An intronic element contributes to splicing repression in

Tsuyoshi Kashima, Nishta Rao, and James L. Manley*

Department of Biological Sciences, Columbia University, New York, NY 10027

Communicated by Michael S. Levine, University of California, Berkeley, CA, January 16, 2007 (received for review October 31, 2006) The neurodegenerative disease spinal muscular atrophy is caused nuclear ribonucleoprotein (snRNP) complex formation (11). How- by mutation of the survival motor neuron 1 (SMN1) gene. SMN2 is ever, even in this case, both exonic and intronic A1 binding sites are a nearly identical copy of SMN1 that is unable to prevent disease, necessary for full repression, perhaps reflecting cooperative inter- because most SMN2 transcripts lack 7 and thus produce a actions between A1 molecules. A third mechanism was elucidated nonfunctional protein. A key cause of inefficient SMN2 exon 7 by studies of of the hnRNP A1 transcript itself splicing is a single nucleotide difference between SMN1 and SMN2 (12, 13). The presence of hnRNP A1 binding sites in both within exon 7. We previously provided evidence that this base surrounding alternative exon 7b is necessary for exclusion of this change suppresses exon 7 splicing by creating an inhibitory ele- exon. Cooperation between the two A1 complexes on these sites is ment, a heterogeneous nuclear ribonucleoprotein (hnRNP) A1- suggested to promote ‘‘looping-out’’ of the intervening RNA, dependent exonic splicing silencer. We now find that another rare including exon 7b, thereby inhibiting splicing. Finally, in Drosoph- nucleotide difference between SMN1 and SMN2, in 7, ila, hrp48, a homolog of hnRNP A1, contributes to splicing repres- potentially creates a second SMN2-specific hnRNP A1 binding site. sion of the P-element third intron by binding to an ESS in the Remarkably, this single base change does indeed create a high- upstream exon, and this repression interferes with 5Ј splice-site affinity hnRNP A1 binding site, and base substitutions that disrupt recognition (14, 15). Splicing repression by hnRNP A1 and the it restore exon 7 inclusion in vivo and prevent hnRNP A1 binding existence of A1-dependent ESSs have been documented in a in vitro. We propose that interactions between hnRNP A1 mole- number of other instances in humans (16–19), but in these cases, the cules bound to the exonic and intronic sites cooperate to exclude mechanism and possible requirement of additional sequences ele- exon 7 and discuss the significance of this exclusion with respect ments that cooperate with the ESSs have not been investigated. to SMN expression and splicing control more generally. Mutations in regulatory elements in premRNAs that affect alternative splicing can give rise to genetic disorders (20). Although alternative splicing ͉ exonic splicing silencer ͉ hnRNP A1 ͉ this frequently involves disruption of positive elements, e.g., ESEs, intronic splicing silencer some mutations also affect splicing by creating negative elements, such as ESSs (21). Splicing of exon 7 in SMN1 and SMN2 transcripts lternative splicing of mRNA precursors is an important mech- constitutes an excellent and clinically important model to investi- Aanism that regulates gene expression and generates increased gate molecular mechanisms of splicing regulation and specifically protein diversity from a limited number of genes. Indeed, expres- the roles of ESEs and ESSs. Homozygous loss of SMN1 is respon- sion of 70% or more of human genes is now estimated to involve sible for the hereditary disease spinal muscular atrophy (22). alternative splicing (1, 2). In higher eukaryotes, alternative splicing Although the SMN1 and SMN2 genes are almost identical, the presence of SMN2 does not prevent development of spinal muscular plays an essential role in diverse cellular functions, contributing to 3 ϩ such basic processes as cell growth, differentiation, and cell death atrophy. A C T transition at position 6 of exon 7 is largely (3, 4). Several types of cis-acting RNA elements and trans-acting responsible for alternative exon 7 splicing (95% of SMN1 transcripts include exon 7, and 80–95% of SMN2 mRNAs lack exon 7; see refs. protein factors help to regulate alternative splicing and do so by Ͼ diverse mechanisms. In general, however, non-spliceosomal pro- 23 and 24). This base change, which is one of only four in 1.25 kb encompassing exon 7 and much of the flanking introns (23), does teins that participate in regulating alternative splicing associate with Ј regulatory elements in and/or introns. Serine/arginine-rich not affect either the exon 7 5 splice site or an ESE in the middle of the exon dependent on the splicing regulator Tra2 (25, 26). proteins (5) are typically involved in positive regulation of splicing, Krainer and colleagues have presented evidence that the ϩ6 base stimulating splicing by interacting with change disrupts an ESE in SMN1 that depends on a specific elements (ESEs) or intronic splicing enhancer elements. In con- serine/arginine-rich protein, ASF/SF2, which they suggest results in trast, negative regulation is promoted most frequently by hetero- SMN2 exon 7 exclusion (27, 28). In contrast, Kashima and Manley geneous nuclear ribonucleoproteins (hnRNPs) (6), which function (29) provided data indicating that the C 3 T change creates an by binding sequences known as exonic splicing silencers (ESSs) or hnRNP A1-dependent ESS that recruits hnRNP A1 to SMN2 but intronic splicing silencers. not SMN1 premRNA, thereby repressing SMN2 exon 7 splicing. Several hnRNP proteins have been identified as key splicing Consistent with the view that a negative element exists in the repressors. Among these, the abundant hnRNP A1 protein has vicinity of ϩ6, Singh et al. (30) showed that sequences flanking this been extensively characterized. The first hnRNP A1-dependent position exert an inhibitory effect on SMN2 exon 7 inclusion. ESS was identified in studies of HIV tat exon 2 repression (7–9). Here we provide additional unexpected insights into the mech- This ESS was found to bind hnRNP A1, and mutations disrupting anism by which SMN2 exon 7 splicing is repressed. We first identify the ESS prevented hnRNP A1 binding and allowed enhanced exon 2 splicing. Several mechanisms have been proposed to explain hnRNP A1-mediated splicing repression. In one, hnRNP A1 binds Author contributions: T.K. and J.L.M. designed research; T.K. and N.R. performed research; to an ESS and through direct protein-protein interactions, recruits T.K., N.R., and J.L.M. analyzed data; and T.K. and J.L.M. wrote the paper. more hnRNP A1 molecules, assembling a complex that inhibits The authors declare no conflict of interest. spliceosome formation (10). In this case, no additional specific Abbreviations: ESE, exonic splicing enhancer element; ESS, exonic splicing silencer; sequence elements are required; a single ESS triggers exon repres- hnRNP, heterogeneous nuclear ribonucleoprotein; NE, nuclear extract; snRNP, small sion in cooperation with A1 self-assembly. In a second scenario, an nuclear ribonucleoprotein. hnRNP A1 binding site is located adjacent to the branch point *To whom correspondence should be addressed. E-mail: [email protected]. sequence near the 3Ј splice site, and A1 directly blocks U2 small © 2007 by The National Academy of Sciences of the USA

3426–3431 ͉ PNAS ͉ February 27, 2007 ͉ vol. 104 ͉ no. 9 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0700343104 Downloaded by guest on September 26, 2021 SmaI SmaI

Exon 6 Exon 7 Exon 8 HP A B XS XS HP E E Wild type: 6.9 kb PstI SMN1 SMN2 Mock SMN2 SMN1 SMN2 SMN1 SMN1 SMN2 mutation XS: 5.0 kb HindIII EcoRI

Deletion mutation HP: Full-length 1.5 kb ∆7

Deletion mutation E: 1.25 kb EcoRI 1 23456789 (390bp) (228bp)

C Exon6 Exon7 Exon8 D ∆Int6 ∆Int7 HP SMN1 HP

SMN2 Mock SMN1 SMN2 SMN1 SMN2 SMN1 SMN2

SMN1-∆Int6 Precursor RNA

SMN2-∆Int6 Full-length SMN1-∆Int7 ∆7 * SMN2-∆Int7 1234567

Fig. 1. Deletion of internal intron 6 and intron 7 sequences minimally affects SMN exon 7 splicing. (A) Schematic diagram of internal deletions of introns 6 and 7 in SMN1/2 plasmids. Black boxes indicate exons, horizontal thick lines indicate introns, and the vertical white line denotes the position of the C 3 T transition in SMN2 exon 7. Restriction enzyme sites that were used for cloning are indicated. Sizes of exons and introns in the shortest constructs, specifically SMN1/2E, are indicated. (B) RT-PCR of RNA isolated from transfected 293 cells with the indicated deletion constructs. Positions of full-length and exon 7-excluded (⌬7) PCR products are indicated on the right. Both spliced products from the full-length SMN1 and SMN2 constructs migrated slightly slower than the corresponding RNAs from the mutants; this slower migration was due to different cloning sites at 3Ј ends. (C) Schematic diagram shows wild type, ⌬Int6, and ⌬Int7 constructs in SMN1 and SMN2 backgrounds. Open boxes indicate exons, thick lines indicate introns, and the vertical line in exon 7 denotes the position of the ESS. (D) SMN1/2HP and SMN1 and SMN2 ⌬int6 and ⌬int7 plasmids (4 ␮g) were transfected into 293 cells. Total RNA was prepared after 48 h and analyzed by using RT-PCR. The position of PCR products corresponding to precursor (unspliced) RNA, full-length, and exon 7-excluded (⌬7) mRNA are indicated on the right. Asterisk indicates an apparent aberrant splicing product.

another single nucleotide difference, at position ϩ100 in intron 7, gradually deleted internal sequences in expression plasmids con-

that has the potential to create a second hnRNP A1 binding site taining exons 6–8 of both SMN1 and SMN2 (see Fig. 1A) and then MEDICAL SCIENCES specific to SMN2. We then provide evidence that this intronic A1 tested the effect of these deletions on SMN2 exon 7 splicing, using binding site is necessary for efficient exon 7 exclusion in vivo.We RT-PCR after transfection in HEK293 cells. The shortest con- also show that this site specifically and strongly binds hnRNP A1 in structs, designated SMN1E and SMN2 E, deleted 5.7 kb of intron 6 vitro and that mutations that disrupt the A1 binding motif prevent and approximately half (217 bp of 444 bp) of intron 7 but displayed protein binding and increase exon 7 inclusion. Our data indicate similar splicing patterns compared with the corresponding full- that the mechanism of SMN2 exon 7 exclusion is more complex than length transcripts (Fig. 1B). Nearly 95% of both full-length and previously thought and provide new insights into splicing control truncated SMN1 transcripts retained exon 7, whereas with SMN2, more generally. exon 7 was Ϸ90% excluded with the full-length transcript and 70% excluded with the truncated version. Thus, significant shortening of Results the long intron 6 had no affect on splicing, but shortening of the 217 Our previous data showed that the C 3 T single nucleotide bases intron 7 led to a slight decrease in exon 7 exclusion (see also difference at position ϩ6inSMN2 exon 7 creates an hnRNP Fig. 2F, lanes 2 and 8). These results indicate that most of the A1-dependent ESS and that this sequence strongly and specifically intronic sequences are not essential for determining exon 7 inclu- binds hnRNP A1 (29). The presence of the ESS is a key mechanism sion or exclusion. in suppression of exon 7 splicing in SMN2 transcripts. Although our data are consistent with the view that this is the sole factor Intron 7 Contains Sequences That Are Necessary for Exon 7 Exclusion. responsible for SMN2 exon 7 exclusion (ref. 29; unpublished data), We next wished to determine whether intronic sequences have any it remains possible that an alternative mechanism, such as loss of essential role in modulating exon 7 splicing. That is, does complete ESE function (27, 28), also contributes to SMN2 exon 7 exclusion. deletion of intron 6 (or intron 7) affect the ability of exon 7 to be However, we do not address this possibility here, but rather describe spliced to exon 8 (or exon 6)? And does the exon 7 ϩ6 base change additional experiments designed to elucidate how the SMN2- influence splicing under these conditions? To investigate, we first specific ESS functions. precisely deleted either intron 6 (⌬int6) or intron 7 (⌬int7) in SMN1/2HP (see Fig. 1C), transfected the plasmids into 293 cells, and Deletion of Most Intronic Sequences Affects Exon 7 Splicing Only again measured exon 7 splicing, using RT-PCR. Deletion of intron Modestly. Alternatively spliced exons are frequently located near 6 resulted in very inefficient splicing of exons 7 and 8 in both SMN1 long introns with nonconsensus 5Ј splice sites and/or a short and SMN2 (Fig. 1D, lanes 4 and 5), suggesting that intron 6 contains polypyrimidine tract at the 3Ј splice site (21). SMN exon 7 is typical (a) positive element(s) necessary for efficient splicing of exons 7 and of this finding, because it is located next to a long intron (intron 6 8. Importantly, though, deletion of intron 7 resulted in efficient encompasses Ϸ6.0 kb) and is flanked by both weak 3Ј and 5Ј splice splicing of exon 6 to exon 7 in SMN2 and SMN1 (lanes 6 and 7). This sites (25, 31). We first wished to determine whether the lengths of finding suggests that intron 7 contains an element required for the flanking introns influence exon 7 splicing. To this end, we SMN2 exon 7 exclusion and motivated us to investigate whether

Kashima et al. PNAS ͉ February 27, 2007 ͉ vol. 104 ͉ no. 9 ͉ 3427 Downloaded by guest on September 26, 2021 B We next wished to determine whether any of the putative hnRNP A A1 binding sites in intron 7 play a role in SMN2 exon 7 exclusion. First, all potential A1 binding sites in intron 7 were mutated (T 3 C, at position ϩ1 of the consensus) in both SMN1E and SMN2E and the mutated constructs were transfected into 293 cells (Fig. 2C). Splicing was again measured by RT-PCR, but in this case by using 32 C D P-dCTP to facilitate quantitation (Fig. 2D). Significantly, exon 7 splicing was enhanced (30% exon 7 inclusion in SMN2E compared with 70% exon 7 inclusion in SMN2E-7m) in the SMN2E context (Fig. 2D, lanes 3 and 5), whereas no effect was observed with SMN1E (lanes 2 and 4). These data suggest that the presence of one or more consensus hnRNP A1 binding sites in intron 7 is necessary for optimal exon 7 exclusion, at least in the SMN2E context.

The SMN2-Specific Intron 7 A 3 G Transition at Position ؉100 Is Necessary for Efficient Exon 7 Exclusion. We next wished to deter- mine which of the consensus hnRNP A1 binding sites is necessary E F for exon 7 exclusion in SMN2E, and especially whether the SMN2- specific site at position ϩ100 is important. To begin to address this question, we first prepared a chimeric construct in which SMN2E intron 7 was precisely replaced with SMN1E intron 7 (SMN2-1E). This process in effect creates a single-base transition, converting SMN2 ϩ100GtoSMN1 ϩ100 A. Plasmids were transfected into 293 cells and exon 7 splicing again measured by quantitative RT-PCR (Fig. 2E). The results revealed that this change dramat- ically reduced exon 7 exclusion, from 71% in SMN2E to 8% in SMN2-1E (compare lanes 3 and 4). To extend this result, we constructed two SMN2E mutant derivatives with single base Fig. 2. Disruption of consensus hnRNP A1 binding sites in intron 7 rescues changes predicted to disrupt the ϩ100 hnRNP A1 binding site (Fig. exon 7 splicing. (A) Location and sequence of consensus hnRNP A1 binding 2E) (14, 26). Both mutations (TAG 3 TCG or TAG 3 TAC; see sites (TAGNNA/T) in SMN1/2E constructs. SMN1/2-common consensus A1 sites are indicated above the diagram, and SMN2-specific consensus sites are Fig. 2E) significantly enhanced SMN2 exon 7 inclusion (Fig. 2F, indicated below the diagram. (B) Position of single nucleotide differences compare lane 3 with lanes 5 and 6), although somewhat less between SMN1 and SMN2 in the SMNE constructs. The exonic ϩ6C3 T and effectively than the intron 7 swap (30/35% vs. 8% exclusion, intronic ϩ100 A 3 G transitions are indicated below the diagram. Other respectively). Thus, our results show that four separate base changes nucleotide differences are shown above the diagram. (C) The schematic altering the core UAG motif of the SMN2-specific hnRNP A1 diagram shows the positions of the T 3 C mutations in the consensus hnRNP consensus in intron 7 each enhanced exon 7 inclusion. A1 sites in intron 7 of the SMN1/2E derivatives. Open boxes indicate exons, We next wished to examine whether intron 7 sequences affect solid lines indicate introns, and the vertical thick line in exon 7 denotes the ESS. exon 7 exclusion in the context of full-length intron 7. Recall that Crosses indicate positions of mutated consensus A1 sites. (D) RNA was pre- Ϸ pared after 48 h transfection of 293 cells and analyzed by quantitative RT-PCR we showed that deleting 50% intron 7 resulted in a slight decrease in the presence of [32P]dCTP. The position of full-length (FL) and exon 7-ex- (from 96% to 71%) in SMN2 exon 7 exclusion (see Figs. 1B and 2D, cluded (⌬7) PCR products are indicated on the right. The percentage of exon lanes 8 and 3, respectively), suggesting that the deleted sequences 7 skipping was calculated from the total of exon 7 inclusion and exclusion in some way enhance exon 7 exclusion. Furthermore, previous products, measured by PhosphorImager, and is indicated below. (E) Diagram studies have suggested that SMN2-specific intron 7 sequences do of mutant and intron 7 ‘‘swap’’ constructs. Open boxes indicate exons, dotted not have a significant effect on exon 7 splicing (refs. 23 and 24; see lines indicate SMN1 introns, solid lines indicate SMN2 introns, and the vertical also ref. 34 and Discussion). We therefore constructed an SMN2 line in exon 7 denotes the position of ESS. The cross indicates TAG mutations intron 7 swap derivative, using the SMN1/2 plasmids, which ϩ HP at position 100 in intron 7 in the SMN2E construct. (F) SMN1/2E, SMN1/2HP, contain full-length intron 7 (see Fig. 2E). SMN1 , SMN2 and and derivative plasmids (4 ␮g) were transfected into 293 cells. RNAs was HP HP prepared after 48 h and analyzed by quantitative RT-PCR. Positions of full- the corresponding SMN2HP derivative containing SMN1 intron 7 length (FL) and exon 7-excluded (⌬7) PCR products are indicated on the right. (SMN2-1HP) were transfected into 293 cells and exon 7 splicing was The percentage of exon 7 skipping for each sample is indicated below. measured by using quantitative RT-PCR (on the right side of Fig. 2F). Consistent with the semiquantitative RT-PCR in Fig. 1B, exon 7 was included in essentially all of the SMN1 transcripts and was specific intron 7 sequences are necessary for SMN2-specific exon 7 excluded from Ϸ96% of the SMN2 transcripts (lanes 7 and 8). Most exclusion. importantly, replacing SMN2 intron 7 with SMN1 intron 7 again significantly enhanced exon 7 inclusion, from Ϸ4% to 40% inclu- Identification of Consensus hnRNP A1 Binding Sites in Intron 7 and sion (lanes 8 and 9). Although less than the increased inclusion Their Role in Exon 7 Exclusion. Intronic and exonic hnRNP A1 observed with the short intron 7-containing transcripts (which went binding sites function together to suppress HIV tat exon 3 splicing from Ϸ29% to 92%; see Fig. 2F, lanes 4 and 5), these results (11, 32). We therefore next searched for the presence of the confirm that the SMN2 intron 7-specific base change at ϩ100 consensus hnRNP A1 binding motif, UAGNNA/U (29, 33), in indeed plays a significant role in exon 7 exclusion. intron 6 and especially intron 7 of SMN1E and SMN2E (Fig. 2A). There are four consensus A1 binding sites in intron 6 and two/three SMN2 Intron 7 Specifically Interacts with hnRNP A1 in HeLa Nuclear in intron 7. Remarkably, SMN2 intron 7 contains a unique con- Extract (NE). We next wished to examine whether hnRNP A1 in fact sensus A1 binding site, reflecting an A 3 G transition at position binds to the SMN2-specific site in intron 7. To this end, we first used ϩ100 (relative to the 5Ј splice site). Given that there are only four 32P-labeled intron 7 (plus the two terminal nucleotides of exon 7) nucleotide differences between SMN1 and SMN2 in the 1.25 kb RNAs prepared from SMN1E and SMN2E in UV crosslinking SMN1/2E constructs (Fig. 2B) (23, 24), it is striking that two of these assays with HeLa NE (ref. 29; see Methods). Wild-type SMN1/2E base changes create consensus hnRNP A1 binding motifs. RNAs and the mutant derivatives containing U 3 C base changes

3428 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0700343104 Kashima et al. Downloaded by guest on September 26, 2021 A B A

B

Fig. 3. hnRNP A1 in HeLa NE UV-crosslinks preferentially to SMN2 intron 7 RNA. (A) Diagram of intron 7 RNAs used in UV crosslinking and immunopre- cipitation assays. Open boxes indicate exons and solid lines indicate intron 7. Solid triangles indicate consensus A1 sites, and open triangles indicate loca- tions of TAG 3 CAG mutations. (B)(Left) SMN1 (lane 1) and SMN2 (lane 2) wild type and mutant (lanes 3 lane 4, respectively) intron 7 RNAs were incubated C with HeLa NE, crosslinked, and analyzed by SDS/PAGE. (Right) UV crosslinked proteins were immunoprecipitated with anti-hnRNP A1 antibodies (lanes 6–10) and analyzed by SDS/PAGE. The position of hnRNP A1 is indicated on the right, and the position of size marker proteins is indicated on the left.

in the putative hnRNP A1 consensus sites (see Fig. 2 A and B) were analyzed (Fig. 3A). With all four RNAs (Fig. 3B, lanes 2–5), the Fig. 4. The SMN2-specific intronic A1 consensus site binds hnRNP A1 strongly prominent crosslinked species was the size expected of hnRNP A1 and specifically. (A) Schematic diagram shows locations of all consensus hnRNP (Ϸ37 kDa) (lanes 2–5), and the identity of the protein was con- A1 binding sites in SMN1/2 intron 7. Partial boxes indicate exonic fragments firmed by immunoprecipitation with anti-hnRNP A1 antibodies and solid lines indicate introns. Filled diamonds indicate positions of consen- sus hnRNP A1 sites. Arrows with filled diamonds indicate RNAs containing a (lanes 7–10). Notably, crosslinking with the SMN2E intron 7 RNA (lanes 3 and 8) was much stronger than with any of the other RNAs. consensus A1 site, and the arrow without a diamond indicates the SMN1- This finding did not reflect anything unusual about this RNA, specific RNA that lacks a consensus site. (B)(Left) UV crosslinking assay with indicated RNAs and HeLa NE. RNAs were incubated with NE, crosslinked, and because crosslinking of background bands was similar with all four MEDICAL SCIENCES ϩ 3 analyzed by SDS/PAGE. The position of hnRNP A1 is indicated on the right. RNAs. This result strongly supports the notion that the 100 A Positions of size marker proteins are indicated on the left. (Right) competition G transition creates a strong hnRNP A1 binding site. UV crosslinking assay. Incubation of labeled RNA (50 fmol) with NE was carried out in the presence of 10-␮M or 0.1-␮M concentrations of the indicated The SMN2-Specific Intron 7 hnRNP A1 Site Strongly and Specifically unlabeled RNAs. The position of hnRNP A1 is indicated on the right, and the Binds hnRNP A1. To extend the results in Fig. 3, we next prepared size marker proteins are identical to the gel in Left.(C)(Left) RNAs containing several 32P-labeled short RNAs (Ϸ23 nt) that contained single the indicated sequences were incubated in NE, crosslinked and analyzed by potential A1 binding sequences from intron 7 (see Fig. 4A) and SDS/PAGE. (Right) UV crosslinked proteins were immunoprecipitated with anti-hnRNP A1 antibodies (lanes 6–10) and analyzed by SDS/PAGE. The posi- tested these in UV crosslinking assays. Strikingly, the RNA that tion of hnRNP A1 is indicated on the right. contained the ϩ100 SMN2-specific site, but none of the others, gave rise to strong crosslinking (Fig. 4B Left). Crosslinking was abolished by an excess of cold SMN2-specific RNA, but not by the corre- ically with hnRNP A1, we suggest that these two hnRNP A1 sponding sequence from SMN1 (Fig. 4B Right), confirming that the protein-RNA interactions cooperate to suppress SMN2 exon 7 enhanced crosslinking indeed reflected stronger RNA binding. splicing. Below, we discuss possible mechanisms by which the two Extending these results, the two point mutations in the core UAG protein-RNA interactions lead to exon 7 exclusion and the impli- sequence that increased SMN2 exon 7 inclusion in vivo (see Fig. 2 cations of our results with respect to SMN expression and to splicing E and F) greatly reduced crosslinking (Fig. 4C). The identity of the control more generally. crosslinked species was again confirmed by immunoprecipitation A key mechanistic implication of our findings is that two distinct, with anti-hnRNP A1 antibodies (Fig. 4C). Taken together, our sequence-specific protein-RNA interactions, involving high-affinity results indicate an SMN2-specific single nucleotide change creates hnRNP A1 binding sites in exon 7 and intron 7, cooperate to a high-affinity hnRNP A1 binding motif that plays a significant role suppress SMN2 exon 7 splicing. hnRNP A1 has been observed to in bringing about maximal exon 7 exclusion. form homodimers by biochemical (35, 12) and structural (36, 37) studies. We suggest that the ability of hnRNP A1 to dimerize Discussion facilitates formation of a loop structure in SMN2 premRNA that In this study, we have provided evidence that an intronic hnRNP A1 suppresses exon 7 splicing. This model is related to the original binding site, in addition to the previously characterized exonic site, loop-out model proposed by Chabot and colleagues (12, 13). In the is necessary for maximal SMN2 exon 7 exclusion. Combined with SMN2 exon 7 case, however, one exonic and one intronic high- our previous data (29), this finding indicates that at least two affinity hnRNP A1 site are necessary for looping, instead of two sequence-specific interactions of hnRNP A1 with splicing silencer- intronic sites in the case of hnRNP A1 exon 7b. In both instances, type elements are necessary for full repression of exon 7 splicing in though, an RNA loop formed by interaction between distant SMN2 transcripts. Given that both the exonic and intronic se- hnRNP A1 molecules (separated by 148 bases in SMN2 premRNA) quences were independently shown to interact strongly and specif- is suggested to facilitate exon exclusion.

Kashima et al. PNAS ͉ February 27, 2007 ͉ vol. 104 ͉ no. 9 ͉ 3429 Downloaded by guest on September 26, 2021 How does the proposed RNA structure repress exon 7 splicing? splicing, for example in intron definition (46, 47). This idea is The hnRNP A1 binding sites do not affect U1 snRNP complex supported by recent studies showing that hnRNP A1 binding motifs assembly at the exon 7 5Ј splice site (T.K. and J.L.M., unpublished are more frequently found in noncoding regions than in coding data), suggesting that the mechanism of exon 7 exclusion involves regions, perhaps playing a role in compaction of long intronic a step subsequent to early spliceosome assembly. This finding is sequence by forming sequential loop structures (48). Consistent consistent with studies with hnRNP A1 premRNA (38, 12) and with with the idea that UAG motifs play important roles in the fidelity other studies proposing a similar mechanism for PTB-dependent of intron recognition and in ensuring efficient splicing, this se- exon suppression (39). Thus, looping out of RNA between occupied quence is frequently lacking in Fugu introns, which offers an hnRNP1 A1 binding sites likely does not affect early snRNP-RNA explanation of why Fugu introns cannot be spliced when introduced interactions but rather subsequent steps, such as cross-talk between into human cells (49). Our results extend these findings by showing U1 and U2 . that an intronic UAG-based site can cooperate with a similar exonic Cooperation between an hnRNP A1-dependent ESS and an sequence to bring about exon exclusion. We suggest that this intronic site has been described in HIV tat exon 3 splicing (11, 32). reflects the incorporation of the exon into an intronic ‘‘network’’ However, in this case the intronic site is located within the poly- that is compacted and defined based on binding and interaction of pyrimidine tract at the 3Ј splice site, which suggests that repression multiple hnRNP A1 molecules (and perhaps other hnRNPs; see is due to competition between hnRNP A1 and U2AF binding to the ref. 48). polypyrimidine tract. In SMN2 exon 7 splicing, there are no hnRNP In summary, our data has identified a second SMN2-specific base A1 binding sites in the vicinity of either proximal 5Ј or 3Ј splice sites, change that creates an hnRNP A1 binding site and provided consistent with the idea that the mechanism of inhibition is distinct unexpected evidence that it plays an important role in exon 7 from that is used in tat exon 3 repression. Our results thus illustrate exclusion. That two of the rare base changes between SMN1 and an important principle: Repressive elements can be localized at SMN2 are located in relatively close proximity and create high- diverse positions relative to splicing signals and lead to inhibition by affinity hnRNP A1 binding sites is intriguing. It is conceivable that distinct mechanisms. the human-specific SMN2 gene provides a buffer that allows Studies have provided evidence that intronic elements are in- up-regulation of SMN protein in cells or tissues with reduced volved both positively and negatively in control of SMN exon 7 hnRNP A1 levels. Although studies examining SMN protein levels splicing (34, 40, 43). However, none of these elements involve in different human tissues have not been reported, it is known that SMN2-specific sequences, and thus they likely contribute to the there are variations in hnRNP A1 protein concentration (50–52). inherent strength of the exon 7 5Ј and 3Ј splice sites. For example, More generally, our results have highlighted the importance of the element intronic splicing silencer (ISS)-N1, identified by Singh interactions between regulatory elements across intron/exon et al. (40), was suggested to interfere with the function of TIA-1, a boundaries and provided new insight into how hnRNP A1 can factor that helps U1 snRNP assembly at weak 5Ј splice sites (41, 42). modulate splicing. This sequence and two other less characterized elements (43, 34) appear to play a more general role in facilitating the efficiency of Methods exon 7 splicing. For example, they may constitute binding sites for Plasmids. All SMN1 and SMN2 derivatives were made from the other hnRNP proteins. In this regard, it is intriguing that hnRNP original full-length (exons 6–8) genomic SMN1 and SMN2 con- F/H binding sites near 5Ј splice sites can cooperate with hnRNP structs described in ref. 29. All deletion constructs (shown in Fig. A1-dependent ESSs to repress a targeted exon (44, 45). All these 1A) were produced by using standard procedures. SMN1/2E con- studies together suggest that the splicing efficiency of SMN 1/2 exon structs were used for most experiments. Multiple point mutations 7 is determined by multiple protein–RNA interactions. (TAG 3 CAG) in intronic sequences were introduced as described Our data has provided evidence that a second rare nucleotide in ref. 53, and single point mutations were also created as described difference between SMN1 and SMN2 contributes to SMN2 exon 7 (29). Precise intron deletion mutations (⌬int6 and ⌬int7) were exclusion. Two studies examined the role of intron 7 sequences by created by back-to-back PCR (29). Briefly, PCR was performed analyzing splicing of transcripts that contained SMN1/SMN2 intron with a reverse primer for exon 6 and a forward primer for exon 7 7 swaps and reached the opposite conclusion (23, 24). However, to create ⌬int6 and a reverse primer for exon 7 and forward primer these experiments used nonquantitative RT-RCR and did not for exon 8 to create ⌬int7, by using Vent DNA polymerase, and consider smaller effects that could be detected by quantitative blunt-ended PCR products were ligated with T4 DNA ligase. To analyses. In fact, a third study that used a more quantitative prepare intron 7 swap constructs, we PCR-amplified full-length RT-PCR analysis did reveal a slight increase in exon 7 inclusion SMN1 intron 7 or the short version from SMN1E intron 7, then when SMN2 intron 7 was replaced with its SMN1 counterpart (34). ligated them with the SMN2 ⌬int7 PCR products described above. It is noteworthy, however, that we observed the strongest effect of All SMN1/2-derived constructs were digested with XhoI and XbaI the SMN2 ϩ100 site in the context of a shortened intron 7, which and subcloned into pcDNA3 (Invitrogen, Carlsbad, CA). also resulted in slightly less efficient exon 7 exclusion (compared We generated all template plasmids for in vitro transcription by with the derivative containing full-length intron 7). A reasonable PCR with a proof-reading DNA polymerase (Pfx DNA polymerase; explanation for both of these observations is that the 225 nucleo- Invitrogen) and amplified intron 7 of SMN1/2E and their derivatives tides deleted in the shortened intron 7 contains sequences that by PCR to add restriction enzyme sites at both ends (HindIII at 5Ј contribute to exclusion, both increasing exclusion in the full-length and XbaI at 3Ј). These were digested with both enzymes, gel- context and partially compensating when the ϩ100 hnRNP A1 site purified and cloned into pGEM4Z (Promega, Madison, WI). For is mutated. We suggest that the sequence elements include addi- short RNA preparation, we slowly annealed double-strand DNA tional hnRNP A1 binding sites. Indeed, this region contains four oligonucleotides (Invitrogen) that contained potential hnRNP A1 potential A1 binding sites, and three of these are capable of binding binding motifs and HindIII and XbaI sites and cloned them into hnRNP A1, two with high affinity (T.K. and J.L.M., unpublished pGEM4Z. All mutations were verified by sequencing (GENEWIZ, data). In any event, our data has established that intron sequences, South Plainfield, NJ). and especially the SMN2-specific ϩ100 transition, contribute to full exon 7 exclusion. Transfection and Splicing Assays. We transiently transfected SMN1/ Recent bioinformatics studies have shown that UAG motifs, the 2-derived plasmids into 293 cells with Lipofectamine 2000 (Invitro- core of the hnRNP A1 binding site, are frequently found in pseudo gen) as described (29). After 48 h, we isolated total RNA and exonic regions within introns in the human genome, leading to the reverse-transcribed 1-␮g RNA, using 0.5 ␮g of oligo (dT)18–20 suggestion that this motif plays a significant role in constitutive primer and SuperScript II RT (Invitrogen). Resultant cDNAs were

3430 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0700343104 Kashima et al. Downloaded by guest on September 26, 2021 PCR amplified with an SMN-specific forward primer and a plas- Reaction mixtures were routinely incubated at 30°C for 30 min or mid-specific reverse primer, and reactions were terminated during for an additional 10 min with 1% Empigen BB when long RNAs the linear phase. PCR products were resolved on 2% agarose gels (intron 7, 269 nucleotides) were used. Samples were then irradiated and visualized by using ethidium bromide staining. For quantitative with UV light by using a Stratalinker (Stratagene, La Jolla, CA), PCR assays, we amplified 2 ␮l of cDNAs in 50 ␮l of standard treated with RNase A (10 ␮g/ml USB; Sigma, St. Louis, MO), and reaction mixtures containing 5 ␮Ci (1 Ci ϭ 37 GBq) [␣-32P]dCTP proteins resolved by 10% SDS/PAGE. For competition assays, we (3,000 Ci/mmol; Amersham, Pittsburgh, PA) for 20 cycles. PCR prepared large amounts of unlabeled RNA by in vitro transcription products were resolved on gels buffered with 5% polyacrylamide with T7 RNA polymerase in 200 ␮l of reaction mixtures and and 90 mM Tris/89 mM boric acid/2 mM EDTA, pH 8.3, exposed gel-purified them (54). The indicated amounts of cold RNA were to x-ray film, and quantitated by using a PhosphorImager (Molec- added to NE, followed by the 32P-labeled RNA. Immunoprecipi- ular Dynamics, Piscataway, NJ). tation was performed as described (29) with the following antibod- ies: 12CA5 for hemagglutinin epitope and 9H10 for hnRNP A1 UV Crosslinking and Immunoprecipitation Assays. We carried out UV (Immuquest, Ingleby Barwick, U.K.) (33). (UV) crosslinking assays as described in ref. 29, with some modi- 32 fications. Intron 7 or short RNAs were synthesized with [ P]CTP We thank J. S. Sharkow for technical assistance, K. Ryan for helping with 32 and [ P]UTP from XbaI-linearized plasmids and gel-purified PhosphorImager scanning, S. Bush and S. Millhouse for discussion, and them. We incubated 40–50 fmol (5 ϫ 105 c.p.m.) of RNA with 5 ␮l I. Boluk for help preparing the manuscript. This work was supported by of HeLa NE plus 100 ␮g of tRNA in 10 ␮l of reaction mixtures grants from the National Institutes of Health and Families of Spinal under splicing conditions, omitting ATP and creatine phosphate. Muscular Atrophy.

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Kashima et al. PNAS ͉ February 27, 2007 ͉ vol. 104 ͉ no. 9 ͉ 3431 Downloaded by guest on September 26, 2021