FIGURE 1.

Cross-linking preserves cellular TCAB1-hTR RNPs and prevents new RNP formation in cell extract. A, cell extract mixing using VA-13 cells expressing combinations of exogenous hTR and ZZF-hTERT. Mixing was performed at 4 and 22 °C for 0.5 or 2 h. Telomerase activity was detected by QTRAP. Values were normalized to VA-13 coexpressing hTR and ZZF-hTERT (n = 3). Note that mature hTR migrates as a doublet under the gel conditions used for Northern blot detection. The U6 snRNA is a control to demonstrate comparable amounts of input extract. B, cell extract mixing using 293T cells expressing combinations of exogenous hTR and F-TCAB1. hTR bound to F-TCAB1 was detected by FLAG IP and Northern blot of hTR. U6 snRNA and tubulin are controls to demonstrate comparable amounts of input extract and also IP specificity. RC, RNA precipitation recovery control. C, a similar experiment was carried out as described in B, except cells were formaldehyde cross-linked prior to cell lysis. hTR bound to F-TCAB1 was detected by FLAG IP followed by RT-qPCR. Input and F-TCAB1-bound hTR levels were each normalized to input RNA polymerase II mRNA and set relative to the triple coexpression sample (n = 3). All lanes were from the same blot; a gap indicates removal of extraneous samples.