FIGURE 1.

L30 binds, but does not repress, the RPL30 5A C9U transcript. (A) Schematic representation of the RPL30 kink-turn bound by L30, wt, and mutant versions, as indicated (SS: splice site, the AUG is at the end of the first exon). Numbers refer to the start of transcription. (B) Effect of mutations in RPL30 on L30 binding. RPL30 transcripts (0.5 pmol, nt 1–123) were incubated with buffer (lanes 1,7,13,19) or with increasing amounts of MBP:L30 (50, 100, 200, 500, and 1000 ng; lanes 2–6, 8–12, 14–18, and 20–24, respectively). Transcripts were wt (lanes 1–6), C9U (lanes 7–12), 5A (lanes 13–18), and 5A-C9U (lanes 19–24). Reactions were analyzed in a 6% acrylamide gel. (C) Both C9U and 5A C9U transcripts fail to accumulate pre-mRNA under conditions of L30 excess. W303 (lanes 1,2) or yJV25 (producing excess L30, lanes 3–6) cells were transformed with pLCUP plasmids (schematized on the bottom) bearing the indicated mutations (top). RNA was extracted, subjected to Northern analysis, and probed with LCUP sequences (D) snRNP coimmunoprecipitation with L30. RPL30 transcripts (nt 1–347) were incubated under splicing conditions with MBP:L30. Reactions were immunoprecipitated with anti-MBP, and pelleted RNA was subjected to Northern analysis to detect RPL30, U1 and U2 snRNA, as indicated. Lane 1, RPL30 +12; lane 2, RPL30 5A C9U; lane 3, RPL30; lane 4, no transcript added; lane 5, no antibody added; lane 6, 1% of the input.