A Duplicated NUCLEOLIN Gene with Antagonistic Activity Is Required for Chromatin Organization of Silent 45S rDNA in <i>Arabidopsis</i>

Durut, Nathalie; Abou-Ellail, Mohamed; Pontvianne, Frédéric; Das, Sadhan; Kojima, Hisae; Ukai, Seiko; Bures, Anne de; Comella, Pascale; Nidelet, Sabine; Rialle, Stéphanie; Merret, Rémy; Echeverrı́a, Manuel; Bouvet, Philippe; Nakamura, Kenzo; Sáez‐Vasquez, Julio

doi:10.1105/tpc.114.123893

Cited by 67 publications

(103 citation statements)

References 44 publications

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“…The reference gene here was an endogenous internal control gene UBQ10 (AT4G05320). qRT-PCR primers for UBQ10 and 18S rRNA were described in Li et al [47] and Durut et al [48]. …”

Section: Methodsmentioning

confidence: 99%

UV crosslinked mRNA-binding proteins captured from leaf mesophyll protoplasts

et al. 2016

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BackgroundThe complexity of RNA regulation is one of the current frontiers in animal and plant molecular biology research. RNA-binding proteins (RBPs) are characteristically involved in post-transcriptional gene regulation through interaction with RNA. Recently, the mRNA-bound proteome of mammalian cell lines has been successfully cataloged using a new method called interactome capture. This method relies on UV crosslinking of proteins to RNA, purifying the mRNA using complementary oligo-dT beads and identifying the crosslinked proteins using mass spectrometry. We describe here an optimized system of mRNA interactome capture for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type often used in functional cellular assays.ResultsWe established the conditions for optimal protein yield, namely the amount of starting tissue, the duration of UV irradiation and the effect of UV intensity. We demonstrated high efficiency mRNA-protein pull-down by oligo-d(T)25 bead capture. Proteins annotated to have RNA-binding capacity were overrepresented in the obtained medium scale mRNA-bound proteome, indicating the specificity of the method and providing in vivo UV crosslinking experimental evidence for several candidate RBPs from leaf mesophyll protoplasts.ConclusionsThe described method, applied to plant cells, allows identifying proteins as having the capacity to bind mRNA directly. The method can now be scaled and applied to other plant cell types and species to contribute to the comprehensive description of the RBP proteome of plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0142-6) contains supplementary material, which is available to authorized users.

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“…The reference gene here was an endogenous internal control gene UBQ10 (AT4G05320). qRT-PCR primers for UBQ10 and 18S rRNA were described in Li et al [47] and Durut et al [48]. …”

Section: Methodsmentioning

confidence: 99%

UV crosslinked mRNA-binding proteins captured from leaf mesophyll protoplasts

et al. 2016

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“…These results suggest that the two atBRX1 proteins have redundant function. In contrast, many other RBFs with multiple orthologs have diverged in their sequence or subcellular localization and only one of the orthologs still has its ancestral function in ribosome biogenesis (Pontvianne et al 2007;Comella et al 2008;Lange et al 2008;Zakrzewska-Placzek et al 2010;Durut et al 2014;Weis et al 2014).…”

Section: Discussion Atbrx1 Proteins Are Important For Developmentmentioning

confidence: 99%

atBRX1-1 and atBRX1-2 are involved in an alternative rRNA processing pathway in Arabidopsis thaliana

Weis

Palm

Missbach

et al. 2015

RNA

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Ribosome biogenesis is an essential process in all organisms. In eukaryotes, multiple ribosome biogenesis factors (RBFs) act in the processing of ribosomal (r)RNAs, assembly of ribosomal subunits and their export to the cytoplasm. We characterized two genes in Arabidopsis thaliana coding for orthologs of yeast BRX1, a protein involved in maturation of the large ribosomal subunit. Both atBRX1 proteins, encoded by AT3G15460 and AT1G52930, respectively, are mainly localized in the nucleolus and are ubiquitously expressed throughout plant development and in various tissues. Mutant plant lines for both factors show a delay in development and pointed leaves can be observed in the brx1-2 mutant, implying a link between ribosome biogenesis and plant development. In addition, the pre-rRNA processing is affected in both mutants. Analysis of the pre-rRNA intermediates revealed that early processing steps can occur either in the 5 ′ external transcribed spacer (ETS) or internal transcribed spacer 1 (ITS1). Interestingly, we also find that in xrn2 mutants, early processing events can be bypassed and removal of the 5 ′ ETS is initiated by cleavage at the P ′ processing site. While the pathways of pre-rRNA processing are comparable to those of yeast and mammalian cells, the balance between the two processing pathways is different in plants. Furthermore, plant-specific steps such as an additional processing site in the 5 ′ ETS, likely post-transcriptional processing of the early cleavage sites and accumulation of a 5 ′ extended 5.8S rRNA not observed in other eukaryotes can be detected.

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“…Also, different A. thaliana ecotypes tested all showed highly variable expression of the variants [41]. Finally, the expression of rDNA variants was found to be tightly regulated throughout early plant development, and the Nucleolin homologs (NUC1 and NUC2) were shown to have a major role in the activation or silencing of certain rDNA variants by affecting the methylation state of different rDNA repeats [44,45].…”

Section: Pre-rrna Processing In Plants Is Secured By Alternative Routesmentioning

confidence: 96%