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. 2011;6(8):e23882.
doi: 10.1371/journal.pone.0023882. Epub 2011 Aug 17.

Matrin 3 binds and stabilizes mRNA

Maayan Salton  1 , Ran Elkon, Tatiana Borodina, Aleksey Davydov, Marie-Laure Yaspo, Eran Halperin, Yosef Shiloh
Affiliations

Matrin 3 binds and stabilizes mRNA

Maayan Salton et al. PLoS One. 2011.

"V体育官网" Abstract

Matrin 3 (MATR3) is a highly conserved, inner nuclear matrix protein with two zinc finger domains and two RNA recognition motifs (RRM), whose function is largely unknown. Recently we found MATR3 to be phosphorylated by the protein kinase ATM, which activates the cellular response to double strand breaks in the DNA. Here, we show that MATR3 interacts in an RNA-dependent manner with several proteins with established roles in RNA processing, and maintains its interaction with RNA via its RRM2 domain. Deep sequencing of the bound RNA (RIP-seq) identified several small noncoding RNA species. Using microarray analysis to explore MATR3's role in transcription, we identified 77 transcripts whose amounts depended on the presence of MATR3. We validated this finding with nine transcripts which were also bound to the MATR3 complex. Finally, we demonstrated the importance of MATR3 for maintaining the stability of several of these mRNA species and conclude that it has a role in mRNA stabilization. The data suggest that the cellular level of MATR3, known to be highly regulated, modulates the stability of a group of gene transcripts VSports手机版. .

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

VSports在线直播 - Figures

Figure 1
Figure 1. Identification of novel MATR3 interactors.
(A) FLAG-MATR3 was expressed in HEK293T cells and immunoprecipitated using anti-FLAG conjugated beads. Immune complexes were separated by SDS-PAGE and visualized with silver staining. Cells with empty vector allowed discrimination between specific and non-specific immunoprecipitation. Bands that appeared specific were identified using mass spectrometry. (B) FLAG-MATR3 was expressed in HEK293T cells and immunoprecipitated using FLAG-conjugated beads. The immune complexes were blotted with the indicated antibodies. (C) Endogenous MATR3 was immunoprecipitated from HEK293T cells, and the immune complexes were treated with RNase A and blotted with antibodies against the indicated proteins.
Figure 2
Figure 2. MATR3 binds RNA via its RRM2 domain.
(A) Schematic presentation of MATR3 domains and deletion of specific domains in different constructs used in this study. (B) HEK293T cells were transfected with the indicated MATR3 expression constructs. FLAG-conjugated beads were used for immunoprecipitation and the immune complexes were blotted with the indicated antibodies.
Figure 3
Figure 3. Validation of RIP-seq results.
(A) HEK293T cells were transfected with the indicated MATR3 constructs and the ectopic MATR3 proteins were immunoprecipitated using FLAG-conjugated beads and blotted with the indicated antibodies. (B) RNA was extracted from the immune complexes, yeast Phe-tRNA was added, qPCR was carried out on reverse transcription products and the Relative Quantification (RQ) is shown as fold-change of signal compared to ΔRRM2 minus the background of the empty vector. The plot represents the mean of three independent experiments and error bars represent SD (*P≤0.05, **P≤0.01, t test).
Figure 4
Figure 4. MATR3 affects the transcript level of mRNAs that bind to it.
(A) Knockdown of MATR3 in U2OS cells: western blotting analysis of total cellular extracts 96 hr after transfection with siGFP or siMATR3. (B) Effect of MATR3 depletion on the expression of 77 human genes. Expression profiles were recorded in U2OS cells knocked-down for MATR3 and in control cells transfected with siGFP, using Affymetrix Human Gene 1.0 ST arrays. Responding genes in the datasets (defined as showing at least 1.7 fold-change in expression level) underwent clustering analysis using the CLICK algorithm . A cluster containing 77 genes was obtained containing genes whose expression level was reduced in MATR3 knocked-down cells (Table S1). The plot represents average expression levels of these genes normalized against their average level of expression in the siGFP samples. (C) Validation of results obtained using microarray analysis. RNA was extracted from U2OS cells knocked-down for MATR3 and from control cells transfected with siGFP was reverse transcribed, and qPCR was carried out. Shown is the relative quantification (RQ) as fold-change compared to the siGFP cells. Two endogenous control transcripts were used: GAPDH and hTBP. The plot represents the mean of three independent experiments and error bars represent SD (*P≤0.05, **P≤0.01, t test). (D) Binding of mRNAs to MATR3. The extent of binding of mRNAs to wild type vs. ΔRRM2 served as a measure of mRNA binding to the protein. RNA was extracted from the MATR3 immune complexes shown in Figs. 3A and Phe-tRNA was added. qPCR was carried out on reverse transcription products and the Relative Quantification (RQ) is shown as fold-change of signal compared to ΔRRM2 minus the background of the empty vector. The plot represents the mean of three independent experiments and error bars represent SD (*P≤0.05, **P≤0.01, t test).
Figure 5
Figure 5. MATR3 stabilizes mRNAs.
(A) Western blotting of total cellular extracts showing the extent of MATR3 knockdown in U2OS cells 96 hr after transfection with siGFP or siMATR3. (B) mRNA half-life was estimated by treating siGFP cells and siMATR3 cells with actinomycin D (2 µg/ml) for the indicated time periods. HLTF, RP56KA4, HNT and GAPDH mRNA levels were measured using qPCR, normalized against 18S rRNA levels and plotted on a scale. The plot represents the mean of three independent experiments and error bars represent SD.
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References

    1. Belgrader P, Dey R, Berezney R. Molecular cloning of matrin 3. A 125-kilodalton protein of the nuclear matrix contains an extensive acidic domain. J Biol Chem. 1991;266:9893–9899. - PubMed
    1. Cohen TV, Hernandez L, Stewart CL. Functions of the nuclear envelope and lamina in development and disease. Biochem Soc Trans. 2008;36:1329–1334. - PubMed
    1. Hisada-Ishii S, Ebihara M, Kobayashi N, Kitagawa Y. Bipartite nuclear localization signal of matrin 3 is essential for vertebrate cells. Biochem Biophys Res Commun. 2007;354:72–76. - PubMed
    1. Hibino Y, Ohzeki H, Sugano N, Hiraga K. Transcription modulation by a rat nuclear scaffold protein, P130, and a rat highly repetitive DNA component or various types of animal and plant matrix or scaffold attachment regions. Biochem Biophys Res Commun. 2000;279:282–287. - PubMed
    1. Hibino Y, Usui T, Morita Y, Hirose N, Okazaki M, et al. Molecular properties and intracellular localization of rat liver nuclear scaffold protein P130. Biochim Biophys Acta. 2006;1759:195–207. - PubMed

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