G-patch domain and KOW motifs-containing protein, GPKOW; a nuclear RNA-binding protein regulated by protein kinase A

Abstract

BackgroundPost-transcriptional processing of pre-mRNA takes place in several steps and requires involvement of a number of RNA-binding proteins. How pre-mRNA processing is regulated is in large enigmatic. The catalytic (C) subunit of protein kinase A (PKA) is a serine/threonine kinase, which regulates numerous cellular processes including pre-mRNA splicing. Despite that a significant fraction of the C subunit is found in splicing factor compartments in the nucleus, there are no indications of a direct interaction between RNA and PKA. Based on this we speculate if the specificity of the C subunit in regulating pre-mRNA splicing may be mediated indirectly through other nuclear proteins.ResultsUsing yeast two-hybrid screening with the PKA C subunit Cbeta2 as bait, we identified the G-patch domain and KOW motifs-containing protein (GPKOW), also known as the T54 protein or MOS2 homolog, as an interaction partner for Cbeta2. We demonstrate that GPKOW, which contains one G-patch domain and two KOW motifs, is a nuclear RNA-binding protein conserved between species. GPKOW contains two sites that are phosphorylated by PKA in vitro. By RNA immunoprecipitation and site directed mutagenesis of the PKA phosphorylation sites we revealed that GPKOW binds RNA in vivo in a PKA sensitive fashion.ConclusionGPKOW is a RNA-binding protein that binds RNA in a PKA regulated fashion. Together with our previous results demonstrating that PKA regulates pre-mRNA splicing, our results suggest that PKA phosphorylation is involved in regulating RNA processing at several steps.

A Rolling Stone Gathers No Moss, but Resistant Plants Must Gather Their MOSes

“…Similarly, the KOW motif from the bacterial elongation factor NusG has shown nucleic acid-binding activity and shares stmctural homology with the tudor protein-protein interaction motif (Steiner et al 2002). Whereas MOS2 has not been experimentally shown to bind RNA, the human M0S2 homolog GPKOW binds RNA in a manner dependent on protein kinase A (Aksaas et al 2011). Furthemiore, the remote yeast homolog SPP2 is an essential protein required for the first RNA cleavage step in pre-mRNA splicing.…”

“…Although MOS2 has not been shown to directly associate with the MAC, human and yeast homologs of MOS2 have been implicated as components of the conserved splieeosome-assoeiated complex (Roy et al 1995;Bessonov et al 2010;Aksaas et al 2011). Additionally, M0S2 has been shown to be required for proper splicing of SNCI (S Xu and Y Zhang, unpubl.).…”

Two Cellular Protein Kinases, DNA-PK and PKA, Phosphorylate the Adenoviral L4-33K Protein and Have Opposite Effects on L1 Alternative RNA Splicing

“…PKA subunits are encoded by four R and C subunit genes, respectively (RIα, RIIα, RIβ, RIIβ, Cα, Cβ, Cγ and PRKX) [21]. Specificity in the cAMP/PKA signal transduction pathway is obtained by tissue-specific expression and targeting the R subunits to A-kinase anchoring proteins (AKAPs) in the cytosol and targeting of the C subunit to C subunit-binding proteins both in the cytosol and nucleus [22]–[28]. PKA is activated upon binding of four molecules of cAMP to the R dimer, releasing the C subunits to phosphorylate relevant substrates on serine and threonine residues in its vicinity [29].…”

A role for the RNA-binding protein MOS2 in microRNA maturation in Arabidopsis

“…MOS2 has homologs in human, mouse, and worm [31,45]. A genome-wide RNAi screen in worm indicated that knock-down of the MOS2 homolog causes embryonic lethality [46].…”