Figure 4.

 hIre1p is a site-specific endoribonuclease. (A) In vitro cleavage of yeast HAC1 mRNA by hIre1p. An in vitro-transcribed ³²P-labeled HAC1 mRNA was incubated with E. coli-expressed GST or GST–Ire1p adsorbed to glutathione beads or with COS-1 cell-expressed hIre1p or hIre1p K599A protein adsorbed to protein A–Sepharose beads. After the indicated period of time, the cleavage products were analyzed by electrophoresis on a 5% denaturing polyacrylamide gel. Schemes on the left depict the predicted cleavage products. Numbers at right indicate predicted base pair size of RNA products expected based on yeast HAC1 mRNA cleavage by yeast Ire1p (Sidrauski and Walter 1997). (B) hIre1p cleaves yeast HAC1 mRNA at residue G661. The HAC1 RNA cleavage site was mapped using in vitro-transcribed HAC1mRNA after incubation with GST,GST–Ire1p, hIre1p, or hIre1p K599A as described in A. The products were reverse transcribed with Superscript II Reverse Transcriptase (Bethesda Research Labs) by use of oligonucleotide primer complementary to the intron of HAC1 RNA. Sequencing ladders on the left represent HAC1 DNA sequence determined with the same primer. (Arrow) Position of primer extended products.