Tips and tricks to probe the RNA-degrading activities of hyperthermophilic archaeal β-CASP ribonucleases.

Abstract

The importance of ribonucleases in posttranscriptional control of gene expression has been established in Eukarya and Bacteria for over a decade. However, this process has been overlooked in Archaea, which are of universal importance to elucidate fundamental biological mechanisms and to study the evolution of life on Earth. Very few ribonucleolytic activities have been reported in Archaea, and RNA metabolism pathways wait to be described. Recently we have identified two major groups of archaeal ribonucleases, aCPSF1 and aRNase J, which are members of the β-CASP metallo-β-lactamase family. Here, we describe in vitro methods to characterize the endo- and exoribonucleolytic activities of hyperthermophilic archaeal β-CASP ribonucleases. The use of various labeled RNA substrates allows defining the specificity of RNA cleavage and the directionality of the exoribonucleolytic trimming activity of the archaeal enzymes which work at high temperature. Elucidating in vitro ribonucleolytic activities is one step toward the understanding of the role of β-CASP ribonucleases in RNA metabolism pathways in archaeal cells.