With the support from the National Natural Science Foundation of China, the research team directed by Prof. Lei Ming (雷鸣) at Shanghai Institute of Precision Medicine, the Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine recently reported the structural insights into precursor tRNA processing mechanism by eukaryotic RNase P, which was published as two research articles in Science (2018, Vol. 362, Issue 6415, eaat 6678) and Cell (2018, Vol 175, Issue 5, P1393-1404).

Ribonuclease P (RNase P) is a ribozyme that processes transfer RNA (tRNA) precursors and is conserved in all three kingdoms of life. Unlike bacterial RNase P, which contains only one small protein cofactor, archaeal and eukaryotic nuclear RNase Ps have evolved considerably more complex protein subunits: five in archaea and 9 to 10 in eukarya. The role of protein components and the reason for the increased complexity of the protein moieties in eukaryotic nuclear RNase P are still poorly understood. It is still largely enigmatic how the pre-tRNA was recognized and processed by RNase P. High-resolution structures of eukaryotic RNase Ps are required to answer these key questions.

Prof. Lei’s group determined the cryo-EM structures of Saccharomyces cerevisiae and human RNase P holoenzymes alone and in complex with pre-tRNA. The structures reveal that all the protein subunits form an interconnected hook-shaped architecture that tightly wraps around the extended single-layered RNA subunit and stabilizes RNase P into a “measuring device”. This “measuring device” employed two fixed anchors to recognize the L-shaped structure rather than specific sequences of pre-tRNA substrates. The binding of pre-tRNA substrate induces a large conformational change in the active center. Two catalytically important magnesium ions in RNase P are coordinated in the catalytic center by a highly conserved uridine and the phosphate backbone of the catalytic RNA, together with the scissile phosphate and the O3′ leaving group of pre-tRNA. Moreover, simulation analysis visualizes the mechanistic details of phosphodiester bond hydrolysis of pre-tRNA, which is a two-Mg2+-ion–mediated SN2 reaction. The structures also provide an evolutionary model depicting how such a simple ribozyme evolved into a much more complex protein components in higher organisms.

This study represents a major step forward for mechanistic understanding of the function of eukaryotic RNase P and support that all RNase P ribozymes share an RNA-based, substrate induced catalytic mechanism of pre-RNA processing.

Cryo-EM structures of yeast and human RNase P bound with pre-tRNA substrate.