Assay for ribosome stimulation of angiogenin nuclease activity

Angiogenin (RNase 5) is an unusual member of the RNase A family with very weak RNase activity and a preference for tRNA. The tRNAs cleaved by angiogenin are thought to have a variety of roles in cellular processes including translation reprogramming, apoptosis, angiogenesis, and neuroprotection. We recently demonstrated that angiogenin is potently activated by the cytoplasmic 80S ribosome. Angiogenin's binding to the ribosome rearranges the C-terminus of the protein, opening the active site for the cleavage of tRNA delivered to the ribosomal A site which angiogenin occupies. Here, we describe the biochemical procedure to test angiogenin's activation by the ribosome using the assay termed the Ribosome Stimulated Angiogenin Nuclease Assay (RiSANA). RiSANA can be used to test the activity of wild-type or mutant angiogenin, or other RNases, against different tRNAs and with different ribosome complexes. For example, given that angiogenin has been implicated in anti-microbial activity, we tested the ability of bacterial 70S ribosomes to stimulate angiogenin activity and found that the E. coli ribosome does not stimulate angiogenin. We also assayed whether angiogenin's closest homolog, RNase 4, could be stimulated by the ribosome, but unlike angiogenin this enzyme was not further activated by the ribosome. The RiSANA assay promises to reveal new aspects of angiogenin mechanism and may aid in the development of new diagnostic tools and therapeutics.