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Although most eukaryotic mRNAs are degraded by exonucleases acting on either end of the molecule, a subset of mRNAs undergo endonuclease cleavage within the mRNA body. Endonuclease cleavage can be activated by cellular stress, extracellular signals, or by ribosome stalling, as might occur at a premature termination codon. Only a few eukaryotic mRNA endonucleases have been identified, and of these, polysomal ribonuclease 1 (PMR1) is the best characterized. A notable feature of PMR1-mediated mRNA decay is that it acts on specific mRNAs while they are engaged by translating ribosomes. This chapter begins with several procedures used to characterize in vivo endonuclease cleavage of any mRNA by any endonuclease. These include approaches for identifying the 5'-end(s) downstream of an endonuclease cleavage site (S1 nuclease protection and primer extension), and a ligation-mediated RT-PCR approach developed in our laboratory for identifying the 3'-ends upstream of a cleavage site. We then describe a number of approaches used to characterize PMR1-mediated mRNA decay in cultured cells. PMR1 participates in a number of different complexes. We show several approaches for studying these complexes, and we describe techniques for isolating and characterizing PMR1-interacting proteins and its target mRNAs. Although the various techniques described here have proven their usefulness in studying PMR1, they can be generalized to studying decay by any other mRNA endonuclease.

Chapter 13 Approaches for Studying PMR1 Endonuclease–mediated mRNA Decay