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      Small RNAs reveal two target sites of the RNA-maturation factor Mbb1 in the chloroplast of Chlamydomonas

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          Abstract

          Many chloroplast transcripts are protected against exonucleolytic degradation by RNA-binding proteins. Such interactions can lead to the accumulation of short RNAs (sRNAs) that represent footprints of the protein partner. By mining existing data sets of Chlamydomonas reinhardtii small RNAs, we identify chloroplast sRNAs. Two of these correspond to the 5′-ends of the mature psbB and psbH messenger RNAs (mRNAs), which are both stabilized by the nucleus-encoded protein Mbb1, a member of the tetratricopeptide repeat family. Accordingly, we find that the two sRNAs are absent from the mbb1 mutant. Using chloroplast transformation and site-directed mutagenesis to survey the psbB 5′ UTR, we identify a cis-acting element that is essential for mRNA accumulation. This sequence is also found in the 5′ UTR of psbH, where it plays a role in RNA processing. The two sRNAs are centered on these cis-acting elements. Furthermore, RNA binding assays in vitro show that Mbb1 associates with the two elements specifically. Taken together, our data identify a conserved cis-acting element at the extremity of the psbH and psbB 5′ UTRs that plays a role in the processing and stability of the respective mRNAs through interactions with the tetratricopeptide repeat protein Mbb1 and leads to the accumulation of protected sRNAs.

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          Most cited references 67

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          Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis.

          The complete sequence of the Arabidopsis thaliana genome revealed thousands of previously unsuspected genes, many of which cannot be ascribed even putative functions. One of the largest and most enigmatic gene families discovered in this way is characterized by tandem arrays of pentatricopeptide repeats (PPRs). We describe a detailed bioinformatic analysis of 441 members of the Arabidopsis PPR family plus genomic and genetic data on the expression (microarray data), localization (green fluorescent protein and red fluorescent protein fusions), and general function (insertion mutants and RNA binding assays) of many family members. The basic picture that arises from these studies is that PPR proteins play constitutive, often essential roles in mitochondria and chloroplasts, probably via binding to organellar transcripts. These results confirm, but massively extend, the very sparse observations previously obtained from detailed characterization of individual mutants in other organisms.
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            Pentatricopeptide repeat proteins: a socket set for organelle gene expression.

            Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that are particularly prevalent in terrestrial plants. Although the PPR protein family was only recognized eight years ago, it is already clear that these proteins have a range of essential functions in post-transcriptional processes (including RNA editing, RNA splicing, RNA cleavage and translation) within mitochondria and chloroplasts. Several PPR proteins have been shown to act as fertility restorer genes in commercially important cytoplasmic male sterility systems. Here, we discuss several recent papers that cover their evolutionary history and molecular mode of action. We use these new data to propose hypotheses for their physiological roles that could explain why PPR proteins are so numerous in terrestrial plants.
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              Improved northern blot method for enhanced detection of small RNA.

              This protocol describes an improved northern blot method that enhances detection of small RNA molecules (<40 nt) including regulatory species such as microRNA (miRNA), short-interfering RNA (siRNA) and Piwi-interacting RNA. Northern blot analysis involves the separation of RNA molecules by denaturing gel electrophoresis followed by transfer and cross-linking of the separated molecules to nylon membrane. RNA of interest is then detected by hybridization with labeled complementary nucleic acid probes. We have replaced conventional UV-cross-linking of RNA to nylon membranes with a novel, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated, chemical cross-linking step that enhances detection of small RNA by up to 50-fold. This requires no specialized equipment, is relatively inexpensive and is technically straightforward. Northern blotting can be done in 2 d, but detection of a specific RNA can vary from minutes to days. Although chemical cross-linking takes longer (15 min to 2 h) than UV cross-linking, improved sensitivity means shorter periods of exposure are required to detect signal after hybridization.
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                Author and article information

                Journal
                Nucleic Acids Res
                Nucleic Acids Res
                nar
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                March 2014
                11 December 2013
                11 December 2013
                : 42
                : 5
                : 3286-3297
                Affiliations
                1Department of Botany and Plant Biology and Institute of Genetics and Genomics in Geneva University of Geneva, CH-1211 Geneva 4, Switzerland and 2Institute of Biology, Molecular Genetics, Humboldt University of Berlin, D-10115 Berlin, Germany
                Author notes
                *To whom correspondence should be addressed. Tel: +41 22 379 6188; Fax: +41 22 379 6868; Email: Michel.Goldschmidt-Clermont@ 123456unige.ch

                These authors contributed equally to the paper as first authors.

                Article
                gkt1272
                10.1093/nar/gkt1272
                3950674
                24335082
                © The Author(s) 2013. Published by Oxford University Press.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

                Page count
                Pages: 12
                Categories
                RNA

                Genetics

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