![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Previous Article | Next Article 
Molecular and Cellular Biology, May 2008, p. 3548-3560, Vol. 28, No. 10
0270-7306/08/$08.00+0 doi:10.1128/MCB.01928-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
,
Cindy L. Will,2,
Michael Grote,2
Reinhard Lührmann,2 and
Mikko J. Frilander1*
Institute of Biotechnology, PL 56 Viikinkaari 9, 00014 University of Helsinki, Helsinki, Finland,1 MPI of Biophysical Chemistry, Department of Cellular Biochemistry, Am Fassberg 11, 37077 Göttingen, Germany2
Received 25 October 2007/ Returned for modification 11 December 2007/ Accepted 7 March 2008
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Intron recognition is achieved via multiple, dynamic RNA- and protein-mediated interactions. RNA-RNA interactions in the two spliceosomes are highly analogous. In the major spliceosome, the first assembly step (generating the E complex) involves the recognition of the 5'ss by U1, while the non-snRNP protein factors SF1, U2AF65, and U2AF35 bind to the BPS, the polypyrimidine tract, and the 3'ss, respectively (for a review, see reference 26). During this stage, U1 base pairs with the pre-mRNA's 5'ss, while U1-associated proteins facilitate 5'ss recognition or stabilize the U1-5'ss complex (for a review, see reference 37). During prespliceosome (A complex) formation, U2 associates stably with the BPS (17), while non-snRNP proteins bridge U1 and U2 snRNPs (40, 41). The U4/U6/U5 tri-snRNP then joins the spliceosome (generating the B complex), which triggers the displacement of U1 from the 5'ss by U6 and additional rearrangements that lead to catalytic core formation (for a review, see references 22 and 33).
The overall assembly pathway of the minor spliceosome is similar. Initially, U11 base pairs with the 5'ss and U12 base pairs with the BPS (16, 35, 42). U11 is displaced from the 5'ss by U6atac after the association of the U4atac/U6atac/U5 tri-snRNP, and this displacement is followed by additional rearrangements that result in a catalytic core with an architecture highly similar to that of the major spliceosome (9, 14, 32). However, intriguing differences during the initial intron recognition step are observed. For example, U12-type E complexes have not been detected. Instead, the 5'ss and BPS are recognized cooperatively by a preformed U11/U12 di-snRNP (10, 36), suggesting that components connecting the 5'ss and the BPS are already present in the di-snRNP prior to prespliceosome formation. The initial base-pairing interactions at the 5'ss in the two spliceosomes are also different. The U1-5'ss interaction spans nucleotides (nt) –2 or –1 through +6 relative to the 5'ss both in mammals and in Saccharomyces cerevisiae (see Fig. 1B and C). In contrast, the first three nucleotides of U12-type introns (RUA) do not engage in base pairing with either U11 or U6atac snRNAs (see Fig. 1A), but their identities are almost 100% conserved (31). Mutations in the +1 and +2 positions block U12-dependent splicing and lead to the activation of cryptic splice sites (2). This observation suggests that a protein interacts with the RUA motif in a sequence-specific manner during intron recognition.
|
Here, we show that the 48K protein makes direct contact with the +2 position of the U12-type 5'ss, demonstrating for the first time an interaction between a U11 protein and the pre-mRNA. In addition, we provide evidence that 48K is essential for the splicing of U12-type introns in vivo and also contributes to U11/U12 di-snRNP formation and stability, potentially by interacting with the di-snRNP interface protein U11-59K.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Plasmids. For the construction of pVps16-i9, the last 70 nt of exon 9, intron 9, and the first 89 nt of exon 10 of the mouse Vps16 gene (Ensembl release 36 accession no. ENSMUSG00000027411; www.ensembl.org) were amplified by PCR from genomic mouse (strain Sv129) DNA. This fragment was then inserted into pCRII-TOPO. For the production of RNA site-specifically labeled with 4-thiouridine (4SU), plasmid pVps16-e9m was constructed, in which nt –70 to +2 (relative to the 5'ss) of pVps16 were modified to contain a T residue solely at position +2, similar to the construction of the previously described P120 cross-linking substrate (8, 9). The mutations CC+5+6GG and A+3G were introduced into pP120 (35) and pVps16-i9 by PCR and verified by sequencing.
Splicing substrates. Capped pre-mRNA substrates uniformly labeled with 32P were produced by in vitro transcription as described previously (9). For cross-linking, substrates containing a 4SU residue and/or a 32P phosphate group at a specific site were constructed using the two-piece RNA ligation protocol (9). Substrates lacking the BPS were truncated 3 nt upstream of the BPS.
In vitro spliceosome assembly and splicing. In vitro splicing was performed as described previously (21), except that the concentration of the U2b oligonucleotide was 2.4 µM. Polyvinyl alcohol (1.0%) was used in all reaction mixtures except those subjected to native-gel analyses and RNase H protection assays. The concentrations of splicing substrates were 2 nM for native-gel analyses, RNase H protection assays, and splicing assays and 10 nM for cross-linking experiments. To deplete ATP, ATP and creatine phosphate were omitted and the reaction mixture was preincubated for 30 min before the addition of the substrate. To deplete Mg2+, MgCl2 was omitted and 2.5 mM EDTA was added. Blocking with 2ome oligonucleotides, RNase H assays, and native-gel analyses were performed as described in reference 10, and Northern blot analyses were performed as described in reference 35.
Cross-linking. For 4SU cross-linking, 5 to 20 µl of a splicing reaction mixture was irradiated for 3 min with a 337-nm N2 excimer laser (PSX-100; Neweks Ltd., Estonia) and then subjected to treatment with a nuclease mixture (0.01 mg of micrococcal nuclease/ml, 0.02 U of RNase T1/µl, 0.1 mg of RNase A/ml) at 37°C for 30 min. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; on a 4 to 12% NuPAGE or 8 to 16% Precise protein gel) and detected by autoradiography or phosphorimager (Fuji FLA-5010) analysis.
IPs. Rabbit antibodies against the U11/U12 25K, 35K, 48K, 59K, and 65K proteins and SF3b49 were raised as described previously (6, 38). Twenty-microliter aliquots of affinity-purified antibody or serum (in the case of anti-48K and anti-SF3b49) were preabsorbed to 20 µl of protein A-Sepharose beads in 200 µl of 100 mM Tris-HCl (pH 8.0)-phosphate-buffered saline (PBS) overnight at 4°C. Beads were washed three times with PBS, and 20 µl of a UV-treated splicing reaction mixture was added. Samples were either left in their native state or treated with RNases and denatured at 95°C for 2 min in the presence of 0.15% SDS prior to immunoprecipitation (IP) (see Fig. 3C). IPs were performed overnight in 0.1% Nonidet P-40 (NP-40)-PBS, after which the beads were washed five times with buffer (10 mM Tris-HCl [pH 8.0], 0.1% NP-40, 150 mM NaCl). Proteins were eluted by adding 20 µl of SDS-PAGE loading buffer supplemented with 4 µl of nuclear extract as a carrier and incubating at 95°C for 10 min. IP of U11/U12 snRNPs from nuclear extracts with SF3b155 antibodies was performed essentially as described previously (39).
|
Yeast two-hybrid assays. Yeast two-hybrid analyses were performed with the Matchmaker 3 two-hybrid system (Clontech). cDNAs encoding full-length or truncated proteins were cloned into pGBKT7 (the bait vector) or pGADT7 (the prey vector). S. cerevisiae strain AH109 was cotransformed with bait and prey plasmids, and the plasmids were selected on agarose plates prepared with minimal synthetic dropout medium lacking leucine and tryptophan by incubation at 30°C for 3 to 5 days. To identify protein-protein interactions, cotransformants were then replicated on plates additionally lacking histidine and adenine (plates containing synthetic dropout medium without Ade, His, Leu, or Trp).
StrepII tag pulldown assays. For pulldown assays, cDNA encoding 59K137-336 was cloned into pEU3-NII-StrepII via PCR-based techniques and 59K137-336 containing a C-terminal StrepII tag was translated in wheat germ extract. Forty microliters of the translated 59K137-336 was incubated with 40 µl of translated 48K for 90 min at 0°C, prior to the addition of 15 µl of Strep-Tactin Sepharose (IBA, Göttingen, Germany) and 200 µl of IPP150 buffer (20 mM HEPES-KOH, pH 7.9, 150 mM NaCl, 1.5 mM MgCl2, 0.05% NP-40). The mixture was incubated with end-over-end rotation for 1 h, and after the beads were washed four times with IPP150 buffer, bound protein was recovered and analyzed by SDS-PAGE.
Cell culture, RNAi, and qRT-PCR analysis of siRNA-targeted mRNAs. Culturing of HeLa S6 cells and the transfection of cells with siRNA duplexes by using Oligofectamine (Invitrogen) were performed essentially as described in reference 38. The targeted regions were not found in any other known genes via BLAST searches of the human genome. To assay for effects on cell growth, HeLa cells were harvested 24, 48, 72, and 96 h after transfection with siRNA and counted with a CASY model TT cell counter according to the instructions of the manufacturer (Schaerfe System, Germany). For 48K rescue experiments, the 48K cDNA with four mutations (silent on the amino acid level) in the region bound by the 139-1 siRNA was cloned into the pcDNA4 expression vector (Invitrogen), generating pcDNA-48K. Twelve hours after siRNA addition, cells were transfected with pcDNA-48K (75 ng per 2.25 x 105 cells) by using Transfectin lipid reagent according to the instructions of the manufacturer (Bio-Rad). Total cellular RNA was isolated using an RNeasy mini kit (Qiagen) and digested with RQ1 DNase (Promega) to remove any contaminating DNA. The level of 48K mRNA remaining after transfection with siRNA BB1 versus siRNA 139-1 was determined by qRT-PCR with an Opticon continuous-fluorescence detector (MJ Research). As an internal control, the level of mRNA encoding glutamate dehydrogenase 2 (which is not spliced) was also determined by qRT-PCR. Small-scale nuclear extracts were prepared essentially as described in reference 19. Nuclear extracts were fractionated on 10 to 30% glycerol gradients, and snRNAs were detected by Northern blotting as described previously (38).
RT-PCR analysis of endogenous pre-mRNA in 48K knockdown cells. The removal of U12- and U2-type introns from pre-mRNA was analyzed by RT-PCR with total cellular RNA from control and 48K knockdown cells after 72 h. First-strand cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) with oligo(dT) primers, followed by 26 cycles (32 cycles with RCD8 and PPP2R2A primers) of amplification using Phusion DNA polymerase (Finnzymes). For details of the analyzed introns and the primers used, see Table S1 in the supplemental material. The RT-PCR products were separated on 2.5 to 3.0% Metaphor agarose (Cambrex) gels.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
The U11/U12 48K protein makes contact with the 5'ss during prespliceosome assembly. To detect proteins interacting with the RUA motif, we performed site-specific cross-linking with WT, A+3G, and CC+5+6GG P120 pre-mRNAs, in which a 4SU residue and a single 32P-labeled phosphate group were placed at the +2 position relative to the 5'ss. Control substrates with 4SU at the –2 position, without 4SU, or with a 3' truncation were also analyzed (Fig. 3A). Splicing and splicing complex formation were not significantly hindered by introducing 4SU at positions +2 and –2 (9, 10) (data not shown).
Splicing was performed with HeLa nuclear extracts, and cross-linking was induced by UV irradiation. RNA was then degraded enzymatically, and cross-linked proteins were separated by SDS-PAGE and visualized by autoradiography. Four bands (corresponding to 
35, 45, 100, and 220 kDa) were reproducibly observed after 40 min of splicing with the P120 substrate containing 4SU at the +2 position (WT+2) (Fig. 3B, lane 1) but not with the substrate lacking 4SU (the WT) (Fig. 3B, lane 2). Of these bands, only the 45-kDa cross-link band was not observed with either the CC+5+6GG substrate or the substrate with 4SU at the –2 position (Fig. 3B, cf. lanes 3 and 4 with lane 1), indicating that the 45-kDa cross-linked protein interacts specifically with the +2 position in a manner dependent on either U11 or U6atac base pairing with the 5'ss. In the absence of SDS, the cross-linked 45-kDa protein was precipitated to various degrees by antibodies against the U11/U12 35K, 48K, and 65K proteins and the SF3b49 protein (Fig. 3C), suggesting that it is a U11/U12 protein. In contrast, after denaturation with SDS, which disrupts all protein-protein and protein-RNA interactions, it was precipitated solely by the 48K antibody (Fig. 3C, lane 6), identifying it as the U11/U12 48K protein.
Similar to that of U11-5'ss cross-linking (42), the efficiency of 48K-5'ss cross-linking was not significantly affected when the splicing reaction mixture was depleted of ATP (Fig. 3D) or Mg2+ (Fig. 3E). However, unlike U11 (8, 42), 48K was not efficiently cross-linked at 0°C (Fig. 3F, lane 1). The 48K cross-linked protein was present in equal amounts after incubation at 30°C for 10 and 20 min (when only the A complex forms), but the amount started to decline by 40 min (when the B complex appears) (10) (Fig. 3F, lanes 2 to 4), suggesting that 48K interacts early during spliceosome assembly in a transient manner. Indeed, blocking the U6atac-5'ss interaction with a 2ome oligonucleotide against U6atac had no effect on 48K-5'ss cross-link formation, suggesting that 48K interacts prior to B complex formation (Fig. 3G). Blocking U12-BPS base pairing with a 2ome oligonucleotide against U12 (Fig. 3G) or truncating the splicing substrate directly upstream of the BPS (Fig. 3H, lane 9) led to a 3- to 4-fold or 2-fold reduction in cross-linking efficiency, respectively. Thus, the 48K-5'ss interaction is not dependent on BPS recognition but may be enhanced by the cooperative recognition of the U12-type 5'ss and the BPS (10). We conclude that 48K makes contact with the 5'ss during the early stages of intron recognition, apparently prior to BPS and 5'ss recognition by the U12 and U6atac snRNAs, respectively.
Strikingly, the A+3G mutation almost completely abolished cross-linking of the 48K protein (yielding a 30- to 60-fold reduction relative to the level observed with the WT+2 construct) (Fig. 3H, lane 7). As this mutation was also shown to inhibit prespliceosome formation (Fig. 2F), the loss of the 48K cross-linked protein correlated with impaired 5'ss recognition, suggesting that the cross-link is not lost due to merely a change in the local orientations of the RNA and the cross-linker. These data suggest that 48K interacts in a sequence-specific manner with the RUA motif either directly or indirectly via an unknown partner that specifically recognizes this motif (see Discussion).
The 48K-5'ss interaction is generally found in U12-dependent spliceosomes. To address the generality of the 48K-5'ss interaction, we constructed a second set of splicing substrates containing a U12-type intron of the GT-AG subtype, derived from the ninth intron of the mouse Vps16 gene. Like the P120 substrates (Fig. 2C and D), the uniformly labeled Vps16 WT substrate was assembled into spliceosomal complexes and was spliced while the A+3G and CC+5+6GG mutant forms were not (data not shown). The efficiency of cross-link formation with the Vps16 substrate was somewhat higher than that with the P120 substrate, resulting in more background bands (Fig. 4). IP with anti-48K antibody revealed that 48K is efficiently cross-linked to the Vps16 WT+2 substrate (Fig. 4, lane 5). In contrast, a large reduction (ca. 50- and 10-fold) in 48K cross-linking was observed with the A+3G and CC+5+6GG mutant substrates, respectively (Fig. 4). Thus, 48K makes contact with the 5'ss of both AT-AC and GT-AG U12-type introns.
|
10 to 15% of the 48K mRNA was present in cells transfected with siRNA 139-1 already after 24 h and at later time points but that the levels of mRNAs encoding other U11 proteins (e.g., 35K and 25K) were not affected by the addition of 139-1 siRNA (data not shown). These results indicate that 48K plays an essential role in the cell, consistent with the idea that it functions in U12-independent splicing.
|
To exclude the possibility that the observed inhibition of U12-type splicing was due to off-target effects of the 48K 139-1 siRNA, cells were transfected 12 h after siRNA addition with a plasmid containing the 48K cDNA with silent point mutations in the region targeted by 139-1 (designated pcDNA-48K). The level of 48K mRNA (72 h after transfection with siRNA) increased in the 48K knockdown cells from 12% in the absence of pcDNA-48K to 41% in its presence, demonstrating the partial rescue of 48K levels (Fig. 5E). Significantly, the splicing of the U12-type introns Mapk12-8, Vps16-9, and Vps16-13 was partially restored upon the addition of pcDNA-48K, as evidenced by the significant decrease in the levels of pre-mRNAs containing these introns in 48K knockdown cells that received the plasmid compared to those in cells that did not (Fig. 5E). In contrast, U12-type splicing in BB1 control knockdown cells was not affected by the addition of pcDNA-48K (Fig. 5E). Taken together, these data demonstrate that 48K knockdown leads to a reduction in the splicing of at least a subset of U12-type introns.
For Mapk12, a faster-migrating band not present in the control reaction mixture was detected in the knockdown cells (Fig. 5B, lane 1). Sequence analysis of this band revealed that it arose by the activation of a cryptic, U2-type 5'ss (Fig. 5D). The activation of cryptic splice sites after 72 h of 48K knockdown was also observed with two other transcripts, namely, those for PPP2R2A and RCD8 (Fig. 5D), neither of which displayed a significant accumulation of pre-mRNA after 48K knockdown. RCD8 transcripts displayed the highest level of cryptic splicing, with 30% of the mRNA in the knockdown cells spliced at cryptic sites (data not shown).
Interestingly, the 5'ss and BPS of the U12-type introns that were most sensitive to 48K knockdown exhibited a number of deviations from the respective U12-type consensus sequences, suggesting that they may be weak sites, while those not affected by 48K knockdown had 5'ss and BPS closely matching the consensus sequences (Fig. 5F). The switch from U12- to U2-dependent splicing does not appear to be an alternative splicing event, as expressed-sequence-tag (EST) database searches revealed no Mapk12, PPP2R2A, or RCD8 ESTs generated from splicing at the observed U2-type splice sites. Thus, 48K plays a critical role in the recognition of U12-type introns in vivo.
The knockdown of 48K leads to a reduction in U11/U12 di-snRNP levels.
To test whether 48K contributes to di-snRNP stability and formation, we prepared nuclear extracts from control or 48K knockdown cells. Extracts were then subjected to glycerol gradient centrifugation, and after fractionation, the sedimentation behavior of snRNPs containing U11 or U12 was determined by Northern blot analysis with the corresponding 32P-labeled probe; as an internal control, the distribution of U2 snRNPs was also analyzed (Fig. 6A to D). No significant difference in the sedimentation behavior of U2-containing snRNPs (i.e., 17S and 12S particles) was observed in extracts from the control versus the 48K knockdown cells (Fig. 6C and D). In contrast, upon 48K knockdown, a smaller percentage of the total U11 snRNA present sedimented in 18S peak fractions 9 to 10 (20 versus 39% in the control as determined by phosphorimager analysis) and a larger percentage sedimented in 12S peak fractions 4 to 5 (27 versus 14% in the control) (Fig. 6A and B), suggesting that the knockdown cells contained 50% less U11/U12 di-snRNP than the control cells. Likewise, more U12 sedimented as a 15S monoparticle in the 48K knockdown cells; however, due to the close migration of 15S and 18S particles, a meaningful quantitation of U12 mono-snRNPs versus di-snRNPs could not be carried out. To provide additional evidence for a reduction in di-snRNP levels after 48K knockdown, we also performed co-IP assays with antibodies directed against the U12-associated SF3b155 protein. Significantly, on average only half as much (50% ± 7%) of the input U11 was precipitated together with U12 by anti-SF3b155 antibodies from the 48K knockdown extracts as from the control extracts (Fig. 6E, cf. lanes 3 and 4 with lanes 7 and 8), consistent with the conclusion that 48K knockdown cells contain 50% fewer U11/U12 di-snRNPs than control cells. Thus, 48K appears to contribute to the formation and/or the stability of the U11/U12 di-snRNP.
|
|
Arg, 48K1-199, and 48K1-92), interacted strongly with 59K (Fig. 7D, lanes 2 to 5). In the absence of amino acids 1 to 92, no interaction was detected (Fig. 7D, lanes 6 and 7), indicating that the N terminus of 48K is necessary and sufficient for interaction with 59K. Interestingly, this region of 48K contains a potential CHHC zinc finger motif, which is highly conserved evolutionarily (see Fig. S1 in the supplemental material). To confirm this interaction, 59K137-336 containing a StrepII tag was translated in vitro and incubated with in vitro-translated 14C-labeled 48K and deletion mutant forms thereof. 59K was then precipitated with Strep-Tactin Sepharose beads, and coprecipitating proteins were analyzed by SDS-PAGE, followed by autoradiography. Consistent with the Far Western results, all 48K mutant proteins containing amino acids 1 to 92 bound to 59K137-336 with efficiencies nearly identical to that of the WT 48K protein (10%), whereas mutant proteins lacking this region did not (Fig. 7E). These results indicate that 48K not only makes contact with the 5'ss of U12-type introns but also interacts with the U11-59K protein and, thus, may possess dual binding affinities. |
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
48K makes contact with the 5'ss in early spliceosomal complexes. We detected an interaction between 48K and the 5'ss (Fig. 3 and 4), consistent with the idea that 48K participates in U12-type 5'ss recognition and that 48K knockdown leads to impaired U12-dependent splicing by preventing this interaction. Furthermore, several lines of evidence support the idea that the 48K-5'ss interaction takes place early during intron recognition. First, 48K could be cross-linked soon after the start of splicing in vitro (Fig. 3F) and seemed to require the U11-5'ss base pairing interaction (see below). Second, 48K-5'ss cross-link formation did not require U6atac-5'ss or U12-BPS base pairing (Fig. 3G) or the presence of the BPS (Fig. 3H), suggesting that the 48K-5'ss interaction precedes U12-BPS base pairing. Finally, 48K knockdown led to the activation of cryptic, U2-type splice sites (Fig. 5D). As commitment to either U2- or U12-type splicing is thought to occur at the time of A complex formation (for a discussion, see reference 14), 48K must act during or prior to this step. 48K is associated with the U11 moiety of the U11/U12 di-snRNP, and thus, it likely is displaced from the 5'ss at a later stage of spliceosome assembly, when the U11 snRNP is thought to dissociate. Indeed, 48K cross-linking was reduced after 40 min of splicing (Fig. 3F), which correlates with reduced U11-5'ss cross-link formation (8). However, at present, we cannot exclude the possibility that 48K remains bound to the 5'ss at later stages of spliceosome assembly.
The 48K-5'ss interaction appears to be greatly enhanced by U11-5'ss base pairing. The CC+5+6GG mutation, which abolishes U11-5'ss base pairing (14), greatly diminished 48K-5'ss cross-link formation (Fig. 3H and Fig. 4). Theoretically, this mutation could also hinder protein interactions with nt +5 and/or +6. However, as the splicing defect caused by the CC+5+6GG mutation could be eliminated by compensatory mutations in U11 and U6atac snRNAs in vivo (14), it appears that there are no critical RNA-protein interactions with these nucleotides. In summary, 48K interacts with the 5'ss during the very first steps of intron recognition, apparently together with or after U11 snRNA.
48K may recognize the 5'ss in a sequence-specific manner. The mutation of the conserved RUA motif (A+3G), which prevents base pairing with the U11 snRNA, abolished both the stable interaction of U11 snRNP with the 5'ss (Fig. 2) and the 48K cross-link (Fig. 3 and 4). This finding suggests indirectly that 48K recognizes the 5'ss in a sequence-specific manner and, further, that the 48K-5'ss interaction helps to stabilize the U11-5'ss interaction. In the major spliceosome, the 5'ss is recognized by the U1-C protein, which can bind the 5'ss on its own and subsequently promotes the U1-5'ss interaction (4, 5, 27). It is not clear whether 48K and U1-C act in mechanistically similar manners. However, the different compositions of U1 and U11 proteins hint at mechanistic differences. Furthermore, in contrast to the U1-C-5'ss interaction, data presented here suggest that the 48K-5'ss interaction occurs either concomitantly with or after U11-5'ss base pairing.
Whether 48K can bind the U12-type 5'ss on its own is currently also unknown; due to the inability to overexpress the WT 48K protein in E. coli, RNA binding studies with purified 48K could not be performed. Recently, serine-arginine-rich (SR) proteins were shown to interact with a U12-type 5'ss (30), and they may possibly mediate the 48K-5'ss interaction either directly or indirectly by modulating U11-5'ss base pairing. Other U11/U12 proteins not found in the major spliceosome may also contribute to 5'ss recognition. Most notably, the U11/U12-20K protein shows some similarity to U1-C, including an N-terminal zinc finger, and it was thus proposed that the 20K protein may be involved in U12-type 5'ss recognition (38). However, 20K was not copurified with U11 monoparticles. Furthermore, we did not detect a 20K-5'ss interaction via cross-linking, at least at the –2 or + 2 position (data not shown). Thus, additional experiments are required to clarify whether proteins in addition to 48K uniquely contribute to 5'ss recognition in the minor spliceosome.
48K plays a critical role in the recognition of U12-type introns. RNAi-induced knockdown of 48K leads to reduced cell growth, indicating that 48K has an essential cellular function (Fig. 5A). This function appears to be related to the recognition of U12-type introns, as 48K knockdown leads to reduced U12-dependent splicing and/or the activation of cryptic splice sites in certain pre-mRNAs containing U12-type introns (Fig. 5). These effects may be due partially to the reduction of U11/U12 di-snRNP levels in 48K knockdown cells (Fig. 6). However, as snRNPs are present in large excess in the cell, it is unlikely that a 50% reduction in di-snRNP levels alone would cause the observed effects. Indeed, residual amounts (<20%) of U4atac snRNA are sufficient to support the splicing of endogenous pre-mRNAs at WT levels (25), suggesting that the reduction of a single spliceosomal component by 80 to 90% does not a priori lead to splicing inhibition.
The removal of some U12-type introns was not significantly affected by 48K knockdown. As residual amounts of 48K mRNA remain after knockdown, some introns appear to be more sensitive to 48K levels than others. Interestingly, 48K knockdown had little or no effect on the removal of introns in which both the 5'ss and BPS matched the consensus sequences, whereas introns with deviations in their 5'ss or BPS were removed less efficiently or no longer recognized as U12-type introns after knockdown. It is likely that the formation and/or stability of the minor prespliceosome is dependent on the combined effects of several sequence-specific interactions at the 5'ss and BPS, including U11-5'ss base pairing, 48K-5'ss interaction, and U12-BPS base pairing, possibly augmented by SR proteins that recognize nearby regulatory elements (13, 30). Thus, 48K knockdown may lead to the defective formation of prespliceosomes solely on introns with suboptimal recognition sequences, especially in the absence of splicing enhancers.
Protein-protein interactions within the U11/U12 di-snRNP. Our knockdown data indicated that 48K contributes to the stability of the U11/U12 di-snRNP (Fig. 6). Interestingly, direct interactions between 48K and integral components of U12 (e.g., SF3b or the 65K and 20K proteins) were not detected, suggesting that 48K may not be located at the interface between the U11 and U12 moieties of the di-snRNP. However, protein-protein interaction studies (Fig. 7) demonstrated that 48K interacts with another U11-associated protein, namely, 59K, and that this interaction involves the central arginine-rich region of 59K and the N-terminal region of 48K. The latter contains a putative zinc finger, a motif shown in several cases to mediate protein-protein interactions (11). Significantly, the C-terminal part of 59K interacts with the U12-65K protein, and thus, both of these proteins lie at the interface of the di-snRNP and likely play an important role in di-snRNP formation and stability (1). Thus, it is conceivable that 48K, via its interaction with 59K, may indirectly affect the association of the U11 and U12 snRNPs in the U11/U12 di-snRNP.
Our studies further suggest that 48K possesses dual binding activities, interacting not only with 59K but also with the 5'ss. At present, the regions of 48K responsible for RNA interactions are not known, as the inability to express WT 48K in significant amounts in E. coli has hampered studies to examine this issue. The sequence of 48K also offers few clues as to which amino acids may be involved in RNA binding, as it contains no known motifs apart from a putative zinc finger. However, there is an arginine-rich sequence at its C terminus (38) which, due to its overall positive charge, may exhibit general affinity for RNA. In addition, a centrally located region of 48K (i.e., amino acids 206 to 256 in humans) is highly conserved from plants to humans (20), suggesting that it may be involved in RNA and/or additional protein interactions.
Intron bridging in the U12-dependent prespliceosome. To enable splicing catalysis, the functional sites of the pre-mRNA, i.e., the 5'ss, the BPS, and the 3'ss, are brought into close proximity during the earliest assembly phases of both spliceosomes (3, 8, 15). For the major spliceosome, SR proteins and the DEAD box protein Prp5 have been implicated in bridging U1 at the 5'ss and U2 at the BPS in E and A complexes (40, 41). In contrast, in the minor spliceosome, U11 and U12 are bridged by integral proteins of the U11/U12 di-snRNP, namely, 59K and 65K, with 65K also directly interacting with the U12 snRNA (1). Our data revealing 48K interactions with the 5'ss and 59K extend this network such that interactions involving 48K, 59K, and 65K would bridge the 5'ss and U12 bound to the BPS (Fig. 8). The link between components interacting with the 5'ss and the BPS is also consistent with the idea that combinatorial interactions at these sites contribute to intron recognition, as discussed above.
|
|
ACKNOWLEDGMENTS |
|---|
This work was supported by grants from the Academy of Finland and Sigrid Jusélius Foundation to M.J.F. and from the Deutsche Forschergruppe (Lu294/12-1), Fonds der Chemischen Industrie, and the Ernst Jung Stiftung to R.L. J.J.T. was supported by the Helsinki Graduate School in Biotechnology and Molecular Biology.
|
FOOTNOTES |
|---|
Published ahead of print on 17 March 2008.
Supplemental material for this article may be found at http://mcb.asm.org/.
These authors contributed equally.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2. Dietrich, R. C., J. D. Fuller, and R. A. Padgett. 2005. A mutational analysis of U12-dependent splice site dinucleotides. RNA 11:1430-1440.
3. Dönmez, G., K. Hartmuth, B. Kastner, C. L. Will, and R. Lührmann. 2007. The 5' end of U2 snRNA is in close proximity to U1 and functional sites of the pre-mRNA in early spliceosomal complexes. Mol. Cell 25:399-411.[CrossRef][Medline]
4. Du, H., and M. Rosbash. 2002. The U1 snRNP protein U1C recognizes the 5' splice site in the absence of base pairing. Nature 419:86-90.[CrossRef][Medline]
5. Du, H., D. F. Tardiff, M. J. Moore, and M. Rosbash. 2004. Effects of the U1C L13 mutation and temperature regulation of yeast commitment complex formation. Proc. Natl. Acad. Sci. USA 101:14841-14846.
6. Dybkov, O., C. L. Will, J. Deckert, N. Behzadnia, K. Hartmuth, and R. Lührmann. 2006. U2 snRNA-protein contacts in purified human 17S U2 snRNPs and in spliceosomal A and B complexes. Mol. Cell. Biol. 26:2803-2816.
7. Elbashir, S. M., J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, and T. Tuschl. 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494-498.[CrossRef][Medline]
8. Frilander, M. J., and X. Meng. 2005. Proximity of the U12 snRNA with both the 5' splice site and the branch point during early stages of spliceosome assembly. Mol. Cell. Biol. 25:4813-4825.
9. Frilander, M. J., and J. A. Steitz. 2001. Dynamic exchanges of RNA interactions leading to catalytic core formation in the U12-dependent spliceosome. Mol. Cell 7:217-226.[CrossRef][Medline]
10. Frilander, M. J., and J. A. Steitz. 1999. Initial recognition of U12-dependent introns requires both U11/5' splice-site and U12/branchpoint interactions. Genes Dev. 13:851-863.
11. Gamsjaeger, R., C. K. Liew, F. E. Loughlin, M. Crossley, and J. P. Mackay. 2007. Sticky fingers: zinc-fingers as protein-recognition motifs. Trends Biochem. Sci. 32:63-70.[CrossRef][Medline]
12. Hall, S. L., and R. A. Padgett. 1996. Requirement of U12 snRNA for in vivo splicing of a minor class of eukaryotic nuclear pre-mRNA introns. Science 271:1716-1718.[Abstract]
13. Hastings, M. L., and A. R. Krainer. 2001. Functions of SR proteins in the U12-dependent AT-AC pre-mRNA splicing pathway. RNA 7:471-482.[Abstract]
14. Incorvaia, R., and R. A. Padgett. 1998. Base pairing with U6atac snRNA is required for 5' splice site activation of U12-dependent introns in vivo. RNA 4:709-718.[Abstract]
15. Kent, O. A., and A. M. MacMillan. 2002. Early organization of pre-mRNA during spliceosome assembly. Nat. Struct. Biol. 9:576-581.[Medline]
16. Kolossova, I., and R. A. Padgett. 1997. U11 snRNA interacts in vivo with the 5' splice site of U12-dependent (AU-AC) pre-mRNA introns. RNA 3:227-233.[Abstract]
17. Krämer, A. 1988. Presplicing complex-formation requires 2 proteins and U2 snRNP. Genes Dev. 2:1155-1167.
18. Lamond, A. I., B. S. Sproat, U. Ryder, and J. Hamm. 1989. Probing the structure and function of U2 snRNP with antisense oligonucleotides made of 2'-OMe RNA. Cell 58:383-390.[CrossRef][Medline]
19. Lee, K. A., and M. R. Green. 1990. Small-scale preparation of extracts from radiolabeled cells efficient in pre-mRNA splicing. Methods Enzymol. 181:20-30.[Medline]
20. Lorkovi
, Z. J., R. Lehner, R. Forstner, and A. Barta. 2005. Evolutionary conservation of minor U12-type spliceosome between plants and humans. RNA 11:1095-1107.
21. McConnell, T. S., S. J. Cho, M. J. Frilander, and J. A. Steitz. 2002. Branchpoint selection in the splicing of U12-dependent introns in vitro. RNA 8:579-586.[Abstract]
22. Nilsen, T. W. 1998. RNA-RNA interactions in nuclear pre-mRNA splicing, p. 279-307. In R. Simons and M. Grunberg-Manago (ed.), RNA structure and function. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
23. Patel, A. A., M. McCarthy, and J. A. Steitz. 2002. The splicing of U12-type introns can be a rate-limiting step in gene expression. EMBO J. 21:3804-3815.[CrossRef][Medline]
24. Patel, A. A., and J. A. Steitz. 2003. Splicing double: insights from the second spliceosome. Nat. Rev. Mol. Cell Biol. 4:960-970.[CrossRef][Medline]
25. Pessa, H., A. Ruokolainen, and M. J. Frilander. 2006. The abundance of the spliceosomal snRNPs is not limiting the splicing of U12-type introns. RNA 12:1883-1892.
26. Reed, R. 2000. Mechanisms of fidelity in pre-mRNA splicing. Curr. Opin. Cell Biol. 12:340-345.[CrossRef][Medline]
27. Rossi, F., T. Forne, E. Antoine, J. Tazi, C. Brunel, and G. Cathala. 1996. Involvement of U1 small nuclear ribonucleoproteins (snRNP) in 5' splice site-U1 snRNP interaction. J. Biol. Chem. 271:23985-23991.
28. Russell, A. G., J. M. Charette, D. F. Spencer, and M. W. Gray. 2006. An early evolutionary origin for the minor spliceosome. Nature 443:863-866.[CrossRef][Medline]
29. Sawasaki, T., T. Ogasawara, R. Morishita, and Y. Endo. 2002. A cell-free protein synthesis system for high-throughput proteomics. Proc. Natl. Acad. Sci. USA 99:14652-14657.
30. Shen, H., and M. R. Green. 2007. RS domain-splicing signal interactions in splicing of U12-type and U2-type introns. Nat. Struct. Mol. Biol. 14:597-603.[CrossRef][Medline]
31. Sheth, N., X. Roca, M. L. Hastings, T. Roeder, A. R. Krainer, and R. Sachidanandam. 2006. Comprehensive splice-site analysis using comparative genomics. Nucleic Acids Res. 34:3955-3967.
32. Shukla, G. C., and R. A. Padgett. 1999. Conservation of functional features of U6atac and U12 snRNAs between vertebrates and higher plants. RNA 5:525-538.[Abstract]
33. Staley, J. P., and C. Guthrie. 1998. Mechanical devices of the spliceosome: motors, clocks, springs, and things. Cell 92:315-326.[CrossRef][Medline]
34. Tarn, W.-Y., and J. A. Steitz. 1996. Highly divergent U4 and U6 small nuclear RNAs required for splicing rare AT-AC introns. Science 273:1824-1832.
35. Tarn, W.-Y., and J. A. Steitz. 1996. A novel spliceosome containing U11, U12 and U5 snRNPs excises a minor class (AT-AC) intron in vitro. Cell 84:801-811.[CrossRef][Medline]
36. Wassarman, K. M., and J. A. Steitz. 1992. The low-abundance U11 and U12 small nuclear ribonucleoproteins (snRNPs) interact to form a two-snRNP complex. Mol. Cell. Biol. 12:1276-1285.
37. Will, C. L., and R. Lührmann. 2006. Spliceosome structure and function, p. 369-400. In R. F. Gesteland, T. R. Cech, and J. F. Atkins (ed.), The RNA world, vol. 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
38. Will, C. L., C. Schneider, M. Hossbach, H. Urlaub, R. Rauhut, S. Elbashir, T. Tuschl, and R. Lührmann. 2004. The human 18S U11/U12 snRNP contains a set of novel proteins not found in the U2-dependent spliceosome. RNA 10:929-941.
39. Will, C. L., C. Schneider, A. M. MacMillan, N. F. Katopodis, G. Neubauer, M. Wilm, R. Lührmann, and C. C. Query. 2001. A novel U2 and U11/U12 snRNP protein that associates with the pre-mRNA branch site. EMBO J. 20:4536-4546.[CrossRef][Medline]
40. Wu, J. Y., and T. Maniatis. 1993. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell 75:1061-1070.[CrossRef][Medline]
41. Xu, Y.-Z., C. M. Newnham, S. Kameoka, T. Huang, M. M. Konarska, and C. C. Query. 2004. Prp5 bridges U1 and U2 snRNPs and enables stable U2 snRNP association with intron RNA. EMBO J. 23:376-385.[CrossRef][Medline]
42. Yu, Y.-T., and J. A. Steitz. 1997. Site-specific crosslinking of mammalian U11 and U6atac to the 5' splice site of an AT-AC intron. Proc. Natl. Acad. Sci. USA 94:6030-6035.
This article has been cited by other articles:
| ||||||||||||||||||||||
denotes a pseudouridine residue, and 3m Gppp indicates the trimethylguanosine cap.
-48K) were separated by SDS-PAGE (lanes 6 to 10) along with 25% of the input (lanes 1 to 5). Molecular weight markers are shown on the left. The 48K band is indicated by an arrow.
