Biomedical subjects
Sean P Ryder
Publications and source records attributed to Sean P Ryder.
Specificity of the STAR/GSG domain protein Qk1: implications for the regulation of myelination.
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.
RNA target specificity of the STAR/GSG domain post-transcriptional regulatory protein GLD-1.
The post-transcriptional regulation of gene expression underlies several critical developmental phenomena. In metazoa, gene products that are expressed, silenced and packaged during oogenesis govern early developmental processes prior to nascent transcription activation. Furthermore, tissue-specific alternative splicing of several transcription factors controls pattern formation and organ development. A highly conserved family of proteins containing a STAR/GSG RNA-binding domain is essential to both processes. Here, we identify the consensus STAR-binding element (SBE) required for specific mRNA recognition by GLD-1, a key regulator of Caenorhabditis elegans germline development. We have identified and verified new GLD-1 repression targets containing this sequence. The results suggest additional functions of GLD-1 in X-chromosome silencing and early embryogenesis. The SBE is present in Quaking and How mRNA targets, suggesting that STAR protein specificity is highly conserved. Similarities between the SBE and the branch-site signal indicate a possible competition mechanism for STAR/GSG regulation of splicing variants.
Biochemical detection of adenosine and cytidine ionization within RNA by interference analysis.
Perturbation of active site functional group pKas is an important strategy employed by protein enzymes to achieve catalysis. There is increasing evidence to indicate that RNAs also utilize functional group pKa perturbation for folding and reactivity. The two best candidates for a functionally relevant pKa perturbation are the N3 of C (pKa 4.2) and the N1 of A (pKa 3.5), either of which could be sufficiently raised to allow protonation near physiological pH. Here we report the synthesis and use of a series of alpha-phosphorothioate tagged cytidine and adenosine analogs whose altered pKas make it possible to efficiently detect functionally relevant protonation events by Nucleotide Analog Interference Mapping. This approach has been used to detect ionization events in several catalytic RNAs, including the group I intron, the Hepatitis Delta Virus (HDV), the hairpin and the Varkud Satellite (VS) ribozymes. The active site residue of the VS ribozyme appears to be ionized in the course of the reaction pathway, which may be indicative of a general acid or base mechanism for catalysis by this RNA.
Comparative analysis of hairpin ribozyme structures and interference data.
Great strides in understanding the molecular underpinnings of RNA catalysis have been achieved with advances in RNA structure determination by NMR spectroscopy and X-ray crystallography. Despite these successes the functional relevance of a given structure can only be assessed upon comparison with biochemical studies performed on functioning RNA molecules. The hairpin ribozyme presents an excellent case study for such a comparison. The active site is comprised of two stems each with an internal loop that forms a series of non-canonical base pairs. These loops dock into each other to create an active site for catalysis. Recently, three independent structures have been determined for this catalytic RNA, including two NMR structures of the isolated loop A and loop B stems and a high-resolution crystal structure of both loops in a docked conformation. These structures differ significantly both in their tertiary fold and the nature of the non-canonical base pairs formed within each loop. Several of the chemical groups required to achieve a functioning hairpin ribozyme have been determined by nucleotide analog interference mapping (NAIM). Here we compare the three hairpin structures with previously published NAIM data to assess the convergence between the structural and functional data. While there is significant disparity between the interference data and the individual NMR loop structures, there is almost complete congruity with the X-ray structure. The only significant differences cluster around an occluded pocket adjacent to the scissile phosphate. These local differences may suggest a role for these atoms in the transition state, either directly in chemistry or via a local structural rearrangement.