Figure 3.

LARP7 prevents MePCE from capping 7SK in vivo and in vitro. (A) NE from either normal (control) or HeLa cells stably expressing shLARP7 was incubated with r7SK and [³H]SAM in capping reactions. The RNA products were isolated, resolved in a polyacrylamide–urea gel, and analyzed by autoradiography (top left) and EB staining (bottom left). The levels of the indicated proteins in NEs of control and LARP7 KD cells were examined by WB in the right panel. (B) Left panels: Purified F-LARP7 (1.2 pmols) and/or MePCE (1.3 pmols) proteins were incubated with r7SK and [³H]SAM in capping reactions. The RNA products were analyzed as in A. (Right panel) F-LAPR7 and MePCE used in the capping assay were examined on a silver-stained SDS–gel. An asterisk indicates a minor contaminating band in the F-LARP7 preparation. (C) The affinity purified F-MePCE free of any associated 7SK snRNP components and the F-LARP7-bound MePCE, which was isolated through anti-Flag immunoprecipitation and then either untreated (−) or treated (+) with MCN were analyzed by WB and NB for the presence of the indicated factors (left panel). These MePCE samples were subsequently analyzed in 7SK capping reactions as in A.