Molecular Analysis of Pcp2/L7 3'UTR and Its Putative Binding Proteins: Unr and Vps36

 
 
 
 

Molecular Analysis of Pcp2/L7 3'UTR and Its Putative Binding Proteins: Unr and Vps36

Show full item record


Title: Molecular Analysis of Pcp2/L7 3'UTR and Its Putative Binding Proteins: Unr and Vps36
Author: Zhang, Rui
Description: Purkinje cell protein 2 (Pcp2 or L7) is highly expressed in cerebellar Purkinje cells and functions as a modulator for G protein-mediated cell signaling. Its mRNA is abundantly localized in the dendrites as well as in the cell bodies. Although the dendritic localization of L7 mRNA has been found to be developmentally regulated, the molecular mechanisms and biological functions of the localization are still unknown. Here we present data to show the cis-acting sequences important for its localization in dendrites, and the proteins that recognize these sequences. First, we show that the L7 3'UTR is critical in the process of L7 mRNA localization in Purkinje cell dendrites. Second, we have partially purified two 3'UTR-binding proteins from cerebellar extracts using FPLC and identified them by MALDI-MS. They are upstream of N-ras (Unr) and vacuolar protein sorting 36 (Vps36). Unr is a cold-shock domain RNA-binding protein with multiple reported functions related to translational control and RNA stabilization. Vps36 is the mammalian homologue of yeast Vps36, a mediator of apical growth and endosome trafficking of receptors in yeast. An RNA binding function for this protein has been reported in Drosophila. Using various RNA-protein binding assays, we confirmed that Unr binds specifically to L7 3'UTR, while Vps36 binding to L7 3'UTR is inconclusive. As direct study in Purkinje cells is difficult due to the lack of suitable cell culture models, we employed an inducible conditional knockout strategy to study the functions of Unr and Vps36 in cerebellar Purkinje cells. We have successfully obtained homologous recombination at the ES cells level for Vps36. We have also generated an inducible cerebellar Purkinje cell-specific CreERT2 transgenic mice. In vivo studies on molecular mechanism of the Purkinje cell-specific expression of L7 has revealed that the 0.9 kb proximal L7 promoter and the structural gene both contribute to this process. Using a minimal promoter test, we found that multiple copies of 0.9 kb fragment can behave as a classical enhancer without the cooperation of the structural gene. However, the full function appears to be dependent on appropriate signals in the L7 structural gene.
Permanent Link: http://rave.ohiolink.edu/etdc/view?acc_num=osu1213363637
http://hdl.handle.net/2374.OX/107149
Date: 2008

Files in this item

Files Size Format View

There are no files associated with this item.

This item appears in the following Collection(s)

Show full item record