FIG. 4.

Two subunits of dyskerin, NHP2, and GAR1 assemble on each molecule of hTR. (A) Schematic of the tandem affinity purification strategy for discriminating subunit stoichiometry. (B) Extracts from 293T cells transfected to express a protein with the tag(s) indicated (Z, F, +, and ZF, where + indicates coexpression of the Z and F tags), wild-type hTR, and the hTR-U64 chimera were subjected to tandem steps of affinity purification. Input cell extracts (2%) and final purified RNP elutions (100%) were supplemented with a recombinant RNA recovery control (RC) prior to RNA extraction. Input and purified samples for the GAR1 panel were from a separate set of transfections and purifications.