RNA-based enzymes are crucial in many important biological processes including translation and RNA processing. While in some RNA-based enzymes the RNA moiety alone is capable of performing catalysis, many of them contain proteins that are required for their function. Our proposal focuses on nuclear Ribonuclease MRP (RNase MRP), which is an excellent model for understanding the interplay of proteins and RNA in ribonucleoprotein complexes. RNase MRP is a eukaryotic ribonucleoprotein complex involved in the maturation of rRNA and the regulation of the cell cycle in yeast. RNase MRP is closely related to RNase P and is highly conserved in eukaryotes. S. cerevisiae RNase MRP contains ten proteins and a 340 nucleotide long RNA component. The similarity between RNase MRP and RNase P strongly suggests that catalysis in RNase MRP is RNA-based;however, as in the case of RNase P, all of RNase MRP proteins are also required for activity in vivo. It is particularly intriguing that this reliance on proteins in the enzymes of the RNase P/MRP family tends to grow in more evolutionary advanced organisms. Little is known about why the enzymes of the RNase P/MRP family acquired more and more proteins as evolution progressed, or why RNase MRP needs proteins when the catalytic RNA moiety seems to be essentially preserved. This lack of knowledge represents a problem since it hinders further understanding of these important enzymes. This proposal focuses on biochemical and structural studies of S. cerevisiae nucleolar RNase MRP and aims to understand how the interactions between the components of this ribonucleoprotein complex affect its activity and specificity.
The specific aims of the proposal are: 1. To determine how protein subunits of RNase MRP interact with the RNA moiety;2. To determine roles of RNase MRP protein components in the enzyme's activity and specificity. We will characterize the RNA-protein interactions in RNase MRP using RNA footprinting, mutational studies, and structural analysis, and determine roles of the protein moiety in the activity of RNase MRP. These experiments will clarify the functions of the proteins in RNase MRP and related enzymes. Relevance to Public Health RNase MRP is an RNA-based enzyme which is required for the survival of eukaryotic cells. Mutations in the RNA component of RNase MRP in humans cause the development of Cartilage Hair Hypoplasia, a severe multi-systemic disorder resulting in dwarfism and immunodeficiency, and a study of RNase MRP has a potential to shed a light on the cause of the disease.
|Krasilnikov, Andrey S (2014) Applying UV crosslinking to study RNA-protein interactions in multicomponent ribonucleoprotein complexes. Methods Mol Biol 1086:193-207|
|Esakova, Olga; Perederina, Anna; Berezin, Igor et al. (2013) Conserved regions of ribonucleoprotein ribonuclease MRP are involved in interactions with its substrate. Nucleic Acids Res 41:7084-91|
|Khanova, Elena; Esakova, Olga; Perederina, Anna et al. (2012) Structural organizations of yeast RNase P and RNase MRP holoenzymes as revealed by UV-crosslinking studies of RNA-protein interactions. RNA 18:720-8|
|Esakova, Olga; Perederina, Anna; Quan, Chao et al. (2011) Substrate recognition by ribonucleoprotein ribonuclease MRP. RNA 17:356-64|
|Perederina, Anna; Khanova, Elena; Quan, Chao et al. (2011) Interactions of a Pop5/Rpp1 heterodimer with the catalytic domain of RNase MRP. RNA 17:1922-31|
|Perederina, Anna; Esakova, Olga; Quan, Chao et al. (2010) Eukaryotic ribonucleases P/MRP: the crystal structure of the P3 domain. EMBO J 29:761-9|
|Esakova, Olga; Krasilnikov, Andrey S (2010) Of proteins and RNA: the RNase P/MRP family. RNA 16:1725-47|
|Lu, Qiaosheng; Wierzbicki, Sara; Krasilnikov, Andrey S et al. (2010) Comparison of mitochondrial and nucleolar RNase MRP reveals identical RNA components with distinct enzymatic activities and protein components. RNA 16:529-37|
|Perederina, Anna; Esakova, Olga; Quan, Chao et al. (2010) Crystallization and preliminary X-ray diffraction analysis of the P3 RNA domain of yeast ribonuclease MRP in a complex with RNase P/MRP protein components Pop6 and Pop7. Acta Crystallogr Sect F Struct Biol Cryst Commun 66:76-80|