Identification and Characterization of Expanded RNA Binding Abilities and Cellular Roles for Nuclear RNase P.

[摘要] Ribonuclease P (RNase P) is an essential endoribonuclease that catalyzes the cleavage of the 5′ leader of pre-tRNAs. In addition, a growing number of non-tRNA substrates have been identified in various organisms. RNase P varies in composition as bacterial RNase P contains a catalytic RNA core and one protein subunit while eukaryotic RNase P has multiple protein subunits with a catalytic RNA core. The more complex composition of eukaryotic RNase P provides unique RNA binding abilities not present in bacterial RNase P. A series of in vitro and in vivo investigations was used to characterize RNA binding with eukaryotic RNase P and how it can translate into cleavage of a diverse set of RNA substrates.In vitro studies established that single stranded RNA binds and strongly inhibits RNase P catalyzed pre-tRNA cleavage. This inhibition was not sequence dependent as multiple mixed sequence RNAs inhibited RNase P similarly to homopolymer RNA, although only mixed sequence RNA was cleaved. Investigation of RNA binding using crosslinking methods indicated that a diverse set of RNA (pre-tRNATyr, polyU50 RNA, and mixed sequence RNA) contacts RNase P near the RNA active site of the enzyme. In addition, 2-3 of the 9 proteins in yeast RNase P crosslink to homopolymer RNA.In vivo studies were used to determine if strong in vitro binding and cleavage translated into new RNase P substrates in vivo. Using cells containing a temperature sensitive RNase P mutation, pre-mRNA and noncoding RNA were shown to accumulate strongly using a strand specific microarray. RNase P’s role appears to be indirect with pre-mRNA accumulation occurring due to a spliceosome assembly defect that exists in the RNase P mutation strain. Also, a variety of noncoding RNAs were shown to accumulate with a subset indicating inverse changes with overlapping coding regions. It was shown that multiple larger antisense RNA accumulate in the cells with the RNase P mutation, consistent with a previously unknown role of RNase P in degrading some of these antisense RNA in vivo.

[发布机构] University of Michigan

[效力级别] Ribonuclease P

[关键词] RNase P;Ribonuclease P;Antisense RNA;Single Stranded RNA;Non-tRNA Substrate;Biological Chemistry;Science;Biological Chemistry