Figure 3

PMR60 associates with PTH mRNA in transfected cells and cleaves PTH in vitro. A. PMR60-PTH mRNA interaction. HEK293 cells were transiently co-transfected with TAP and Myc-tagged PMR60⁰ (catalytically inactive) or with pcDNA3 (vector) and expression plasmids for hPTH and luciferase. PMR60⁰ was affinity-purified followed by Tev protease cleavage. Left panels: Immunoblot (IB) analysis of the input (10% of the IPed fraction) and bound fractions using either anti-Myc antibody (left gel) or anti-luciferase antibody (right gel) to demonstrate transfection efficiency. A dot indicates nonspecific cross-reaction of anti-luciferase antibody with PMR60⁰. The decreased size of the recovered PMR60⁰ (bound) results from Tev protease cleavage of the TAP tag. Right panels, RT-PCR analysis of RNA recovered from input and bound fractions, assayed for PTH (top gel) and luciferase (bottom gel) mRNAs. Similar results were obtained in 2 independent experiments. B. A PMR60 enriched fraction cleaves PTH mRNA in vitro. PMR60 was affinity-purified from cytoplasmic extracts of HEK293 cells transiently transfected with catalytically active PMR60 as in A. Top panel: IB analysis using anti-Myc antibody showing increasing concentrations of purified PMR60 used in the cleavage assay. Bottom panel: Uniformly radiolabeled rPTH or GH mRNAs without or with increasing amounts of PMR60 analyzed by agarose gel electrophoresis and autoradiography. C. 3'-end labeled rat PTH mRNA was treated with the amounts of PMR60 as in B. Cleavage products were analyzed by urea-PAGE and autoradiography to detect smaller intermediate products. The arrows mark the intact end-labeled PTH transcript and a single 3' cleavage product.