Nat Struct Mol Biol 15: 213C215 [PMC free article] [PubMed] [Google Scholar]Anderson K, Moore MJ 1997. were allowed to form, and biotinylated 2-OMe RNA oligonucleotides were annealed to these accessible regions. To allow localization by EM of the bound oligonucleotide, first antibiotin antibodies and then protein A-coated colloidal gold were additionally bound. EM analyses allowed us to map the position of exon and intron nucleotides near the cleaved 5 splice site, as well as close to the anchoring site just upstream of the branch adenosine. The identified positions in the C complex EM map give first hints as to the path of the pre-mRNA splicing intermediates in an active spliceosomal C complex and further define a possible location for its catalytic center. and with the longer oligoribonucleotides used for EM localization are indicated by IL-1RAcP black lines. The biotin positions of the three 2-O-methyl-RNA oligonucleotides used for EM localization (label intron anchoring site, label intron 5 splice site, and label exon 1) are each indicated with a green asterisk. The individual results of all the RNase H cleavage experiments performed with 100 oligonucleotides are shown in Supplemental Figure S1. The RNase H screen showed that most of exon 1 and the analyzed intron sequences are accessible in assembled C complexes. In exon 1, a long accessible stretch was found to begin 48 nt upstream of the cleaved 5 splice site and to end with a short (6-nt) inaccessible sequence 23 nt before the 5 MS2 hairpins (Fig. 2; Supplemental Fig. S1). Closer to the 5 splice site, an additional accessible stretch 15 nt in length extended to the 29th nt upstream of the 3 end of exon 1. Within the intron lariat, a large accessible region, starting 26 nt downstream from the 5 end of the intron (connected to the 2-OH of the branch point) and ending 44 nt before the branch-point adenosine, was found (Fig. 2; Supplemental Fig. S1). There was no indication of any additional accessible regions. We did not analyze the polypyrimidine tract downstream from the branch site, as its inaccessibility to DNA-directed RNAase H cleavage has already been established, (??)-BI-D at least in C complexes assembled on a pre-mRNA (AdML-GG) that contained exon 2 and a mutated 3 splice site (Ilagan et al. 2009), and its sequence redundancy would, in any case, hamper a site-specific localization. As only 17, mostly nonoverlapping, 15- to 18-nt-long oligos were used to cover the whole pre-mRNA in the aforementioned study, exact accessibility borders could not be determined. Nonetheless, protected regions were found in exon 1 and the intron that are compatible with our results. In addition, Ilagan et al. (2009) found that the 3 splice site and exon 2 were largely accessible to RNAse H cleavage. Using a finer RNase H cleavage grid (12-nt-long oligos (??)-BI-D having a 6-nt overlap), Reichert et al. (2002) identified safeguarded sequences of exon 1 in C complexes put together on two different pre-mRNAs. They found, similar to (??)-BI-D our results, the 1st 25C27 nt of exon 1 upstream of the 5 splice site are completely safeguarded, and with one of the pre-mRNAs used, that an additional 15-nt-long region further upstream is also safeguarded. Therefore, all three RNA chains radiating from your branch point appear well safeguarded (??)-BI-D over at least a length of 25 nt within the C complex. Taken collectively, these results suggest that a general feature of the C complex is definitely that its catalytic center (comprising the branched adenosine, cleaved 5 splice site, and also the polypyrimidine tract) is largely buried within (??)-BI-D the complex. The mostly unprotected nature of the 3 splice site and exon 2 found by Ilagan et al. (2009) suggests that this 3 region of the pre-mRNA might be quite flexible within the C.