Both the P3-like domain and the CS2 bulge sites on TLC1 are required for association of Est1 with the telomerase RNP. (A) Model showing the telomerase RNP and the MS2 binding stems–loops (dark red) at the 3′ end of the RNA. These will be bound by the MS2 core protein (MS2CP) fused with Protein A (ProA). A detailed view of Tlc1's Stem IVc is shown on the left. The green nucleotides (nt 601–611) represent the CS2a element and the area within the green rectangular outline is the P3-like domain. The blue nucleotides (nt 646–659) represent the CS2 bulge, and the blue rectangular outline represents the previously determined Est1 binding site. (B) Western blot after IP of telomerase components via the MS2 RNA stems and Protein A tagged MS2CP. Extracts from NLYH128 containing both a WT TLC1 genomic copy and the indicated plasmid variants of the MS2-tagged TLC1 were used. Lanes 1–5: 4% of the inputs. Lanes 6–10: 20% of the IP fractions. The corresponding tagged proteins were revealed using Myc- (Est1 and Est2) and HA- (Pop1, Pop6) specific antibodies. Strains of lanes 2 and 7 express a WT copy of TLC1 on the plasmid. (C) Northern blot analysis of coimmunoprecipitated RNAs using IgG beads and extracts of NLYH128 (TAP-tagged MS2CP) transformed with the various TLC1-tagged plasmids. Lanes 1, 4, 7, 10, 13: IN, inputs (2.5%). Lanes 2, 5, 8, 11, 14: IP, immunoprecipitates (20%). Lanes 3, 6, 9, 12, 15: FT, flowthroughs (2.5%). The strain used for lanes 4–6 expresses an untagged version of TLC1 on the plasmid; this RNA runs ahead of the tagged versions and is not visible on this part of the northern blot. The top part of the blot was hybridized with a TLC1-specific probe and the bottom part was hybridized with a NME1-specific oligo.
