Our scientific objective is to understand how proteins modulate the function of ribonucleoprotein (RNP) enzymes through structural changes to their associated catalytic RNA. This goal is highly relevant to public health due to the growing appreciation for the roles of RNPs in tissue complexity and human diseases. In this proposal, we will use RNase P as a model to test our postulate that the versatility of RNPs is due to protein- mediated structural changes in their RNA cores. Although the primary function of RNase P is 5??-maturation of precursor tRNAs, recent findings suggest an expanded functional mission that includes biogenesis of eukaryotic non-coding RNAs. Eukaryotic and archaeal RNase P consist of a catalytic RPR (RNase P RNA) and multiple (4-10) RPPs (RNase P Proteins), unlike the simpler bacterial version (1 RPR + 1 RPP). Because all RPRs are active on their own in vitro, the need for multiple archaeal and eukaryotic RPPs is unclear. We found from step-wise reconstitutions of archaeal RNase P that its assembly intermediates comprising partial suites of five RPPs and the RPR exhibit activity and fidelity of processing in between the RPR alone or the full holo- enzyme (RPR + all RPPs). These findings motivate our central hypothesis that binding of RPPs to specific RPR regions independently and collectively mediates RNA structural changes essential for assembly and catalysis. We will address this hypothesis with two specific aims to delineate structure-function relationships of intermediates en route to assembly of the full RNP: (1) Dissect the structural basis for the distinct roles of archaeal RPPs in aiding RPR catalysis, and (2) map the assembly landscape of archaeal RPPs on the RPR. To study how RPPs guide the RPR towards its functional state, we propose an innovative combination of site- specific and global structural methods coupled to direct functional readouts.
In Aim 1, we will probe archaeal RPR structural changes induced by different suites of RPPs at nucleotide resolution using SHAPE-Seq (selective 2??-hydroxyl acylation analyzed by primer extension sequencing), a high throughput method to probe RNA structures. Inferences from SHAPE-Seq, linking structural changes to functional outcomes, will be guided by the RNA-protein contact sites obtained from tethered-nuclease mapping and validated using assays of RPR mutants.
In Aim 2, we will survey the hierarchy and cooperation during RNase P assembly with bulk and single molecule fluorescence kinetic studies. RPP-mediated alterations in RPR conformational sampling will be studied using fluorescence resonance energy transfer, and changes in RPR topology will be uncovered with small angle x-ray scattering and native mass spectrometry. Although activity versus fidelity tradeoffs have shaped the adaptive landscape of many enzymes, we expect our work to provide insights into how multiple RPPs allowed archaeal/eukaryotic RNase P to maintain robust cleavage without compromising processing fidelity on a broad range of substrates. This study will contribute to a framework for understanding the mechanistic basis of RNA-protein cooperation in RNPs and how dysfunctioning RNPs lead to disease.
A number of genetic diseases (e.g., neurodegeneration) are associated with defective processing of transfer RNAs, which are needed to decode the genetic blueprint. One of the enzymes critical for this processing depends on cooperation between a catalytic RNA and multiple protein subunits. To understand this enzyme's assembly and catalysis, we will study how the protein subunits affect the structure and function of its essential RNA core, thus contributing to a mechanistic appreciation that could help elucidate the causes of disease.
| Chen, Tien-Hao; Sotomayor, Marcos; Gopalan, Venkat (2018) Biochemical Studies Provide Insights into the Necessity for Multiple Arabidopsis thaliana Protein-Only RNase P Isoenzymes. J Mol Biol : |
| Lyon, Seth; Gopalan, Venkat (2018) A T7 RNA Polymerase Mutant Enhances the Yield of 5'-Thienoguanosine-Initiated RNAs. Chembiochem 19:142-146 |
| Gopalan, Venkat; Jarrous, Nayef; Krasilnikov, Andrey S (2018) Chance and necessity in the evolution of RNase P. RNA 24:1-5 |
| Lai, Lien B; Tanimoto, Akiko; Lai, Stella M et al. (2017) A novel double kink-turn module in euryarchaeal RNase P RNAs. Nucleic Acids Res 45:7432-7440 |
