Interaction of Exon1 with the U6/U2 complex. (A) The uridine at position 5 of Exon1 can be cross-linked to U6 nucleotide 54 in the AGC triad. Left panel: the cross-linked band requires Exon1 for its formation. The RNA species present in the cross-linking reaction are indicated on the top. Arrow points to the cross-link. Middle panel: an identical cross-linked species forms when Exon1 carries the radiolabel instead of U6. Arrow points to the location of the cross-linked species. Arrowhead marks the site of uncross-linked U6. Location of uncross-linked Exon1 is shown to the left. Asterisks indicate the species carrying the radioactive label. Right panel: alkaline hydrolysis mapping of the cross-linked nucleotides in U6. Ctrl lanes contain untreated U6 or purified cross-linked species. Lanes marked Alk contain alkaline hydrolysis reactions. An RNase T1 digestion reaction is loaded into the lane marked T1. XL: purified cross-linked species containing a radioactive label at the 5′ end of U6. Arrow points to the site of stop in the hydrolysis ladder. Locations of G49 and 54 in the RNase T1 digestion lane are shown. (B) The U6/U2 complex with the bound substrates. The base-pairing interactions between U6 and Exon2 are shown. The highlighted regions in U6 mark the invariant sequences. The highlighted regions in the substrates are the “exonic” sequences. Exon1 is shown in boldface letters. Numbers indicate the human numbering system for U6 and U2 or the position from the 5′ end in the case of the substrates. Thunderbolts mark the nucleotides which can be cross-linked to each other. (C) Exon1 does not bind to the U6 ISL in isolation. Location of 5′-labeled Exon1 is shown. The concentration of ISL added to each lane is indicated on top. The left-most lane contains the U6/U2 complex instead of ISL. Arrow points to the complex formed between Exon1 and U6/U2.
