Fig. 3. GLD-1 is pulled out of crude extracts by tagged SCR RNA. (A) Proteins were pulled out of crude extracts using digoxigenin (dig)-labeled RNAs that included the entire 61 nt glp-1 SCR (total RNA size, including vector sequence, was 104 nt). Precipitated proteins were subjected to UV cross-linking to a ³²P-labeled RNA containing 34 nt of the wild-type glp-1 SCR (the GRE/GDE region in Fig. 2; total RNA probe was 68 nt). 58 and 30 kDa (p58 and p30) proteins labeled by the probe are indicated. Dig-labeled RNAs were either wild type, had the M7 mutation in the GRE and GDE (see Fig. 2), or a 13 nt mutation (M9) at the 3' end of the SCR, downstream of the 34 nt GRE/GDE region (not shown). (B) Proteins pulled out by wild-type dig-SCR RNA were UV cross-linked to ³²P-labeled probes that had 34 nt of wild-type or mutant SCR sequences (refer to Fig. 2). The ratios of p58 to p30 varied from prep to prep, possibly due to proteolysis (data not shown). (C) Competition of wild-type ³²P-labeled probe by unlabeled wild-type or mutant RNAs, as assayed by UV cross-linking. Unlabeled RNAs were added at 10- and 500-fold molar excess of labeled probe. Lanes 1 and 8 have no competitor RNA added. (D) A western blot of proteins pulled out by dig-tagged wild-type (WT), M7 or M9 SCR RNAs (71 nt) probed with GLD-1 antibodies. Protein samples used for this blot were from the same samples as used for UV cross-linking in A. The arrow marks the position of the single band detected in total worm homogenate (data not shown).

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