Site-specific crosslinking of mammalian U11 and U6atac to the 5′ splice site of an AT–AC intron

A rare class of introns with AT-AC at their termini recently has been identified in metazoan genes. Splicing of these introns requires a different set of small nuclear ribonucleoprotein particles (snRNPs) (U11, U12, U5, and U4atac/U6atac) compared with the snRNPs (U1, U2, U5, and U4/U6) required for splicing the majority of pre-mRNA introns, but otherwise little is known regarding the excision of AT-AC introns. Here we use site-specific 4-thiouridine (4SU) crosslinking analysis to dissect the mechanism of 5' splice site recognition during in vitro splicing of the AT-AC intron from the P120 pre-mRNA. Upon irradiation with 365-nm UV light, three P120 substrates, each with a single 4SU substitution near the 5' splice site (at position +2, +4, or +7), produce two early ATP-independent crosslinks with similar kinetics. For one of the substrates, P120-4SU+2, a third ATP-requiring crosslink forms as the two early crosslinks diminish. RNase H digestion coupled with Northern blotting indicates that the two early crosslinks generated with P120-4SU+2 contain the U11 small nuclear RNA. Reverse transcription-PCR followed by cloning and sequencing demonstrates that the third crosslink involves U6atac. The dynamic appearance of the three crosslinks correlates with the kinetics of the splicing reaction and suggests that the 5' splice site is recognized first by U11 and then by U6atac. Our results argue that the splicing of AT-AC introns is mechanistically similar to the splicing of the major class of introns and that the U11 and U6atac snRNPs in the AT-AC spliceosome fulfill analogous roles to U1 and U6, respectively, in the major spliceosome.