CmRBP50 Protein Phosphorylation Is Essential for Assembly of a Stable Phloem-mobile High-affinity Ribonucleoprotein Complex

Abstract

RNA-binding proteins (RBPs) form ribonucleoprotein (RNP)complexes that play crucial roles in RNA processing for gene regulation. The angiosperm sieve tube system contains a unique population of transcripts, some of which function as long-distance signaling agents involved in regulating organ development. These phloem-mobile mRNAs are translocated as RNP complexes. One such complex is based on a phloem RBP named Cucurbita maxima RNA-binding protein 50 (CmRBP50), a member of the polypyrimidine track binding protein family. The core of this RNP complex contains six additional phloem proteins. Here, requirements for assembly of this CmRBP50 RNP complex are reported. Phosphorylation sites on CmRBP50 were mapped, and then coimmunoprecipitation and protein overlay studies established that the phosphoserine residues, located at the C terminus of CmRBP50, are critical for RNP complex assembly. In vitro pull-down experiments revealed that three phloem proteins, C. maxima phloem protein 16, C. maxima GTP-binding protein, and C. maxima phosphoinositide-specific phospholipase-like protein, bind directly with CmRBP50. This interaction required CmRBP50 phosphorylation. Gel mobility-shift assays demonstrated that assembly of the CmRBP50-based protein complex results in a system having enhanced binding affinity for phloem-mobile mRNAs carrying polypyrimidine track binding motifs. This property would be essential for effective long-distance translocation of bound mRNA to the target tissues.

Polypyrimidine Tract Binding Protein Homologs from Arabidopsis Are Key Regulators of Alternative Splicing with Implications in Fundamental Developmental Processes

“…Thus, PTB3 might not play a major role in AS control in Arabidopsis, but rather have other functions in RNA metabolism. In line with this hypothesis, a pumpkin homolog of At-PTB3 was shown to be part of a phloem-mobile ribonucleoprotein ), the assembly of which requires phosphorylation of the PTB protein (Li et al, 2011).…”

Overexpression of Arabidopsis Plasmodesmata Germin-Like Proteins Disrupts Root Growth and Development

“…Purification of recombinant proteins from ZYMV viral vector-infected pumpkin leaves was performed as described previously (Ma et al, 2010;Li et al, 2011). Briefly, ZYMV-based constructs were coated onto gold particles (1 µm) and bombarded onto pumpkin cotyledons using a Helios gene gun system (Bio-Rad).…”

“…For pull-down assays, GFP, Nt-NCAPP1DN 1-22 , PDGLP1, and PDGLP1DSP were subcloned into a Zucchini yellow mosaic virus (ZYMV)-based viral vector as described previously (Ma et al, 2010;Li et al, 2011). GST and Cm-PP16-GST were amplified from pGWB24/CmPP16 (Li et al, 2011) and inserted into the modified ZYMV viral vector without a c-MycX4-His 6 tag (Lin et al, 2002).…”

“…GST and Cm-PP16-GST were amplified from pGWB24/CmPP16 (Li et al, 2011) and inserted into the modified ZYMV viral vector without a c-MycX4-His 6 tag (Lin et al, 2002).…”

Selective Targeting of Mobile mRNAs to Plasmodesmata for Cell-to-Cell Movement

“…The crucial roles of RBPs in the subcellular 361 localization of mobile mRNAs may be mediated by bridging mobile mRNAs to 362 endomembrane or cytoskeleton systems or be associated with motor proteins (Takizawa 363 et al, 1997;Schnorrer et al, 2000;Pohlmann et al, 2015). Indeed, analysis of RBPs in 364 phloem exudates identified multiple putative systemic RBPs involved in 365 phloem-mediated mRNA transport (Ham et al, 2009;Li et al, 2011 …”