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. 2000 Sep;20(18):6659–6667. doi: 10.1128/mcb.20.18.6659-6667.2000

FIG. 4.

FIG. 4

Analysis of a mutated SL1 RNA containing part of SL2 stem-loop III. (A) Schematic diagram of SL1 RNA with mutations. The predicted secondary structure of the C. elegans SL1 RNA is shown with the mSL1 mutation and the portion of SL1 changed to the equivalent region of SL2 boxed. Nucleotide positions marked with an asterisk remain unchanged in the mutated SL1 RNA. The arrow marks the trans-splice site. (B) Expression of mutated SL1 RNAs. Expression of mutated SL1 RNAs was assayed by primer extension with ddC using a primer that anneals to the SL1 RNA just 3′ of the trans-splice site. The band at the +2 position corresponds to endogenous SL1 RNA, while the band at +6 corresponds to mSL1 RNA. Primer extension from two strains carrying the SL2 mutated C17G RNA is included as a control. (C) trans splicing of Stem 3 sub mSL1 to gpd-3 mRNA. The ability of the mutated SL1 RNAs to trans splice to SL2-accepting mRNAs was determined by primer extension with ddC from the gpd-3 mRNA, as in Fig. 2B. The predicted primer extension terminations at +9, +6, and +5 correspond to endogenous SL2, mSL1, and mSL2, respectively. No trans splicing of the mSL1 or Stem 3 sub mSL1 mutated RNAs to gpd-3 was observed. (D) trans splicing of Stem 3 sub mSL1 to gpd-2 mRNA. The ability of the mutated SL1 RNAs to trans splice to an SL1-accepting mRNA was determined by primer extension with ddC, using a primer that anneals to gpd-2 mRNA just 3′ of the trans-splice site. The bands at the +6 and +2 positions correspond to mSL1 and endogenous SL1 trans splicing, respectively. The bands at the +9 and +13 positions correspond to endogenous SL2 trans-splicing (46) and unprocessed RNA, respectively. trans splicing of mSL2 at +5 can be seen in the C17G lanes upon longer exposure (data not shown). (E) trans-splicing efficiency of mSL1 and Stem 3 sub mSL1 to gpd-2 mRNA. trans-splicing efficiencies (trans splicing/expression) were calculated as described in Materials and Methods. The samples were normalized to the endogenous SL1 trans-splicing levels. Means and standard deviations are shown.