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Arginine methylation mediated by the Arabidopsis homolog of PRMT5 is essential for proper pre-mRNA splicing - PubMed

  • ️Fri Jan 01 2010

Arginine methylation mediated by the Arabidopsis homolog of PRMT5 is essential for proper pre-mRNA splicing

Xian Deng et al. Proc Natl Acad Sci U S A. 2010.

Abstract

Protein arginine methylation, one of the most abundant and important posttranslational modifications, is involved in a multitude of biological processes in eukaryotes, such as transcriptional regulation and RNA processing. Symmetric arginine dimethylation is required for snRNP biogenesis and is assumed to be essential for pre-mRNA splicing; however, except for in vitro evidence, whether it affects splicing in vivo remains elusive. Mutation in an Arabidopsis symmetric arginine dimethyltransferase, AtPRMT5, causes pleiotropic developmental defects, including late flowering, but the underlying molecular mechanism is largely unknown. Here we show that AtPRMT5 methylates a wide spectrum of substrates, including some RNA binding or processing factors and U snRNP AtSmD1, D3, and AtLSm4 proteins, which are involved in RNA metabolism. RNA-seq analyses reveal that AtPRMT5 deficiency causes splicing defects in hundreds of genes involved in multiple biological processes. The splicing defects are identified in transcripts of several RNA processing factors involved in regulating flowering time. In particular, splicing defects at the flowering regulator flowering locus KH domain (FLK) in atprmt5 mutants reduce its functional transcript and protein levels, resulting in the up-regulation of a flowering repressor flowering locus C (FLC) and consequently late flowering. Taken together, our findings uncover an essential role for arginine methylation in proper pre-mRNA splicing that impacts diverse developmental processes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

AtPRMT5 methylates certain RNA processing-related proteins. (A) Identification of in vivo substrates of AtPRMT5. The 2-DE stained by Coomassie blue is shown on the top. Methylation status of proteins was detected with indicated antibodies (lower three panels). Spots marked by circles indicate the disappearance of methylation in atprmt5 mutants. Arrows indicate the corresponding proteins identified by MS. (B) Methyltransferase activity of GST and GST-AtPRMT5 with GST-AtGRP7, GST-AtGPR8 fusion proteins. (C) Methyltransferase activity of GST-AtPRMT5 with AtSmD1a, AtSmD1b, AtSmD3a, AtSmD3b and AtLSm4 fusion proteins. Autoradiography indicates AtPRMT5 methylates these AtSm and AtLSm proteins. “+” represents GST-AtPRMT5; “–” represents GST control. (D) In vivo methylation status of AtSmD1b and AtLSm4 in Col and atprmt5. AtSmD1b-GFP and AtLSm4-GFP immunoprecipitated by anti-GFP monoclonal antibody were immunoblotted with SYM10 antibody for arginine methylation and anti-GFP polyclonal antibody for loading controls.

Fig. 2.
Fig. 2.

AtPRMT5 is required for pre-mRNA splicing. (A) Four examples of mRNAs with splicing defects detected by RNA-seq. Annotated gene structures are shown (Top), with thick lines representing exons and thin lines representing introns. Wiggle plots representing the normalized reads coverage in a logarithmic scale (log2) are shown in blue for Col (Middle) and in green for atprmt5 mutants (Bottom). The red frames indicate the retained introns. (B) Validation of the intron retention events in 12 genes by RT-PCR. The upper and lower bands represent the unspliced and spliced forms, respectively. The wiggle plots of genes marked in red are shown in A. The diagrammatic presentation of primer position is shown in the top panel. The dashed lines in the primers represent primers crossing introns that are normally spliced to avoid genomic DNA contamination.

Fig. 3.
Fig. 3.

Gene ontology terms enriched in genes with splicing defects in atprmt5 mutants.

Fig. 4.
Fig. 4.

Analysis of splicing defects in RNA processing-related flowering regulators in atprmt5, atprmt10-1, se-1, and cbp80/abh1-753 mutants. The bands marked by arrows represent the forms with retained introns.

Fig. 5.
Fig. 5.

AtPRMT5 regulates FLK splicing and flowering time. (A) RNA blot and RNA-seq analysis of the expression and splicing patterns of FLK, with ACTIN as a control. The annotated gene structure and wiggle plots of FLK from Col and atprmt5 are shown (Right). (B) Schematic representation of wild-type FLKL (wtFLKL) and mutated FLKL (mFLKL). In mFLKL, the splice sites were mutated, as shown by the arrows. (C) Expression levels of FLK and FLC (Upper) and average total leaf number at flowering of flk-1 transgenic plants with wtFLKL and mFLKL (Lower) under long-day photo-period conditions. ACTIN was used as a loading control and error bars show SDs. (D) Reduction of FLK protein levels in atprmt5 mutants. Immunoblotting was performed using anti-FLK antibody, with Tubulin as a loading control. Lines “1–5” and “3–3” represent individual lines for AtPRMT5 rescued with the AtPRMT5 transgene. (E) RNA blot analysis of FLC mRNA levels in Col, atprmt5-1, atprmt5-2, flk-1, and atprmt5-2 flk-1 mutants.

Fig. 6.
Fig. 6.

A diagram illustrating the essential role of AtPRMT5 in pre-mRNA splicing.

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References

    1. Bedford MT, Clarke SG. Protein arginine methylation in mammals: Who, what, and why. Mol Cell. 2009;33:1–13. - PMC - PubMed
    1. Liu C, Lu F, Cui X, Cao X. Histone methylation in higher plants. Annu Rev Plant Biol. 2010;61:395–420. - PubMed
    1. Ren J, et al. Methylation of ribosomal protein S10 by protein-arginine methyltransferase 5 regulates ribosome biogenesis. J Biol Chem. 2010;285:12695–12705. - PMC - PubMed
    1. Zhou Z, et al. PRMT5 regulates Golgi apparatus structure through methylation of the golgin GM130. Cell Res. 2010;20:1023–1033. - PubMed
    1. Jansson M, et al. Arginine methylation regulates the p53 response. Nat Cell Biol. 2008;10:1431–1439. - PubMed

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