Chloroplast ribonucleoproteins are associated with both mRNAs and intron-containing precursor tRNAs.
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Tobacco chloroplasts possess five conserved ribonucleoproteins (cpRNPs). To elucidate the function of cpRNPs we analyzed their localization and target nucleic acid molecules in chloroplasts. Immunoprecipitation of the stromal extract and Northern analysis revealed that cpRNPs are associated in vivo with not only various species of chloroplast mRNAs but also intron-containing precursor (pre-) tRNAs. This observation strongly suggests that cpRNPs are involved in RNA processing, including mRNA stability and pre-tRNA splicing. (+info)
Polyadenylation of three classes of chloroplast RNA in Chlamydomonas reinhadtii.
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Three classes of RNA, represented by atpB and petD mRNAs, Arg and Glu tRNAs, and 5S rRNA, were found to exist in polyadenylated form in Chlamydomonas reinhardtii chloroplasts. Sequence analysis of cDNA clones derived from reverse transcriptase-polymerase chain reaction protocols used to select polyadenylated RNAs revealed that, at least for the mRNAs and tRNAs, there are three apparent types of polyadenylation. In the first case, the poly(A) tail is added at or near the mature 3' end, even when this follows a strong secondary structure. In the second case, the tail is added to pre-mRNA or pre-tRNA, suggesting a possible competition between polyadenylation and RNA-processing pathways. Finally, in all cases, the poly(A) tail can be added internally, possibly as a part of an RNA-decay pathway. The tails found in Chlamydomonas chloroplasts differ from those of spinach chloroplasts in adenine content, being nearly homopolymeric (>98% adenine) versus 70% in spinach, and are similar in length to those of Escherichia coli, being mostly between 20 and 50 nt. In vitro assays using a Chlamydomonas chloroplast protein extract showed that a 3' end A25 tail was sufficient to stimulate rapid degradation of atpB RNA in vitro, with a lesser effect for petD, and only minor effects on trnE. We therefore propose that polyadenylation contributes to mRNA degradation in Chlamydomonas chloroplasts, but that its effect may vary. (+info)
Kinetics of photoacclimation in response to a shift to high light of the red alga Rhodella violacea adapted to low irradiance.
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The unicellular rhodophyte Rhodella violacea can adapt to a wide range of irradiances. To create a light stress, cells acclimated to low light were transferred to higher irradiance and the kinetics of various changes produced by the light shift were analyzed. The proton gradient generated by excess light led to a non-photochemical quenching of the chlorophyll fluorescence and some photoinhibition of photosystem II centers was also produced by the light stress. After the shift to higher irradiance, the mRNA levels of three chloroplast genes that encode phycoerythrin and phycocyanin apoproteins and heme oxygenase (the first enzyme specific to the bilin synthesis) were negatively regulated. A change in the amount of thylakoids and in the total pigment content of the cells occurred during light acclimation after a light stress. The change in the size of the phycobilisome was limited to dissapearance of the terminal phycoerythrin hexamers in some of the rods. The ability of R. violacea to photoacclimate depends both on large changes in thylakoid number and pigment content and on smaller changes in the antenna size of photosystem II. (+info)
Complete 5' and 3' end maturation of group II intron-containing tRNA precursors.
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Higher plant chloroplasts provide the only experimentally validated example of functional tRNA genes that are disrupted by group II introns. Here, precursor transcripts for tRNA(Gly)(UCC), tRNA(Val)(UAC), and tRNA(Ala)(UGC) were investigated for processing of 5' leader and 3' trailer sequences in vivo. Use of intron-specific primer pairs and inclusion of a barley chloroplast splicing mutant specifically allowed us to evaluate the potential effect of intervening sequences that disrupt tRNA secondary and tertiary structures. The data suggest that (1) neither integrity of the dihydrouridine nor the anticodon domain is required for the nucleotidyltransferase-mediated addition of 3'-terminal CCA; (2) interruption of these two structural elements by group II introns does not interfere with nucleotide-specific 5' maturation by RNase P; (3) processing intermediates of chloroplast tRNAs can be 3' polyadenylated; and (4) plastid DNA-encoded proteins are not required for 3' and 5' maturation of plastid tRNAs. (+info)
Intron-specific RNA binding proteins in the chloroplast of the green alga Chlamydomonas reinhardtii.
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Mitochondria and chloroplasts both contain group II introns which are believed to be the ancestors of nuclear spliceosomal introns. We used the mitochondrial group II intron rI1 from the green alga Scenedesmus obliquus for biochemical characterization of intron-specific RNA binding proteins. rI1 is correctly spliced from a chloroplast precursor RNA when integrated into the chloroplast genome of Chlamydomonas reinhardtii. Glycerol gradients revealed the sedimentation profile of transcripts containing intron rI1 in native C. reinhardtii extracts and in deproteinized RNA preparations, thus indicating the association of rI1 containing transcripts with high molecular weight ribonucleoprotein complexes in vivo. Furthermore, the specific binding of a 61 kDa protein and a 31 kDa protein with the conserved domain IV was demonstrated using a set of intron derivatives for in vitro RNA binding experiments. We propose that we have biochemically characterized 'general splicing factors', which enable the successful splicing even of mitochondrial introns in chloroplasts. (+info)
The nucleus-encoded HCF107 gene of Arabidopsis provides a link between intercistronic RNA processing and the accumulation of translation-competent psbH transcripts in chloroplasts.
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To understand the functional significance of RNA processing for the expression of plastome-encoded photosynthesis genes, we investigated the nuclear mutation hcf107 of Arabidopsis. The mutation is represented by two alleles, both of which lead to a defective photosystem II (PSII). In vivo protein labeling, in vitro phosphorylation, and immunoblot experiments revealed that the psbB gene product (CP47) and an 8-kD phosphoprotein, the psbH gene product (PsbH), are absent in mutant plants. PsbH and PsbB are essential requirements for PSII assembly in photosynthetic eukaryotes, and their absence in hcf107 is consistent with the PSII-less mutant phenotype. RNA gel blot hybridizations showed that the hcf107 mutation specifically impairs the accumulation of some but not all oligocistronic psbH transcripts that are released from the pentacistronic psbB-psbT-psbH-petB-petD precursor RNA by intergenic endonucleolytic cleavage. In contrast, neither the levels nor the sizes of psbB-containing RNAs are affected. S1 nuclease protection analyses revealed that psbH RNAs are lacking only where psbH is the leading cistron and that they are processed at position -45 in the 5' leader segment of psbH. These data and additional experiments with the cytochrome b(6)f complex mutant hcf152, which is defective in 3' psbH processing, suggest that only those psbH-containing transcripts that are processed at their -45 5' ends can be translated. Secondary structure analysis of the 5' psbH leader predicted the formation of stable stem loops in the nonprocessed transcripts, which are unfolded by processing at the -45 site. We propose that this unfolding of the psbH leader segment as a result of RNA processing is essential for the translation of the psbH reading frame. We suggest further that HCF107 has dual functions: it is involved in intercistronic processing of the psbH 5' untranslated region or the stabilization of 5' processed psbH RNAs, and concomitantly, it is required for the synthesis of CP47. (+info)
Misreading of termination codons in eukaryotes by natural nonsense suppressor tRNAs.
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Translational stop codon readthrough provides a regulatory mechanism of gene expression that is extensively utilised by positive-sense ssRNA viruses. The misreading of termination codons is achieved by a variety of naturally occurring suppressor tRNAs whose structure and function is the subject of this survey. All of the nonsense suppressors characterised to date (with the exception of selenocysteine tRNA) are normal cellular tRNAs that are primarily needed for reading their cognate sense codons. As a consequence, recognition of stop codons by natural suppressor tRNAs necessitates unconventional base pairings in anticodon-codon interactions. A number of intrinsic features of the suppressor tRNA contributes to the ability to read non-cognate codons. Apart from anticodon-codon affinity, the extent of base modifications within or 3' of the anticodon may up- or down-regulate the efficiency of suppression. In order to out-compete the polypeptide chain release factor an absolute prerequisite for the action of natural suppressor tRNAs is a suitable nucleotide context, preferentially at the 3' side of the suppressed stop codon. Three major types of viral readthrough sites, based on similar sequences neighbouring the leaky stop codon, can be defined. It is discussed that not only RNA viruses, but also the eukaryotic host organism might gain some profit from cellular suppressor tRNAs. (+info)
FUGOID: functional genomics of organellar introns database.
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FUGOID is a web-based, taxonomically broad organelle intron database that collects and integrates various functional and structural data on organellar (mitochondrial and chloroplast) introns. The main information provided by FUGOID includes intron sequence, subclass, resident ORF, self-splicing capability, host gene, protein factor(s) involved in splicing, mobility, insertion site, twintron, seminal references and taxonomic position of host organism. It is implemented in a relational database management system, allowing sophisticated, user-friendly searching, data entry and revision. Users can access the database by any common web browser using a variety of operating systems. The main page of the database is available at http://wnt.cc.utexas.edu/~ifmr530/introndata/main.htm. (+info)