Institute of Biotechnology, Program on Developmental Biology, PL56 (Viikinkaari 9), 00014 University of Helsinki, Finland
Received 4 February 2005/ Accepted 8 March 2005
| ABSTRACT |
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| INTRODUCTION |
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Distinct U2- and U12-dependent spliceosomes facilitate the removal of the two different intron types. These two spliceosomes differ in their composition of small nuclear ribonucleoproteins (snRNPs), but their overall assembly pathways and catalytic mechanisms are similar. The U1, U2, U4, and U6 small nuclear RNAs (snRNAs) are specific to the U2-dependent spliceosome, whereas U11, U12, U4atac, and U6atac snRNAs are functionally analogous components in the U12-dependent spliceosome. Both spliceosomes utilize the U5 snRNA (42, 56).
A widely accepted paradigm of spliceosome assembly states that snRNPs and protein components associate with the pre-mRNA in a stepwise manner (3, 4, 16). In the U2-dependent spliceosome, this process starts with intron recognition in complex E (the commitment complex in yeast), in which the 5'ss is recognized by the U1 snRNP, whereas the BPS, polypyrimidine tract (PPT), and 3' splice site (3'ss) are recognized by the protein factors SF1/BBP, U2AF65, and U2AF35, respectively (1, 36, 37, 44, 68, 75). In the first ATP-dependent stage (complex A, or prespliceosome), SF1/BBP at the BPS is replaced by the U2 snRNP (43, 74). A spliceosome (complex B) is formed upon entry of the U4/U6.U5 tri-snRNP (20, 24). Additional rearrangements of RNA-RNA interactions (39, 50), and changes in protein composition (33) result in the final catalytically active spliceosome (complex C) in which the U2 and U6 snRNAs together with the pre-mRNA form the catalytic core (6, 17, 32). In the catalytic core, U5 snRNA and Prp8/220-kDa protein interact with exon sequences near the 5'ss (38, 49, 57, 69), while U6 and U2 snRNAs base pair with the 5'ss and BPS, respectively, and with each other to achieve a structure that can carry out likely RNA-based catalysis (21, 25, 59, 60, 74). During the assembly, U1 and U4 snRNPs are released from the final spliceosome prior to catalysis. However, this traditional view of spliceosome assembly has been challenged recently by the identification of a penta-snRNP structure in yeast cell extracts that exhibits splicing activity (52). These data suggest that individual spliceosomal complexes that have been most commonly analyzed by native gels may represent dissociation products of the dynamic preassembled penta-snRNP complex in different conformational stages (3, 40). Although this question is not yet fully resolved, the identification of the U5/5' exon interactions in early spliceosomal complexes (35, 49) and the detected interaction between U1 and U5 snRNPs (1, 2) suggest that larger spliceosomal complexes may indeed be involved already in intron recognition, an idea that is consistent with the penta-snRNP model of assembly.
The assembly of the U12-dependent spliceosome follows the sequential assembly model of the U2-dependent spliceosome (41), although the data are also compatible with the dynamic penta-snRNP model. As in the assembly process described above, the 5'ss and BPS of a U12-type intron initially base pair with U11 and U12 snRNAs, respectively, to form a prespliceosome complex A (14, 23, 55, 72). In contrast to the U2-dependent system, a complex E has not been described, but rather at the earliest stage a preformed U11/U12 di-snRNP (61, 63) cooperatively recognizes U12-type introns. In particular, the U12-BPS interaction requires a prior U11-5'ss interaction (10). Following initial intron recognition, the entry of the U4atac/U6atac.U5 tri-snRNP to the nascent spliceosome promotes global structural rearrangements that lead to catalytic core formation. As in the U2-dependent spliceosome, the U6atac and U12 snRNAs are base paired both together and with the 5'ss and BPS in the catalytic core, respectively, thus holding the critical 5'ss and BPS regions in proximity for the first step of catalysis (9, 19, 48, 54).
In this study, we investigated in vitro recognition of the 5'ss using site-specific cross-linking methods. Unexpectedly, we captured an interaction between the 5' end-most region of the U12 snRNA and the 2 position of the P120 splicing substrate. This transient interaction takes place early during spliceosome assembly. The region of the U12 interacting with the 2 position also forms the catalytically important U12/U6atac helix Ib in the catalytic core. We also captured a similar interaction using a cross-linking substrate derived from an unrelated SCN4a splicing substrate. Our data suggest that this region of U12 snRNA is positioned near the 5'ss before the tri-snRNP-specific interactions occur, further suggesting that catalytically important structures may start forming at the earliest stages of spliceosome assembly.
| MATERIALS AND METHODS |
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Splicing substrates. Capped, full-length P120 splicing substrates were produced by in vitro transcription as described before (9). The chimeric P120-SCN4a substrate contains a P120 5' exon and intron plus 3' exon sequences from the SCN4a substrate (15). The construction of P120 substrates with two 4SU residues (P120[2/+2]) has been described previously (9, 11). P120 substrates containing a single 4SU substitution (P120[2] or P120[+2]) were constructed essentially as was P120[2/+2]. The 4SU-containing SCN4a cross-linking substrates were constructed essentially as the P120 cross-linking substrate described above. The SCN4a cross-linking substrates contain 5' exon, intron, and 3' exon sequences derived from SCN4a-ENH1 splicing substrate (15), but the 5' exon has been truncated and is similar in size as in P120 splicing substrate (41 versus 46 nucleotides) and modified to contain a single 4SU at the 2 position and a short sequence complementary to 5' end of U12 snRNA. The P120 splicing substrates containing mutations in the site interacting with U12 snRNA and the substrates containing duplicated 5' splice sites were constructed by PCR and verified by sequencing.
Splicing reactions. In vitro splicing reactions were performed as described before (10) using 32P-labeled P120 substrate. Splicing of the chimeric P120-SCN4a substrate was accomplished as described before (67), except that 500 nM U11-14 2ome oligonucleotide was used to inhibit the U2-dependent spliceosome. Cross-linking reactions included 10 nM trace-labeled 4SU-containing P120 substrates or 20 nM SCN4a substrates; for the P120 substrates the cross-linking reaction conditions were as described previously (9), while with the SCN4a substrates the cross-linking reactions are carried out in splicing conditions described by Wu and Krainer (67), except that the substrate concentration was 20 nM. Native gel analysis was performed as described before (10) using 25 µg/ml heparin in the loading buffer, unless otherwise indicated.
Cross-linking and identification of cross-linked RNA species. The conditions for performing and processing psoralen and 4SU cross-linking reactions were described previously (9, 10). In time course experiments and in mapping (see Fig. 2B and C and 4A), a saturating concentration (20 nM) of splicing substrate was used to maximize amount of the cross-linked product. In other reactions the substrate concentration was 5 to 10 nM. A 365-nm UV lamp (UVL-225D; UVP, Cambridge, United Kingdom) or a 337-nm N2 excimer laser (PSX-100; Neweks Ltd., Tallinn, Estonia) was used to excite the 4SU residues during cross-linking reactions. Cross-linked RNAs were separated in denaturing 5 to 8% polyacrylamide gels and detected by Northern blotting (9, 54).
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| RESULTS |
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To confirm the identity of the 4SU-containing substrates, blots from the above experiments were stripped and reprobed for U5 snRNA (U5 interacts with the 2 site) and U11 snRNA (U11 interacts with the +2 site). As expected, P120[2/+2] produced cross-links that were detected with both the U5- and U11-specific probes (Fig. 2A, lane 4, middle and upper panel), whereas with P120[2] only the U5-specific cross-links were observed (lane 10, upper panel). Furthermore, both substrates produced U12-specific cross-links that had virtually identical intensities and responded similarly to various 2ome oligonucleotides used to block spliceosome assembly (see below). Therefore, based on RNase H targeting and the use of splicing substrates containing a single 4SU residue, we conclude that U12 snRNA is in close proximity with the 2 position of the P120 splicing substrate during spliceosome assembly in nuclear extracts.
To further investigate the requirements for the U12/5' exon interaction, we arrested spliceosome assembly at various stages using 2ome oligonucleotides complementary to either the P120 splicing substrate or the snRNA components of the U12-dependent spliceosome (see Materials and Methods). Both P120[2/+2] and P120[2] were used to test the effect of the 2ome oligonucleotides, and both substrates displayed identical U12 snRNA-specific cross-linking patterns (Fig. 2A). The most intriguing results were obtained by including a P120 5' exon oligonucleotide, which stimulated U12/5' exon cross-link formation by approximately three- to fivefold compared with the control (Fig. 2A, compare lanes 4 and 5 and 10 and 11). Furthermore, the U12/5' exon cross-link was insensitive to the U6atac1-20 oligonucleotide (compare lanes 4 and 6 and 10 and 12). Together the data above suggest that the U12/5' exon interaction could take place early during spliceosome formation, since both the U6atac1-20 and the 5' exon 2ome oligonucleotides also prevent the U6atac/5'ss interaction (9 and data not shown) and either abolish or reduce the extent of the U5/5' exon interaction (Fig. 2A, U5 panel, lanes 4 and 5 and 10 and 11).
In contrast, each of the two U12-specific oligonucleotides used in this experiment prevented the formation of the U12/5' exon cross-link. The lack of cross-link with the U12-BPS oligonucleotide (Fig. 2A, lanes 8 and 14) was most likely due to inhibition of prespliceosome formation, suggesting that the U12/5' exon interaction requires prior U12/BPS interaction. This is supported by similar negative cross-linking results from experiments with a 3'-truncated P120[2] substrate lacking the BPS, 3'ss, and the 3' exon (data not shown). The above results suggest, together with ATP requirement for cross-link formation (Fig. 2B), that the U12-5' exon interaction takes place after the intron recognition but prior to tri-snRNP entry to the spliceosome. The observed inhibition of the cross-link formation with the U121-16 oligonucleotide (Fig. 2A, lanes 7 and 13), which prevents the formation of the U12/U6atac helix I but not the U12/BPS interaction (Fig. 1B, lane 5), is in contrast most likely due to a direct or indirect blockage of the U12 region that interacts with the 5' exon (see below).
An important question is whether the U12/5'exon interaction takes place in functional spliceosomal complexes, in which both the 5'ss and BPS have been simultaneously recognized by the spliceosomal components. We have previously used DNA oligonucleotide-directed RNase H hydrolysis to show that the 5'ss and BPS are recognized by U11/U12 di-snRNP (10), which renders those sites resistant to RNase H hydrolysis. Here we used a similar method to ask if the U12/5' exon cross-link is resistant to a prior RNase H hydrolysis at the 5'ss and BPS. Short DNA oligonucleotides directed to 5'ss, intron, or BPS were added to the splicing reactions prior to the UV irradiation step, and the cross-link formation was subsequently investigated by Northern blotting (see Fig. 2C). We found that the U12/5' exon cross-link is resistant to the RNase H hydrolysis at the 5'ss and BPS, even when both sites were targeted simultaneously (Fig. 2D, upper panel, lanes 2, 3, 5, and 6). In contrast, the DNA oligonucleotide targeting the intron sequence resulted in a loss of the cross-link signal (lane 4). The reduced cross-linking intensity in the presence of DNA oligonucleotides targeting the 5'ss or BPS in comparison to the control reaction (compare lane 2 to lanes 3, 5, and 6) is most likely due to competition between oligonucleotide versus U11/U12 complex binding to the pre-mRNA, since we can detect a similar reduction in the A complex intensity in native gel analyses following the RNase H treatment with 5'ss- and BPS-specific oligonucleotides (data not shown).
The efficiency of the RNase H hydrolysis with each oligonucleotide was confirmed by probing the same blot for P120 splicing substrate (Fig. 2D, lower panel). The level of RNase H resistant P120 signal (maximum,
5% of the input) in lanes 3, 5, and 6 agrees with our earlier study (10), and suggests that while only a minor population of the splicing substrates have been assembled into RNase H resistant spliceosomal complexes, the majority, if not all, of the U12/5' exon interactions are taking place in that population. We conclude that the U12/5' exon cross-link takes place in spliceosomal complexes in which both the 5'ss and BPS have been recognized by the components of the spliceosome, most likely by U11 and U12 snRNPs, respectively.
The U12 snRNA-5' exon interaction occurs in the prespliceosome and is transient. To investigate the timing of the U12-5' exon interaction, we compared the kinetics of the U12-specific cross-link formation with the other snRNA-pre-mRNA interactions occurring in the vicinity of the 5'ss. The time course of various snRNA/pre-mRNA cross-links was followed using the P120[2/+2] substrate by hybridizing the same Northern blot with probes complementary to the individual snRNA components of the U12-dependent spliceosome. Maximal U12/5' exon cross-linking was detected after 20 min of incubation at 30°C (Fig. 3A, lane 2), after which the cross-link slowly decreased. Comparison of U12/5'exon cross-link formation with the tri-snRNP-specific U6atac/5'ss and U5/5' exon cross-links suggests that the U12-5' exon interaction takes place prior to the formation of the tri-snRNP-specific interactions, which level off after 60 min. More detailed kinetic data were obtained by repeating the above experiment with a P120[2/+2] substrate containing only an 8-nucleotide (nt) 5' exon (Fig. 3B). This substrate does not interact with U6atac, and the appearance of the U5/5'ss cross-link is delayed and its formation is less efficient (9). Using this truncated splicing substrate, the U12-specific cross-link was readily detected at the 10-min time point (Fig. 3B, lane 2). In contrast, only a faint U5/5' exon cross-link was visible after 20 min (lane 3), indicating that the U12/5' exon interaction occurs prior to the U5- or U6atac-specific interactions with pre-mRNA. Interestingly, the U12/5' exon cross-link intensity was not reduced with the truncated substrate at the later time points, suggesting that the reduction of the U12-5' exon interaction at the later time points may be linked to the entry of the tri-snRNP.
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The kinetics of the U12-5' exon interaction were further investigated using the oligonucleotide block-release method (9), which synchronizes the spliceosome assembly, thereby increasing the precision with the kinetic measurements. Since the U12/5' exon cross-link intensity was enhanced with the 5' exon oligonucleotide, we tested the outcome after the release from the blockage. Compared with the control time course (Fig. 3C, lanes 1 to 6), the presence of the blocking oligonucleotide enhanced cross-link formation (lanes 7 and 8; note that the long, tailed oligonucleotides sometimes smear the bands containing cross-links). When the blocking oligonucleotide was released, the U12/5' exon cross-link signal disappeared almost completely (lanes 9 to 11). These data, together with the time course experiment above (Fig. 3A and B), suggest that the U12-5' exon interaction not only occurs early but also is transient.
5' exon oligonucleotide increases the stability of the complex A. Since the U12-5' exon interaction appears to take place early during spliceosome assembly, we next tested whether the 5' exon 2ome oligonucleotide affects spliceosomal complex formation. To distinguish the various complexes in a native gel, we performed the analysis using unlabeled P120 substrate followed by Northern blotting. Interestingly, similar to the U12/5' exon cross-link formation, the 5' exon oligonucleotide increased the intensity of the complex A band (containing both U11 and U12 snRNAs) by three- to fivefold as compared with the control reaction (Fig. 4A, compare lanes 2 and 3 and 5 and 6). In contrast, the intensity of the complex B band was reduced approximately twofold (lanes 8 and 9 and 11 and 12).
The increased intensity of the complex A suggests that more spliceosomal complexes form in the presence of the 5' exon oligonucleotide. This result contradicts our cross-linking data, especially the U11/5'ss and U12/BPS cross-links, both of which should have also been stimulated with the 5' exon oligonucleotide if the number of prespliceosomes had increased (Fig. 2A; see also Fig. 2 and 4 in reference 9). To resolve this apparent inconsistency, we asked if the stability of prespliceosomes instead of their number was increased under the native gel conditions. To test this possibility, we took advantage of our earlier observation that increased heparin concentration in the native gel loading buffer dissociates the U11 snRNP from the complex A (10). We titrated the reactions with heparin in the presence of either the 5' exon oligonucleotide or an unrelated 2ome oligonucleotide of equal length. The assay was performed with both a 32P-labeled P120 substrate and an unlabeled substrate followed by Northern blotting to confirm the loss of the U11 snRNP (Fig. 4B). We found that the presence of the 5' exon oligonucleotide increased the stability of the complex A, resulting in approximately twofold higher tolerance to heparin concentration (Fig. 4C). We conclude that the native gel analyses with 5' exon oligonucleotide support the cross-linking time course analyses which indicated that the U12-5'exon interaction takes place at the prespliceosome stage.
Mapping the cross-link within U12 snRNA. The relatively fast gel mobility of the U12-specific cross-link (compare the migration of U12/BPS and U12/5'exon cross-links in Fig. 1B, lane 4) and the sensitivity to the U121-16 2ome oligonucleotide suggest that the 5' end of the U12 snRNA may interact with the 2 position of the substrate. To map the location of the specific cross-link within U12 snRNA, the P120[2] substrate was used together with the 5' exon 2ome oligonucleotide to maximize the yield of cross-linked species. A large-scale reaction was performed, followed by specific 3' end labeling (see Materials and Methods) and gel purification of the U12-specific cross-linked products. A 3'-end-labeled un-cross-linked U12 snRNA was also gel purified for control reactions.
Standard primer extension blockage analysis did not reveal any clear intermediate stops within the cross-linked U12 snRNA (not shown). Therefore, we localized the cross-link site by partially hydrolyzing the 3'-end-labeled cross-linked RNAs using ribonucleases T1 and PhyM, which hydrolyze RNA after G residues or after C and U residues, respectively. Since the cross-linked U12 snRNA was 3' end labeled, the partial hydrolysis was expected to reproduce the hydrolysis pattern of an un-cross-linked U12 snRNA as long as linear RNA fragments were produced. When hydrolysis occurs on the 5' side of the cross-linked site, similarity with the control reaction should disappear because of the decreased gel mobility of the resulting branched fragments.
Partial hydrolysis with RNase T1 yielded the most detailed results. The partial hydrolysis pattern of the cross-linked RNA (Fig. 5A, lane 3) was virtually identical to that of the control U12 snRNA (lane 1); even the cleavage at G3 near the very 5' end of the U12 snRNA was recognized, and only the full-length U12 snRNA band was missing or greatly reduced. These data suggest that the cross-link must be located upstream of the G3 residue, within the first two 5'-most nucleotides of the U12 snRNA. Supporting results were also obtained with RNase PhyM, but due to the reduced activity of this RNase in the reaction buffer the resulting bands were very weak. This method was validated with control reactions containing previously mapped psoralen cross-links (U12/BPS and U12/U6atac Ia), which were found to produce the expected results (not shown).
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Detection of the U12-5' exon interaction requires base pairing. To test the contribution of the proposed U12/5'exon base pairing for the cross-link formation, we constructed a P120[2] cross-linking substrate in which the proposed base pairing was weakened by inverting the GC dinucleotide located upstream of the 4SU residue (P120-CG [Fig. 6A]). When such a substrate was used in cross-linking reactions, the U12/5' exon cross-link was lost and not observed even in the presence of the 5' exon oligonucleotide (Fig. 6B), suggesting that the base-pairing interaction is indeed needed for the detection of the U12/5'exon cross-link. Thus, the results with P120 splicing substrate support the view that the 5' end of U12 snRNA could be stabilized near the 5'ss by base-pairing interactions which in turn allowed us to capture the two RNAs in proximity of each other via cross-linking.
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Each SCN4a cross-linking substrate described above was incubated under splicing conditions and cross-linked, and the resulting cross-links were separated in 5% and 8% gels (Fig. 6C and D, respectively) followed by detection by Northern blotting with U12-specific probes. The P120[2] cross-linking substrate was used as a control (Fig. 6C and D, lane 7).
A slowly migrating band which has very similar gel mobility as the U12/5'exon cross-link produced with P120[2] substrate was detected in both 5% and 8% PAGE gels in reactions containing SCN4a[2] (Fig. 6C and D, lane 2) but not in those containing SCN4a-CG substrate (Fig. 6C and D, lane 5; note the similar nonlinear migration of the SCN4a and P120 specific cross-links in 5% and 8% PAGEs in panels C and D, respectively). The SCN4a-specific cross-link bands were missing from the reactions that were not UV irradiated (Fig. 6C and D, lane 1) or which contained a 2ome oligonucleotide complementary to the 5' end of the U12 snRNA (U121-16 in Fig. 6C and D, lane 3), further suggesting that an authentic U12/5'exon cross-link was indeed detected with the SCN4a[2] cross-linking substrate. Due to the lower cross-linking efficiency it was not possible to map the SCN4a cross-link location on U12 snRNA similarly as with the P120[2] substrate. However, the similar gel mobility of the SCN4a- and P120-specific cross-link bands in both 5% and 8% gels and the sensitivity of the cross-link formation to a mutation that reduces the extent of the 5' exon/U12 base-pairing potential further suggests that the location of the cross-link within the U12 snRNA is similar with both P120 and SCN4a cross-linking substrates. We conclude that the proximity of the U12 snRNA with the 5'ss can be detected with other splicing substrates besides P120, but efficient detection by cross-linking requires stabilization via base-pairing interaction between 5' exon and U12 snRNAs.
The effect of U12/5'exon base pairing on splicing activity. To evaluate if the U12-5' exon interaction could affect splicing activity in vitro, we mutated the nucleotides on P120 substrate that are involved in U12/5' exon base pairing. We did not detect any major changes in splicing activity in our in vitro splicing experiments with P120 substrates containing mutations in the 5' exon region binding to U12 snRNA (see Fig. S1B in the supplemental material; Frilander and Meng, unpublished data). Therefore, to ensure an accurate comparison with the wild-type substrate containing wild-type sequences near the 5'ss, the effects of mutations were studied using modified P120 splicing substrates which contained two functional 5'ss that were separated by 20 nucleotides from each other (Fig. 7A). Each construct contained mutations either in the intron-proximal or in the intron-distal 5'ss that prevent the U12-5' exon base-pairing interactions, while the other site contained a wild-type sequence. We used two different types of mutations to confirm that the possible effects would be specific to the disruption of the U12/5'exon base pairing (Fig. 7A).
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| DISCUSSION |
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Mapping of the cross-link on U12 snRNA to the very end of the molecule suggests that the U12 snRNA forms a base-pairing interaction with the 5' exon sequences of the P120 splicing substrate which could promote the observed cross-link formation by aligning the two molecules (Fig. 5). This is supported by experiments using additional P120 and SCN4a cross-linking substrates in which the U12/5'exon cross-link was observed only when a sufficient base-pairing potential upstream of the 4SU residue was present while the mutations that weakened the base pairing resulted in the loss of cross-link formation (Fig. 6). Since the proposed U12/5' exon helix has a melting temperature of approximately 10 to 12°C, it should not be stable under the experimental conditions unless stabilized by protein factors and/or oriented near the 5'ss by the architecture of the U12 snRNA in the complex A. We have not found evidence for specific stabilizing protein factors by 4SU-cross-linking (J. Turunen and M. Frilander, unpublished data), suggesting that the U12 snRNA may be positioned near the 5'ss solely by the favorable architecture within complex A. Consistently, we observed no U12/5' exon cross-links when the spliceosome assembly was blocked in conditions that lack ATP or contain a 2ome oligonucleotide that blocks the U12-BPS interaction or in reactions that contain a cross-linking substrate lacking a functional BPS, even though in each case the 5' end of the U12 snRNA should be free to interact with the 5' exon sequences. These results, and the observed resistance of the cross-link to DNA oligonucleotide-directed RNase H degradation using a BPS-specific DNA oligonucleotide (Fig. 2D), indicate that the U12-5'exon interaction takes place in functional spliceosomal complexes which probably position the 5' end of the U12 near the 5'ss. On the other hand, the loss of cross-linking signal when the base-pairing interaction was weakened using both P120 and SCN4a cross-linking substrates suggests a proximity, instead of rigid alignment, of the U12 snRNA and 5'ss in complex A. Detection of cross-link signal using two unrelated substrates suggests that such a proximity may be a general property of the system, but base pairing with the substrate would transpire only if suitable and sufficiently long complementary sequences are present within the physical range of the U12 snRNA.
The U12-5' exon base-pairing interaction suggests a model for the previously observed reversible inhibitory effect of the 5' exon 2ome oligonucleotide (9). The 5' exon oligonucleotide forms, together with the 5' end of the U12 snRNA, a continuous double-stranded structure which can prevent binding of necessary splicing factors (such as Prp8, EJC complex proteins, or specific helicases [28, 30, 58]) near the 5'ss and thus prevent the exchange of U11 to U6atac at the 5'ss and results in the weaker U5/5' exon and U12/U6atac Ib cross-link signal (9). The rapid recovery of splicing activity after the removal of the 5' exon oligonucleotide suggested that this arrested complex containing U12-5' exon interaction represented an on-pathway assembly intermediate (9).
The sequences upstream of the U12-type 5'ss in human, Arabidopsis, or Drosophila genes are not conserved (29, 46, 73), suggesting that cross-link formation between U12 and the P120 5' exon was facilitated in part by fortuitous sequence complementarity, which does not necessarily serve any essential role in intron recognition or splicing. This is supported by our in vitro splicing experiments in which splicing substrates containing mutations in the region binding to U12 snRNA did not show any significant differences in splicing activity as compared to control reactions (Frilander and Meng, unpublished; see Fig. S1B in the supplemental material). In a competition situation with splicing substrates containing two tandem 5'ss, we found that mutations which disrupt the U12/5'exon base pairing do change the relative splicing efficiency but only at the distal site (Fig. 7). We found reproducible 1.5-fold stimulation of distal splice site usage with proximal site mutations (Fig. 7, substrates 1 and 3) and similarly approximately 30% inhibition of distal splice site usage with mutations at the distal site (Fig. 7, substrates 2 and 4). In contrast, the effects at the proximal site were either neutral or slightly negative, possibly due to an interference with the two closely spaced 5'ss as reported earlier with the U2-dependent spliceosome (5, 7). As the effects of U12-5' splice site interaction on splicing are relatively small, we conclude that this interaction reflects mostly the molecular architecture of the early spliceosomal complexes instead of playing any major, if any, regulatory role.
Protection of the U12/5' exon cross-link in the RNase H experiment with BPS-specific DNA oligonucleotides (Fig. 2D) indicates a simultaneous interactions of U12 with both the BPS (10) and the 5' exon sequences. This simultaneous interaction allows to estimate the distance between the 5'ss and BPS in the complex A, given that there are 13 nucleotides between the base-paired sites in U12 snRNA (Fig. 8). Assuming that the RNA is extended and has a 3.1-Å base-to-base spacing, the distance between the 5'ss and the BPS sequences should not exceed 40 to 50 Å. This indicates that, at least in the complex A, the 5'ss and BPS binding elements of the U11/U12 di-snRNP are close in space, perhaps forming a discrete structure for the recognition of the two signals in the pre-mRNA. Electron microscopy studies of the U2-dependent spliceosome provide support to this interpretation as the distance measured above is similar to the diameter of a single protuberance in U1 snRNP (51), while the diameters of individual U1 and U2 snRNPs range from 80 to 120 Å (26, 51) and the diameter of a U2-type complex A is
250 Å (12).
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Localization of the site of cross-link on U12 snRNA to the very 5' end of the molecule (Fig. 5) provides insight to the possible significance of the U12-5' exon interaction. In the catalytic core of the U12-dependent spliceosome, the very 5' end of the U12 snRNA forms helix Ib together with U6atac snRNA (48, 54). This and the other RNA-RNA interactions found from the catalytic core of the U12-dependent spliceosome are similar to the interactions described in the U2-dependent spliceosome, with U2 and U6 snRNA base paired together as originally proposed by Madhani and Guthrie (32) based on yeast mutation data. The role of helix Ib has been investigated primarily on the U2-dependent spliceosome in which it has been shown to be essential for the catalysis of splicing (18, 32, 64; but see reference 8), possibly functioning in the second step while the U2 and U6 snRNA would adopt an alternative ribozyme-like conformation for the first step (45, 53). Consistent with the catalytic role of the U2/U6 helix Ib, genetic interactions that link the 5'ss and helix Ib, either directly (Ib/5'ss interaction [31]) or indirectly (Ib/U5 interaction [70]) have been described in yeast. Several studies have identified cross-bridging interactions that link 5'ss and BPS and can thus bring the U2 in the vicinity of the 5'ss for the formation of the catalytic core structure. These include SR proteins and other factors bridging 5'ss and BPS at the E-complex stage (1, 65), the Prp5 protein directly bridging U1 and U2 snRNPs (71), the U2-U6 helix II interaction that can be observed in HeLa nuclear extracts by psoralen cross-linking even in the absence of pre-mRNA (17, 60), and most recently, a preformed penta-snRNP structure that could serve as a physical link between the components involved in intron recognition (34, 52). However, even though the presence of bridging interactions between 5'ss and BPS at the time of intron recognition has been well documented in the U2-dependent spliceosome, the orientation and positioning of the individual components, including U2 snRNA, in these structures is mostly unknown. Our detection of U12 snRNA in the proximity of the 5'ss during the early phase of the spliceosome assembly argues that at least in the U12-dependent spliceosome the helix Ib region of the U12 snRNA may be positioned near the 5'ss already at the time of the prespliceosome formation, thus resulting in the bridging of the 5'ss and BPS by a single spliceosomal component and further suggesting that the molecular architecture present in the spliceosome catalytic core may start forming at the earliest stages of the spliceosome assembly.
| ACKNOWLEDGMENTS |
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This work was supported by the Academy of Finland (grants 50116 and 50527 to M.J.F.).
| FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/. ![]()
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