Resurrecting the Activity of the RNA Subunit of Human RNase P

The RNA subunit of human nuclear RNase P, H1 RNA, has low catalytic activity in the absence of its protein cofactors. To improve H1 RNA activity, we introduced minor structural changes toward the bacterial consensus into its catalytic (C) domain. Incorporating a bacterial-like P4-J19/4 element increased its ribozyme activity ∼two fold. Changes toward bacterial-like P2-P3-J3/4 or P5-P15 elements individually impaired its activity, but increased activity ∼five fold when combined with a bacterial P4-J19/4 element, suggesting that several core elements act cooperatively to enhance the catalytic performance of H1 RNA. Neither fusion of the H1 RNA C-domain to the bacterial S-domain nor substrate tethering to H1 RNA resulted in detectable cleavage activity, indicating that low H1 RNA activity is not merely a consequence of defective S-domain function. Pb²⁺-probing and UV melting profiles indicate an inherently unstable global structure of H1 RNA that is sensitive to structural alterations. The parental H1 RNA and its two to five fold more active variants could be activated by the Escherichia coli RNase P (RnpA) protein at low (10 mM) Mg²⁺ concentration, indicating that bacterial RnpA and eukaryotic Pop5 protein subunits recognize C-domain core elements common to all RNase P RNAs.