Using site-specific incorporation of a single 32
P-label and the photoactivatable cross-linking reagent s4
U, we have explored the association of factors with the 3′ splice site region of the yeast actin pre-mRNA during nuclear pre-mRNA splicing. It is unknown at precisely which stage of spliceosome assembly the 3′ splice site of introns is first recognized in yeast, although it is clear that cleavage of the 5′ splice site and formation of the lariat intermediate can occur in the absence of a 3′ splice site (27
). In mammalian splicing extracts, a 3′ splice site AG is required prior to the first catalytic step if no long polypyrimidine tract is present (36
) and the 3′ splice site AG can be cross-linked to an unidentified 100 kDa protein in pre-spliceosomes (21
). Recent experiments have established that in metazoan introns, the 3′ splice site AG is recognized in a sequence-specific manner during pre-spliceosome formation by U2AF35
). Although no homolog of U2AF35
exists in Saccharomyces cerevisiae
), the experiments presented here demonstrate that in the yeast pre-spliceosome, the 3′ splice site is contacted in an ATP-dependent manner by an unidentified ~122 kDa protein. Notably, this interaction is independent of the distance between the 3′ splice site and branch site, as it occurs with both the ACT47
UAG↓) as well as ACT13
UAG↓) substrates (Fig. A). This independence suggests the 3′ splice site of yeast introns may be specifically contacted much earlier than previously envisaged (27
). Whereas cross-linking of 100 kDa mammalian protein to the 3′ splice site AG in pre-spliceosomes is sensitive 3′ splice site mutations (21
), cross-links to the ~122 kDa yeast pre-spliceosome protein are not (Figs and ), and suggest these factors may be unrelated. Experiments to identify and further characterize this ~122 kDa component of the yeast pre-spliceosome are currently in progress.
Previous cross-linking studies have established that both Prp16p and Prp8p are closely associated with the 3′ splice site following the first catalytic step (7
). Our experiments have shown that an additional ~140 kDa species is associated with intron sequences at the 3′ splice site following the first catalytic step (Figs –). Our identification of this ~140 kDa band as Prp22p is based upon several lines of evidence. First, the observed size (~140 kDa) is consistent with that predicted for Prp22p (130 kDa) cross-linked to an 18–24 nt RNase T1 fragment. Second, the ~140 kDa species is efficiently immunoprecipitated by antibodies directed against Prp22p. Finally, while depletion of Prp22p from splicing extracts results in loss of cross-linking to the ~140 kDa species, addition of purified Prp22p results in the restoration of cross-linking to a species with slightly decreased mobility consistent with the presence of a His-tag on the recombinant protein.
studies have defined at least two distinct stages that occur prior to the second catalytic step. The first stage involves Prp17 and Prp16p and is ATP-dependent (13
), while the second stage is ATP-independent and involves Slu7p, Prp18p and Prp22p (10
). ATP-hydrolysis by Prp16p in the presence of Slu7, Prp18p and Prp22p leads to a conformational change in the spliceosome that results in the protection of the 3′ splice site region from oligonucleotide-mediated RNase H cleavage (10
). These protection experiments have utilized an actin intron with a 3′ splice site mutation in order to circumvent the normally rapid conversion of lariat intermediates to mature mRNA (37
). Our experiments have shown that Prp22p cross-links to the 3′ splice site (Figs –) in a manner dependent on Prp16p (Fig. ). The strong cross-linking of Prp22p to non-functional 3′ splice sites suggests that the protection of the 3′ splice site region from oligonucleotide-mediated RNase H cleavage previously observed may be a direct consequence of the interaction of Prp22p with the 3′ splice site region. With our cross-linking substrates (Fig. ), we have not detected cross-linking of Slu7p to the 3′ splice site region as has been previously reported (D.S.McPheeters, unpublished data; 16
). This apparent discrepancy, as well as differences in cross-links to the 3′ splice site between yeast and mammalian systems (7
), is most likely due to differences in the photochemistry of the cross-linking reagents used. This, and differences in splicing substrates, may also explain our failure to detect the previously observed increase in Prp8 cross-linking following addition of Prp16p to a Prp16p-depleted extract (16
). Whatever the reason for these differences, it is clear that use of a variety of cross-linking reagents, substrates, and other approaches will be necessary to fully uncover the many interactions involved in 3′ splice site selection.
It has been proposed that prior to the second catalytic step, Slu7p, Prp18p and Prp22p may act to either form a bridge, or stabilize interactions of factors bound to the branch site and distal 3′ splice site sequences (10
). The dependence of the biochemical requirement in the second step for these factors on the distance between the branch site and 3′ splice site suggests they are involved in an early, as opposed to late, step in 3′ splice site selection. The recent finding that metazoan extracts depleted of the human homolog of Slu7p (hSlu7) utilize incorrect 3′ splice sites is consistent with such multiple steps being involved in 3′ splice site recognition (38
). It is unknown if or how any of these factors contribute directly to sequence specific recognition of the consensus YAG↓ sequence at the 3′ splice site. Our data show that Prp22p can be cross-linked to varying extents to a variety of different 3′ splice site sequences, including UAG↓, CAG↓, CAA
↓ and AC
G↓. The ability of Prp22p to cross-link to several different 3′ splice site sequences suggests that this association itself is largely non-sequence specific and likely occurs in part of a larger complex (see below). Although Prp22p is not required for the second step of splicing with our ACT13
UCAG↓) substrate, we have repeatedly observed weak cross-linking of Prp22p to this, and related substrates (Fig. ). Inhibition of the second step by 3′ splice site mutations in the ACT13
substrates, or alteration of the branch site-3′ splice site spacing and intervening sequence in the ACT47
UAG↓) substrate, results in greatly enhanced cross-linking of Prp22p (Figs –). This increase in cross-linking of Prp22p upon inhibition of the second step suggests that the weak cross-linking of Prp22p reflects a normal, but transient association of Prp22p with functional 3′ splice sites prior to the second step. It is also possible that the increased cross-linking of Prp22p to substrates unable to undergo the second step represents a non-specific interaction of Prp22p with sequences downstream of the branch site in substrates lacking functional 3′ splice sites. However, such non-specific interaction of Prp22p seems unlikely given the absence of Prp22p cross-linking to exon sequences in a substrate with a mutant 3′ splice site (Fig. ).
Current evidence suggests the intron and/or exon sequences flanking the 3′ splice site may be recognized prior to the second step by a complex consisting of the U5 snRNA, Prp8p, Prp16p, Slu7p, Prp18p and Prp22p. With functional 3′ splice sites, cross-linking of Prp22p is weak because this 3′ splice site recognition complex assembles only transiently before the 3′ splice site is bound by the active site of the spliceosome. We suggest the strong cross-linking of Prp22p to the mutant 3′ splice sites of our ACT13
substrates, or the non-mutant 3′ splice site of the ACT47
substrate, represents the ‘dead-end’ assembly of this intermediate complex involved in 3′ splice site recognition. In the ACT13
substrates, the next AG dinucleotides downstream from the normal 3′ splice site are 58 and 92 nt, respectively, from the branch site adenosine. Such spacing is near the upper bound of that normally observed in yeast introns (4
), and is much greater than the optimal in vivo
spacing of 18–22 nt determined by Luukkonen and Seraphin (5
). In the absence of an appropriately spaced YAG, a closely related sequence located within a reasonable distance from the branch site may direct assembly of 3′ splice site recognition complexes. In agreement with this interpretation, correct 3′ splice site choice in yeast is frequently maintained even when mutant 3′ splice site sequences are presented (5
). In a separate study, we have found very efficient and correct in vivo
use of mutant 3′ splice sites that are spaced 12 nt from the branch site adenosine (J.S.Chang and D.S.McPheeters, manuscript in preparation). Mutations in Prp8p (18
), as well as the U5, U2 and U6 snRNAs (40
; J.S.Chang and D.S.McPheeters, manuscript in preparation) can activate the use of mutant 3′ splice sites. In total, these data are consistent with a hierarchy of multiple, redundant interactions involved in 3′ splice site selection.
Our experiments have shown that the association of Prp22p with the mutant 3′ splice site of the ACM13
(ACG↓) substrate is not likely to extend into the 3′ exon prior to the second catalytic step (Fig. ). Following the second catalytic step, ATP-hydrolysis by Prp22p is required for release of mature mRNA from the spliceosome even when the branch site to 3′ splice site spacing is short (10
). We have found that an ATPase deficient version of Prp22p (K512A) fails to accumulate cross-links to a substrate proficient in the second step (Fig. A), suggesting that the association of Prp22p with intron sequences at the 3′ splice site changes following completion of the second step. Following the second step, the RNA helicase activity of Prp22p has been proposed to break contacts made by the U5 snRNA and exon sequences adjacent to the 3′ splice site (10
). In light of this proposal, one possible interpretation of our data is that the interaction of Prp22p with the 3′ splice site may shift from intron to exon sequences following the second catalytic step. Further experiments will be necessary in order to test this hypothesis.