Experiments in cell cultures have been useful for investigating a number of RNA endonucleases. However, endonuclease decay intermediates are often challenging to study in cellulo, as decay intermediates are rapidly degraded by exoribonucleases. Thus, cell-free assays have been critical for assessing endonuclease kinetics. Here, we describe such an in vitro assay to analyze endoribonuclease activity using recombinant proteins and end-radiolabeled RNA oligonucleotides. Specifically, we detail a protocol for assaying the endoribonuclease activity and kinetics of the human MARF1 protein.
Keywords: Endoribonuclease; MARF1; NYN; PIN; mRNA decay.