Global Impact of Arginine Methylation on Splicing in Arabidopsis

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Posttranslational protein modification by arginine (R) methylation plays a key role in the cell. R methylation emerges as a common modification of many RNA-binding proteins, but its impact on RNA processing in higher plants is largely unknown. Protein arginine methyl transferases (PRMTs) that catalyze these modifications are conserved from plants to humans and are connected to many physiological and pathological processes, including cancer, acting at least in part through their effect on RNA-binding proteins involved in alternative splicing. We aim to evaluate how posttranslational modification of splicing factors by R methylation affects their function in pre-mRNA splicing and thus impacts physiological processes in plants. As a paradigm we chose Arabidopsis thaliana PRMT5. PRMT5 mutants show a global defect in alternative splicing and have an aberrant circadian clock. In particular, alternative splicing of the clock gene PSEUDORESPONSE REGULATOR 9 (PRR9) is altered, suggesting that aberrant splicing of PRR9 contributes to slowing down the clock. We will focus on the PRMT5 targets LSM4, GRP7 and GRP8. LSM4 is a component of the small nuclear ribonucleoprotein complexes that are part of the spliceosome. Lsm4 mutants are affected in a suite of AS events and show aberrant circadian rhythms, suggesting that a component of the spliceosome affects clock function. The glycine-rich RNA-binding proteins GRP7 and GRP8 regulate alternative splicing and play a regulatory role in the circadian system, innate immunity, and flowering. Plants with altered PRMT5 or GRP7 levels show both common and distinct changes in splicing. Thus, we hypothesize that PRMT5 may affect splicing of some of its target transcripts and its impact on the circadian clock and flowering partly by modulating GRP7 and GRP8 activity. We will determine the relevance of R methylation by PRMT5 for the LSM4, GRP7, and GRP8 in vivo function. We will mutate the methylable R residues so that the modified proteins can no longer be methylated or mimic a constitutively methylated variant. A comparison of splicing events affected in grp7, lsm4 and prmt5 mutants genome-wide will reveal if and to which extent PRMT5 control of splicing involves GRP7 and LSM4. These data will be used to construct a network illustrating hierarchical interactions between PRMT5 and its targets. Furthermore, it is unknown how the arginine methylation marks deposited on RNA-binding proteins by the PRMT writers are recognized in Arabidopsis. We will test TUDOR proteins for a role as readers of the methylation marks in GRP7, GRP8, and LSM4. An interaction of splicing-relevant PRMT5 targets with a TUDOR domain protein would provide a hint for a regulation of the activity of SFs by interpreting the output of PRMT5 activity. This would add a largely unexplored level of control to alternative splicing in plants and contribute to defining the functional output of Tudor domains in plants, a largely unexplored topic.