Purification Of MRNA Binding Proteins And Molecular Mechanism Of CmRBP50 Based Ribonucleoprotein Complex Formation In Pumpkin Phloem Sap

Phloem is an important part of plant vascular system, which not only plays key roles in translocating assimilated carbohydrates, but also serves an information superhighway in coordinating with plant growth and development as it is rich in signal moleculars such as hormones, redox oxygen species, Ca2+, RNAs and proteins. Plant vascular system is also important for plants in response to environmental changes, thus understanding phloem long-distance substances and their functions would help us in improving agricultural productivity. Pumpkin(Cucurbita maxima cv Big Max) is one of the best model plants to study vascular biology as it has distinct vascular system and is easy to collect phloem sap. At present, its phloem transcriptome and proteome have been investigated, but little is known about those thousands of RNAs and proteins, still many questions wait for further study. Our previous work identified a RNA binding protein CmRBP50 which formed a ribonucleoprotein (RNP) complex in pumpkin phleom sap. This is the first RNP identified in plants which can undergo long-distanced movement, however, the molecular mechanism remained to be characterized. In this paper, pumpkin was used as material, and molecular biology, biochemistry techniques were applied to study the putative mRNA binding proteins and the molecular mechanism of CmRBP50 based RNP formation. Results were obtained as follows:1. Proteomic analysis of putative mRNA binding proteins in pumpkin phloem sap. Poly(U) affinity column and LC-MS/MS analysis were applied and 29 putative mRNA binding proteins were identified from pumpkin phloem sap. They were RNA metabolism, translation or protein metabolism, transport or cell structure, energy production or metabolism as well as some unknown functions related.2. We examined the phosphorylation sites on CmRBP50 and the importance of phosphorylation to the formation of CmRBP50 based RNP complex. Firstly, protein overlay assay was applied to confirm the importance of phosphorylation to CmRBP50 based RNP complex formation, and then four phoshoserines 223,438,440 and 444 were mapped as in vivo phosphorylation sites on CmRBP50. Point mutations and Zucchini yellow mosaic virus (ZYMV) viral system were used to clone and purify CmRBP50 and its mutant proteins S223A (Serine 223 to Alanine), STripleA (Serine 438,440,444 to Alanine), SQuadA (Serine 223,438,440,444 to Alanine). Protein overlay assay and co-immunoprecipitation experiments confirmed that the above phosphorylation sites were essential for CmRBP50 based RNP complex formation, especially the three serines on C terminus. The replacement of these three serines to alanine abolished the binding to its phloem interaction partners efficiently. Finally, we cloned and expressed putative interacting proteins of CmRBP50, in vitro pull-down experiments confirmed the interaction between CmRBP50 and CmPP16, GTPbP as well as PSPL, and additionally the phosphorylation on C-terminus was essential for their binding.3. Protein kinases for CmRBP50 were analyzed and discussed. In gel kinase assay were applied to detect possible kinases for CmRBP50 from pumpkin vascular bundle proteins and phloem sap proteins. We found a nucleoside diphosphate kinase, a serine threonine kinase from vascule proteins and a protein kinase adkl, a protein kinase Csa000515 and a glycogen synthase kinase-3 from phloem sap analyzed by LC-MS/MS. Glycogen synthase kinase-3 was targeted as the candidate kinase for further study.By studying the mRNA binding proteins in pumpkin phloem sap and the molecular mechanism of the first identified long-distance translocatable CmRBP50 based RNP complex, this paper will help us to understand CmRBP50 mediated physiological functions in phloem sap, and to study the other RNPs in phloem sap. Eventually this paper will not only help us to reveal the mechanism of communication and signal transduction pathways between distal organs, to increase the knowledge of plant physiology, ecology and horticulture, but also provide us a useful tool to improve stress defense, quantity and quality of vegetables in agricultural production, which has significant scientific and applicable meaning.