Globaler Einfluß von Arginin-Methylierung auf Spleissen bei Arabidopsis
Zusammenfassung der Projektergebnisse
In this project, we aimed at determining the impact of protein arginine methylation by PRMT5 on the function of Arabidopsis thaliana Lsm4 and AtGRP7. In the course of this project we optimized our previous RIP-qPCR protocol to detect candidate in vivo targets of RNA-binding proteins so that we could perform an unbiased global screen for in vivo targets by RIP coupled to high throughput sequencing (16). Application of the RIP-seq strategy to plants expressing YFP-tagged Lsm4 unveiled 982 in vivo targets of Lsm4. Some of these target transcripts are differentially expressed or alternatively spliced in lsm4-1 mutant plants, pointing to a functional role of Lsm4 binding to these transcripts. To determine a potential role of R methylation on the function of the circadian clockregulated RNA-binding protein AtGRP7 we have generated protein variants that can no longer be methylated by mutating R141 to lysine (K) or alanine (A). Upon transformation of the grp7-1 loss of function mutant with genomic construct and variants with R141 mutated to A or K, the late flowering phenotype was complemented irrespective of R141. In turn, constitutive overexpression of wt AtGRP7 protein as well as of AtGRP7 R141K, AtGRP7 R141A and AtGRP7 R141F accelerated the transition of flowering in a manner dependent on the degree of overexpression, indicating that R141 and, by extension, its methylation by PRMT5 is not critical for flowering time control by AtGRP7. At the molecular level, mutation R141 to A did not have a large impact on in vitro binding of recombinant AtGRP7-GST fusion protein or on the nucelo-cytoplasmic distribution. A prerequisite to study AtGRP8 function was the availability of a true loss-of-function mutant. For this, we designed three different sg RNAs for CRISPR/Cas9 mutagenesis. Several atgrp8 mutants with deletions of variable size were obtained which do not express AtGRP8 protein. While these mutants have been very useful to generate grp7-1 grp8 double mutants in our other DFG funded projects and obtain prmt5 grp7-1 grp8 triple mutants in this project, the complementation of the grp8 mutants with genomic GRP8 constructs and R141A and R141K versions we have generated in this project has been put on hold until we find conditions under which evidence for a functioned role of AtGRP7 R141 can be obtained. Although we do not have strong evidence that PRMT5 affects AtGRP7 or AtGRP8 function through dimethylation of R141, triple mutants revealed that PRMT5, AtGRP7 and AtGRP8 may have an additive effect on flowering time. Altogether, we did not see much effect of mutating methylable R residues in Lsm4 and AtGRP7 under regular growth conditions but our data so far indicate that the R residues can be of importance under stressful conditions.
Projektbezogene Publikationen (Auswahl)
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(2015) Time to network: The molecular blueprint of the circadian timing system in plants. In: Rhythms in Plants, Mancuso, S (ed), 257-276
Danisman S, Mateos J, Staiger D
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(2018) Beyond transcription – fine tuning of circadian timekeeping by posttranscriptional regulation. Genes 2018, 9(12), 616
Mateos JL, de Leone MJ, Torchio J, Reichel M, Staiger D
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(2021) RNA-Binding Protein Immunoprecipitation and High-Throughput Sequencing. In: Arabidopsis Protocols, Sanchez-Serrano, JJ and Salinas J (eds.), Springer New York, 453-461
Köster T, Staiger D
