Specificity of the STAR/GSG domain protein Qk1: Implications for the regulation of myelination

Abstract

Inadequate formation and maintenance of myelin is the basis for several neurodegenerative disorders, including leukodystrophy and multiple sclerosis. In mice, oligodendrocyte differentiation and subsequent formation of myelin requires the Quaking gene. Mutation of this gene leads to embryonic lethality or to a trembling phenotype characteristic of dysmyelination. Quaking encodes Qk1, a member of the highly conserved STAR/GSG family of RNA-binding proteins that function as master developmental regulators in higher eukaryotes. Qk1 has been implicated in the regulation of alternative splicing, stability, and translation control of mRNAs that code for myelin structural components in glial cells. We have used quantitative gel mobility shift and fluorescence polarization assays to define the nucleotide sequence specificity of the Qk1 STAR/GSG domain, and to probe the interaction between Qk1 and the 3-untranslated region (UTR) of myelin basic protein (MBP) mRNA. The results show that Qk1 recognizes a hexanucleotide consensus element that is similar although not identical to the specificity determinant recognized by the Caenorhabditis elegans STAR/GSG protein GLD-1. Several consensus sites are present in the 3-UTR of MBP mRNA. The highest affinity site is located within the RNA localization region, suggesting a possible role for Qk1 in restricting MBP mRNA to the myelin compartment.

Quantitative approaches to monitor protein–nucleic acid interactions using fluorescent probes

“…1). The apparent equilibrium dissociation constant is 26 6 14 nM, identical to the value previously determined by radioactive EMSA (25 6 4 nM) (Ryder and Williamson 2004). However, it is worth noting that the apparent cooperativity seems greater by F-EMSA than by radioactive EMSA (n app = 1.8 6 0.4 versus 1.0 6 0.2), which could be due to the increase in RNA concentration needed to detect binding, or to limitations of detection at the edge of the binding transition.…”

“…FP is a useful technique to study the thermodynamic and kinetic properties of a protein-nucleic acid interaction (LeTilly and Royer 1993;Aviv et al 2003;Ryder and Williamson 2004). FP takes advantage of the change in the tumbling properties of a fluorescent ligand upon binding to a larger macromolecule: in our case, a fluorescent DNA or RNA probe and a nucleic acid-binding protein.…”

“…Due to the challenges associated with using radioisotopes, fluorescent probes have become a favorable alternative in many biochemical assays (Royer and Scarlata 2008;LeTilly and Royer 1993;Aviv et al 2003;Ryder and Williamson 2004;Hardin and Batey 2006;Besse et al 2009;Kohn et al 2010). With modern instrumentation, the detection of fluorescent probes now rivals that of 32 P-labeled probes.…”

Transposition of two amino acids changes a promiscuous RNA binding protein into a sequence-specific RNA binding protein

“…Homodimerization significantly enhances the RNA binding affinity of the STAR/GSG proteins (Chen and Richard 1998;Chow et al 2000;Ryder and Williamson 2004). Lacking the QUA1 domain, BBP/SF1 does not homodimerize; instead, it interacts with Mud2 in yeast or U2AF65 in human, and these proteins bind cooperatively to the branchpoint sequence and adjacent polypyrimidine tract at the 39 end of the intron Berglund et al 1998a).…”

Quaking, an RNA-Binding Protein, Is a Critical Regulator of Vascular Smooth Muscle Cell Phenotype

“…5,6 Importantly, QkI null mice are embryonic-lethal because of an inability of immature mural cells to migrate and differentiate into vascular smooth muscle cells (VSMCs) effectively, resulting in perturbed investment and stabilization of nascent vessels in the yolk sac vasculature. [7][8][9] In adults, VSMCs of the artery wall contract and provide vascular tone.…”