References 29 publications

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“…Therefore, the 28RNP is not associated with polysomes and is primarily located in the soluble fraction of the chloroplast where only a small fraction of psbA RNA is localized. This is in agreement with the hypothesis that the 28RNP is involved in the 3' end processing of RNA, which is a rapid and short-time process in vivo, as was shown in experiments in which 3' end RNA precursors were electroporated into intact chloroplasts (G. Schuster, P. Klaff, and W. Gruissem, unpublished data; Adams and Stern, 1990).…”

Section: Localization Of the 28rnp In The Chloroplastsupporting

confidence: 90%

RNA-Binding Characteristics of a Ribonucleoprotein from Spinach Chloroplast

Lisitsky

Liveanu²,

Schuster³

1995

Plant Physiol.

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A chloroplast (nuclear-encoded) RNA-binding protein (28RNP) was previously purified from spinach (Spinacia oleracea). This 28RNP was found to be the major RNA-binding protein co-purified during the isolation scheme of 3' end RNA-processing activity of severa1 chloroplastic genes. To learn more about the possible involvement of 28RNP in the 3' end RNA-processing event, we investigated the RNA-binding properties and the location of the protein in the chloroplast. We found that recombinant Escherichia coliexpressed 28RNP binds with apparently the same affinity to every chloroplastic 3' end RNA that was analyzed, as well as to RNAs derived from the 5' end or the coding region of some chloroplastic genes. Differences in the RNA-binding affinities for some chloroplastic 3' end RNAs were observed when the recombinant 28RNP was compared with the "native" 28RNP in the chloroplast-soluble protein extract. In addition, we found that the 28RNP i s not associated with either thylakoid-bound or soluble polysomes in which a great portion of the chloroplast rRNA and mRNA are localized. These results suggest that the native 28RNP binds specifically to certain RNA molecules in the chloroplast in which other components (possibly proteins) and/or posttranslational modifications are involved i n determining RNA-binding specificity of the 28RNP.

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Section: Localization Of the 28rnp In The Chloroplastsupporting

confidence: 90%

RNA-Binding Characteristics of a Ribonucleoprotein from Spinach Chloroplast

Lisitsky

Liveanu²,

Schuster³

1995

Plant Physiol.

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“…time (min) 0 0.25 1 Although the expected products were obtained from the 5' half of the tandem IR construct, we never observed a second IR-containing molecule in processing reactions with uniformly labeled substrates, as might be expected from cleavage between the two IRs in atpBA8D. This suggested either that the putative endonuclease cleavage product was in fact derived by exonucleolytic degradation or that the 3' cleavage product was unstable.…”

Section: Methodsmentioning

confidence: 70%

3'end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a two-step process.

Stern

Kindle

1993

Mol. Cell. Biol.

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Inverted repeat (IR) sequences are found at the 3' ends of most chloroplast protein coding regions, and we have previously shown that the 3'IR is important for accumulation of atpB mRNA in Chlamydomonas reinhardtii (D. B. Stern, E.R. Radwanski, and K. L. Kindle, Plant Cell 3:285-297, 1991). In vitro studies indicate that 3' IRs are inefficient transcription termination signals in higher plants and have furthermore defined processing activities that act on the 3' ends of chloroplast transcripts, suggesting that most chloroplast mRNAs are processed at their 3' ends in vivo. To investigate the mechanism of 3' end processing in Chlamydomonas reinhardtii chloroplasts, the maturation of atpB mRNA was examined in vitro and in vivo. In vitro, a synthetic atpB mRNA precursor is rapidly cleaved at a position 10 nucleotides downstream from the mature 3' terminus. This cleavage is followed by exonucleolytic processing to generate the mature 3' end. In vivo run-on transcription experiments indicate that a maximum of 50% of atpB transcripts are transcriptionally terminated at or near the IR, while the remainder are subject to 3' end processing. Analysis of transcripts derived from chimeric atpB genes introduced into Chlamydomonas chloroplasts by biolistic transformation suggests that in vivo processing and in vitro processing occur by similar or identical mechanisms.

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“…In chloroplast, such stability is mainly influenced by the presence of 5# untranslated regions, or UTRs (Eibl et al, 1999;Zou et al, 2003), nucleus-encoded factors (Lezhneva and Meurer, 2004), and 3#UTRs (Adams and Stern, 1990;Chen and Stern, 1991), without which rapid degradation or low accumulation of primary transcripts has been observed. The role of plastid 3#UTRs differs from the role of its bacterial counterparts by being more involved in transcript stability and less involved in the effective termination of transcription (Stern and Gruissem, 1987).…”

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confidence: 99%

Characterization of Heterologous Multigene Operons in Transgenic Chloroplasts. Transcription, Processing, and Translation

2005

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The first characterization of transcriptional, posttranscriptional, and translational processes of heterologous operons expressed via the tobacco (Nicotiana tabacum) chloroplast genome is reported here. Northern-blot analyses performed on chloroplast transgenic lines harboring seven different heterologous operons revealed that polycistronic mRNA was the predominant transcript produced. Despite the lack of processing of such polycistrons, large amounts of foreign protein accumulation was observed in these transgenic lines, indicating abundant translation of polycistrons. This is supported by polysome fractionation assays, which allowed detection of polycistronic RNA in lower fractions of the sucrose gradients. These results show that the chloroplast posttranscriptional machinery can indeed detect and translate multigenic sequences that are not of chloroplast origin. In contrast to native transcripts, processed and unprocessed heterologous polycistrons were stable, even in the absence of 3# untranslated regions (UTRs). Unlike native 5#UTRs, heterologous secondary structures or 5#UTRs showed efficient translational enhancement independent of cellular control. Abundant read-through transcripts were observed in the presence of chloroplast 3#UTRs but they were efficiently processed at introns present within the native operon. Heterologous genes regulated by the psbA (the photosystem II polypeptide D1) promoter, 5# and 3#UTRs have greater abundance of transcripts than the endogenous psbA gene because transgenes were integrated into the inverted repeat region. Addressing questions about polycistrons, and the sequences required for their processing and transcript stability, are essential in chloroplast metabolic engineering. Knowledge of such factors would enable engineering of foreign pathways independent of the chloroplast complex posttranscriptional regulatory machinery.Plastid genes in higher plants are mainly organized as operons, of which more than 60 have been described in the tobacco (Nicotiana tabacum) chloroplast genome (Sugita and Sugiura, 1996). These may group genes of related or unrelated functions, the former being the most common (Barkan, 1988;Rochaix, 1996). Most of these genes are transcribed into polycistronic precursors that may be later processed and modified to render the transcripts competent for translation (Eibl et al., 1999;Barkan and Goldschmidt-Clermont, 2000;Monde et al., 2000b).The processing mechanisms for translation regulation in chloroplast genes of higher plants are still largely unknown. The general consensus is that most native primary transcripts require processing to be functional (Barkan, 1988;Zerges, 2000;Meierhoff et al., 2003) and that posttranscriptional RNA processing of primary transcripts represents an important control of chloroplast gene expression (Hashimoto et al., 2003;Nickelsen, 2003). However, it is believed that more than one pathway may be involved in transcript processing (Danon, 1997;Choquet and Wollman, 2002).For example, several studies have shown that the regul...

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Control of mRNA stability in chloroplasts by 3′ inverted repeats: effects of stem and loop mutations on degradation ofpsbA mRNAin vitro

Cited by 85 publications

References 29 publications

RNA-Binding Characteristics of a Ribonucleoprotein from Spinach Chloroplast

RNA-Binding Characteristics of a Ribonucleoprotein from Spinach Chloroplast

3'end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a two-step process.

Characterization of Heterologous Multigene Operons in Transgenic Chloroplasts. Transcription, Processing, and Translation

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