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Fig. 8. SRp38 can interact with the peptidyl transferase domain of 28s ribosomal RNA. (A) RNA immunoprecipitation was performed using Flag-tagged wild-type SRp38 or a mutant SRp38 (SRp38*), in which all four conserved phenylalanines and tyrosines in the RNA-binding domain were converted to alanines (underlined), which should abrogate sequence-specific binding. (B) Immunoprecipitation of S11 sequence with SRp38. Control, Flag-SRp38- or Flag-SRp38*-expressing embryos were lysed and immunoprecipitated with anti-Flag (M2) agarose beads. IP products were then subjected to RT-PCR for S11 sequence. S11 was not immunoprecipitated in control uninjected embryos (lane 1) but was efficiently brought down in the wild-type SRp38 injected embryos (lane 2). A significantly smaller amount of S11 was also brought down with mutant SRp38*; this may reflect interactions of S11 with SRp38 partner proteins. A proportion of the samples were also used for western blotting to determine efficiency of initial protein expression (input) and immunoprecipitation (IP). (C) Nucleotide sequence of S11. (D) S11 maps to a portion of Domain V of Xenopus 28s ribosomal RNA, modeled after Gutell Lab Comparative RNA Web Site (http://www.rna.icmb.utexas.edu/). (E) S11 is expressed in a punctate pattern in the tadpole. Sense probe was used as a control for background. (F) Embryos injected with SRp38 (S) compared to mutant SRp38 (S*) show some decrease in the activity of a luciferase plasmid reporter. At the one-cell stage, embryos were uninjected (C), injected with SRp38 (S) or mutant SRp38 (S*). Embryos were allowed to divide and each set (C, S, S*) of embryos were injected with 50 pg luciferase DNA. Embryos were cultured and harvested for luciferase readings at stage 21. Error bars represent 95% confidence intervals. (G) ³⁵S-methionine incorporation is mildly decreased in SRp38 (S)-injected embryos. Embryos were injected at one cell stage with 250 pg luciferase, SRp38 or SRp38* mRNA. At stage 17, 0.1 mCi/ml ³⁵S-methionine was added to the culture media. Embryos were subsequently lysed, proteins were acetone precipitated and incorporated ³⁵S-methionine was counted. Left: from stage 17 to 21, there were no significant differences. By stage 28 (right) there was a small difference between SRp38- and SRp38*-injected embryos. Error bars represent 95% confidence intervals.

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