PRP4KA, a Putative Spliceosomal Protein Kinase, Is Important for Alternative Splicing and Development in Arabidopsis thaliana.

Tatsuo Kanno,Tuan-Nan Wen,Peter Venhuizen,Maria Kalyna,Wen-Dar Lin,Antonius J M Matzke,Phebe Chiou,Marjori Matzke

doi:10.1534/genetics.118.301515

Abstract

Splicing of precursor messenger RNAs (pre-mRNAs) is an essential step in the expression of most eukaryotic genes. Both constitutive splicing and alternative splicing, which produces multiple messenger RNA (mRNA) isoforms from a single primary transcript, are modulated by reversible protein phosphorylation. Although the plant splicing machinery is known to be a target for phosphorylation, the protein kinases involved remain to be fully defined. We report here the identification of pre-mRNA processing 4 (PRP4) KINASE A (PRP4KA) in a forward genetic screen based on an alternatively spliced GFP reporter gene in Arabidopsis thaliana (Arabidopsis). Prp4 kinase is the first spliceosome-associated kinase shown to regulate splicing in fungi and mammals but it has not yet been studied in plants. In the same screen we identified mutants defective in SAC3A, a putative mRNA export factor that is highly coexpressed with PRP4KA in Arabidopsis Whereas the sac3a mutants appear normal, the prp4ka mutants display a pleiotropic phenotype featuring atypical rosettes, late flowering, tall final stature, reduced branching, and lowered seed set. Analysis of RNA-sequencing data from prp4ka and sac3a mutants identified widespread and partially overlapping perturbations in alternative splicing in the two mutants. Quantitative phosphoproteomic profiling of a prp4ka mutant detected phosphorylation changes in several serine/arginine-rich proteins, which regulate constitutive and alternative splicing, and other splicing-related factors. Tests of PRP4KB, the paralog of PRP4KA, indicated that the two genes are not functionally redundant. The results demonstrate the importance of PRP4KA for alternative splicing and plant phenotype, and suggest that PRP4KA may influence alternative splicing patterns by phosphorylating a subset of splicing regulators.

Highlights

PRECURSOR messenger RNA splicing, which entails removal of introns and joining of exons, is an essential step in the expression of most eukaryotic genes
In a forward genetic screen for mutants showing modified splicing of an alternatively spliced GFP reporter gene in Arabidopsis, we recovered loss-of-function mutations in the genes encoding the dual-specificity protein kinase PRP4KA and the putative messenger RNA (mRNA) nuclear export factor SAC3A. Both the prp4ka and sac3a mutants were identified by their GFPweak phenotypes, which are due—at least in part—to diminished splicing efficiency of GFP pre-mRNA
PRP4KA and SAC3A have not been identified in any prior forward genetic screen in Arabidopsis or studied previously for their roles in premRNA splicing in plants. It is unclear why this particular screen repeatedly retrieved mutants defective in these two genes, but the findings clearly demonstrate the contributions of PRP4KA and SAC3A to GFP pre-mRNA splicing and to GFP expression

Summary

Introduction

PRECURSOR messenger RNA (pre-mRNA) splicing, which entails removal of introns and joining of exons, is an essential step in the expression of most eukaryotic genes. Trans-acting splicing factors include serine/arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs), which respectively bind exonic and intronic cis-regulatory elements, which are termed splicing enhancers and silencers (Barta et al 2008; Matera and Wang 2014). SR proteins, which are present in organisms with more complex splicing patterns (fission yeast, plants, and metazoans), feature one or two RNA recognition motifs at their N terminus and an arginine/serine-rich (RS) domain at their C terminus. Kinases found previously to be important for phosphorylating SR proteins in Arabidopsis include SR protein kinases (de la Fuente van Bentem et al 2006; Rosembert 2017), Cdc2-like or LAMMER-type kinases (Golovkin and Reddy 1999; Savaldi-Goldstein et al 2000), and mitogen-activated protein kinases (Feilner et al 2005; de la Fuente van Bentem et al 2006, 2008)

Methods

Results

Conclusion