Est2 binding is stabilized upon Pop1 binding to the RNP via the P3-like domain. (A) Western blot (top) and northern-IP (bottom) of the corresponding immunoprecipitation samples after pulldown of HA-tagged Pop1. (Lane 1) NLYH494 strain expressing an untagged version of Pop1. (Lanes 2–6) NLYH125 strain expressing HA3-Pop1 and the indicated TLC1 alleles (except for lane 2 that has no TLC1). The indicated tagged proteins were revealed using Myc (Est2) or HA (Pop1) antibodies. Immunoprecipitated RNAs were visualized by hybridizing the northern membrane with TLC1- and NME1-specific probes. (B) Telomerase activity assay performed on the same immunoprecipitated extracts as in A. Indicated samples were treated with RNase to verify RNA-dependent activity (lanes marked with +). 0 + 32P: 32P-end-labeled substrate oligo. Loading ctrl: 12-nt labeled oligo. Relative activity/RNA ratios for the WT, WT + 3, and CS2Δ samples were of 1.0, 1.1, and 1.0, respectively (n = 2). The relative band intensities of the extension products were quantified and values were divided by the total RNA content verified by northern blots (data not shown). These were then corrected by HA3-Pop1 protein signal shown in A, middle panel. The obtained values were adjusted relative to the WT Tlc1, which was set as 1.
