A KH-Domain RNA-Binding Protein Interacts with FIERY2/CTD Phosphatase-Like 1 and Splicing Factors and Is Important for Pre-mRNA Splicing in Arabidopsis

Abstract

Eukaryotic genomes encode hundreds of RNA-binding proteins, yet the functions of most of these proteins are unknown. In a genetic study of stress signal transduction in Arabidopsis, we identified a K homology (KH)-domain RNA-binding protein, HOS5 (High Osmotic Stress Gene Expression 5), as required for stress gene regulation and stress tolerance. HOS5 was found to interact with FIERY2/RNA polymerase II (RNAP II) carboxyl terminal domain (CTD) phosphatase-like 1 (FRY2/CPL1) both in vitro and in vivo. This interaction is mediated by the first double-stranded RNA-binding domain of FRY2/CPL1 and the KH domains of HOS5. Interestingly, both HOS5 and FRY2/CPL1 also interact with two novel serine-arginine (SR)-rich splicing factors, RS40 and RS41, in nuclear speckles. Importantly, FRY2/CPL1 is required for the recruitment of HOS5. In fry2 mutants, HOS5 failed to be localized in nuclear speckles but was found mainly in the nucleoplasm. hos5 mutants were impaired in mRNA export and accumulated a significant amount of mRNA in the nuclei, particularly under salt stress conditions. Arabidopsis mutants of all these genes exhibit similar stress-sensitive phenotypes. RNA-seq analyses of these mutants detected significant intron retention in many stress-related genes under salt stress but not under normal conditions. Our study not only identified several novel regulators of pre-mRNA processing as important for plant stress response but also suggested that, in addition to RNAP II CTD that is a well-recognized platform for the recruitment of mRNA processing factors, FRY2/CPL1 may also recruit specific factors to regulate the co-transcriptional processing of certain transcripts to deal with environmental challenges.

C-terminal domain (CTD) phosphatase links Rho GTPase signaling to Pol II CTD phosphorylation in Arabidopsis and yeast

“…1C). This gene was first identified as CPL1 or FRY2, which encodes a Pol II CTD phosphatase involved in stress responses and acting as a regulator of gene expression (18,19,(22)(23)(24)(25). The cae2-1 mutation resulted in three types of splicing products of CPL1 mRNA in cae2-1 CA1-1, and sequencing of the three cDNA molecules showed all of which differed from the WT CPL1 transcript ( Fig.…”

“…Given the role of CTD Ser5 phosphorylation in the Pol II control of gene expression (18,19,(22)(23)(24), we performed transcriptome analysis to determine whether ROP2 and CPL1 affected expression of a similar set of downstream genes. RNAsequencing analysis revealed that 1,333 genes were differentially expressed (with a twofold cutoff) in cae2-1, CA1-1, or cae2-1 CA1-1 compared with WT (SI Appendix, Fig.…”

“…In Arabidopsis, there are several members of CTD phosphatases with sequence homology to yeast Fcp1 phosphatase (18)(19)(20)(21). Among these members, CPL1 has been extensively studied in stress response and gene expression regulation (18,19,(22)(23)(24)(25). CPL1 has been demonstrated to act as a Ser5 phosphatase in vitro, but its upstream regulatory pathway remains to be revealed.…”

The RNA Polymerase II C-Terminal Domain Phosphatase-Like Protein FIERY2/CPL1 Interacts with eIF4AIII and Is Essential for Nonsense-Mediated mRNA Decay in Arabidopsis

“…Compared with the wild type, 420 AS events including 151 retained introns, 171 alternative 39 splice sites, 96 alternative 59 splice sites, and 2 cassette exons were overrepresented in the fry2-1 mutant (P < 0.001, a 4-fold upregulation) ( Figure 5A). A previous study has documented that a great portion of introns cannot be removed in the fry2-1 mutant as a result of the role of FRY2 in pre-mRNA splicing via interactions with the splicingrelated factors HOS5 and SR40/SR41 (Chen et al, 2013). Therefore, we excluded intron retention from this analysis.…”

“…In addition, FRY2 can dephosphorylate HYPONASTIC LEAVES1 and is essential for accurate miRNA processing (Manavella et al, 2012). Recently, we found that FRY2 contributes to the regulation of pre-mRNA splicing via interacting with the KH-domain RNA binding protein HOS5 and two splicing factor proteins, RS40 and RS41 (Chen et al, 2013). In addition, FRY2 has been suggested to inhibit transcription by preventing mRNA capping and the transition from transcription initiation to elongation (Jiang et al, 2013).…”