Human Molecular Genetics, 2002, Vol. 11, No. 17 2037-2049
© 2002 Oxford University Press
hnRNP-G promotes exon 7 inclusion of survival motor neuron (SMN) via direct interaction with Htra2-ß1
Institute of Human Genetics, University of Bonn, Bonn, Germany
Received May 7, 2002; Accepted June 18, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Proximal spinal muscular atrophy (SMA) is a common motor neuron disease caused by homozygous loss of the survival motor neuron gene (SMN1). SMN2, a nearly identical copy of the gene and present in all SMA patients, fails to provide protection from SMA, due to the disruption of an exonic splicing enhancer (ESE) by a single translationally silent nucleotide exchange, which causes alternative splicing of SMN2 exon 7. Identification of splicing factors that stimulate exon 7 inclusion and thereby produce sufficient amounts of full-length transcripts from the SMN2 gene is of great importance for therapy approaches. Here, by use of in vivo splicing assays, we identified the protein hnRNP-G and its paralogue RBM as two novel splicing factors that promote the inclusion of SMN2 exon 7. Moreover, hnRNP-G and RBM non-specifically bind RNA, but directly and specifically bind Htra2-ß1, an SR-like splicing factor which we have previously shown to stimulate inclusion of exon 7 through a direct interaction with the AG-rich ESE in SMN2 exon 7 pre-mRNA. By using deletion mutants of hnRNP-G, we show that the specific protein–protein interaction of hnRNP-G with Htra2-ß1 mediates the inclusion of SMN2 exon 7 rather than the non-specific interaction of hnRNP-G with SMN pre-mRNA. Additionally, we show for the first time that recombinant trans-acting splicing factors such as hnRNP-G and Htra2-ß1 are also effective on endogenous SMN2 transcripts and increase the endogenous SMN protein level. Finally, we suggest a model of how the exon 7 mRNA processing is regulated by the splicing factors identified so far.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Proximal spinal muscular atrophy (SMA) is an autosomal recessive, neurodegenerative disorder characterized by progressive loss of alpha-motor neurons in the spinal cord, causing secondary muscle weakness and atrophy of the voluntary muscles. With an incidence of about 1 in 6000 live births and a carrier frequency of 1 in 40, SMA is the second most common childhood neuromuscular disease in the Caucasian population and the leading hereditary cause of infant mortality worldwide (1,2). The survival of motor neuron gene (SMN) is the SMA-determining gene (3). It is duplicated on chromosome 5q13, giving rise to a telomeric (SMN1) copy and a centromeric (SMN2) copy. SMA is mainly caused by homozygous absence (deletions, gene conversions) or rare intragenic mutations of the SMN1 gene (4). In humans, the SMN protein is ubiquitously expressed, but decreased protein levels of SMN correlate with phenotypic severity of SMA (5,6). SMN2 fails to provide protection from SMA, due to a single translationally silent C–T transition in exon 7 (7). This substitution disrupts an exonic splicing enhancer (ESE). A part of this ESE bears a heptamer motif which, destroyed by the nucleotide exchange, fails to facilitate specific recruitment of the splicing factor SF2/ASF and thus fails to ensure 3' splice site recognition (8). Finally, this leads to exon 7 skipping and to abundant production of a shorter isoform, SMN2
7. The truncated transcript encodes a biochemically defective protein with less stability and reduced self-oligomerization activity (9,10). The remaining full-length SMN protein produced by SMN2 is not sufficient for viability of motor neurons. Remarkably, in the presence of SMN1, homozygous loss of SMN2 has no phenotypic effect (3). However, increased SMN2 copy numbers do modulate the phenotypic severity in SMA patients and in SMN2 transgenic mice (11–14). The accurate removal of introns from pre-mRNA and joining of exons is performed by the spliceosome, a macromolecular complex (15) that recognizes 5' and 3' splice sites, canonical sequences at the exon/intron borders, as well as auxiliary splicing elements, such as ESEs (16). The spliceosome consists of several small nuclear ribonucleoprotein particles (snRNPs) and a large number of additional factors, collectively referred to as non-snRNP proteins (17–19). One group of non-snRNP factors important in constitutive and alternative splicing comprises the serine–arginine (SR) proteins (20–22). The SR-like protein Htra2-ß1, a human homolog of the Drosophila alternative splicing factor Tra2 (23,24), has been shown to bind to a GAA repeat within the ESE in SMN2 exon 7 and to restore the splicing pattern of SMN2 mRNA by significantly increasing the level of full-length SMN2 transcript to some 80% (25).
It is most likely that further factors, other than SR and SR-like proteins, mediate exon inclusion through direct or indirect association with the SMN exon 7 pre-mRNA or interactions with ESE-associated splicing factors. Proteins containing RNA-binding motifs should be considered, and in particular hnRNP proteins, because these are associated very early with nascent pre-mRNA. Pre-mRNA or heterogeneous nuclear RNA (hnRNA) is present in the cell nucleus as a complex with a discrete set of proteins, including the major core hnRNP proteins of the A, B and C groups, as well as a more heterogeneous population of proteins designated D–U (26). These latter proteins have the ability to form complexes with the core hnRNP proteins and many of them share a highly conserved RNA-binding domain (RBD), which consists of 80–100 amino acids bearing two hallmark consensus sequences: the RNP-2 motif (six amino acids) and the RNP-1 motif (eight amino acids) with RNA-binding capacity (27–29). HnRNP proteins are thought to be involved in the packaging, splicing, post-transcriptional processing and turnover of all pre-mRNAs (30,31).
By use of in vivo splicing assays, we identified the hnRNP-G protein and its paralogue RBM as two novel trans-factors that stimulate the inclusion of SMN2 exon 7, depending on the amount of transfected splicing factors. HnRNP-G is a ubiquitously expressed nuclear protein of unknown function that binds nascent pre-mRNA (32,33) and which is encoded by the RBMX gene localized on the X chromosome (34). The hnRNP-G protein contains at the N-terminus one RNP-consensus RBD, and at the C-terminus a domain rich in serine, arginine and glycine bearing three RGG boxes modified by O-linked N-acetylglucosamine (35,36). Its paralogue, the RBMY gene (for RNA-binding motif gene, Y chromosome), evolved on the Y chromosome from an originally X–Y identical ancestral gene (34,37). It encodes the testis-specific RBM protein that is involved in nuclear RNA processing during spermatogenesis and is expressed only in the nuclei of male germ cells. RBM contains an RBD, and four tandemly repeated sequences, each of 37 amino acids (SRGY boxes), that are rich in SR/RS dipeptides characteristic of the SR proteins (21,38). The cDNAs of hnRNP-G and RBM show 
60% homology (34). We show that both hnRNP-G and RBM bind mRNA non-specifically in vitro, regardless of a particular binding motif. However, both proteins directly interact in vivo with the SR-like protein Htra2-ß1 which simultaneously binds specifically to exon 7 RNA and promotes inclusion of exon 7. Htra2-ß1 and hnRNP-G/RBM act in concert during pre-mRNA processing by stabilizing each other's function. This is the first time that hnRNP-G has been found to play a specific role as a trans-acting splicing factor during pre-mRNA splicing, which differs from its general involvement with other hnRNP proteins during pre-mRNA processing. Additionally, it is shown for the first time that the influence of recombinant trans-acting splicing factors such as hnRNP-G and Htra2-ß1 is also effective on endogenous SMN2 transcripts and increases the endogenous SMN protein level.
Finally, we propose a model in which disruption of the ESE in the SE1 subdomain of SMN exon 7 can be overcome by over expression of factors such as Htra2-ß1, hnRNP-G and SRp30c, which depend on the SE2 sequence in the exon and stimulate the expression of increased amounts of full-length SMN.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The SR-like protein Htra2-ß1 was previously reported to bind an ESE in SMN exon 7 and to stimulate restoration of FL-SMN2 by regulating alternative splicing of SMN2 exon 7 (25). To identify factors that support the stimulating effect of Htra2-ß1, we looked for proteins that interact with Htra2-ß1. An interaction of Htra2-ß1 with hnRNP-G and RBM has recently been shown by a yeast-two hybrid screen as well as by in vitro binding studies (39). Therefore, we tested the effects of the hnRNP-G protein and its paralogue RBM for their ability to (1) interact with Htra2-ß1 in vivo in HEK293 cells, and (2) promote the inclusion of SMN2 exon 7 in vivo.
hnRNP-G and RBM interact in vivo with Htra2-ß1
In order to demonstrate the interaction of Htra2-ß1 with hnRNP-G and RBM in HEK293 cells by co-immunoprecipitation, a polyclonal antibody was raised against both human and murine Htra2-ß1. The anti-Htra2-ß1 antibody specifically detected endogenous as well as recombinant GFP-Htra2-ß1 fusion protein, as shown by western blot analysis using cell lysates from human HEK293 and murine NIH3T3 fibroblasts (Fig. 1A).
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For binding studies, V5-tagged expression constructs of hnRNP-G, RBM and Htra2-ß1 were generated and transiently transfected into HEK293 cells. Correct expression of V5-tagged fusion proteins was confirmed by western blot analysis (Fig. 1B, lanes 2–5) prior to incubation of the lysates with the anti-Htra2-ß1 antibody. Immunoprecipitation of endogenous Htra2-ß1 from nuclease-treated cell lysates and analysis by western blotting with monoclonal antibody V5 clearly detected recombinant hnRNP-G-V5 and RBM-V5 (Fig. 1B, lanes 6 and 7). These results demonstrate that both hnRNP-G and RBM are associated with Htra2-ß1 in vivo in HEK293 cells.
hnRNP-G and RBM specifically facilitate the inclusion of SMN2 exon 7
To address the question of whether or not FL-SMN2 can be restored by hnRNP-G and RBM, in vivo splicing assays were performed. Htra2-ß1, the effect of which on SMN2 exon 7 alternative splicing has been reported (25), was included as a positive control. In vivo splicing assays were performed in human HEK293 cells by co-transfection of increasing amounts (0–5 µg) of mammalian expression constructs encoding hnRNP-G, RBM or Htra2-ß1 with 1 µg of the pSMN2 minigene. Importantly, the pSMN2 minigene (Fig. 2A) recapitulates the splicing pattern of the endogenous SMN2 gene (7). pSMN2 primarily produces dramatically reduced full-length transcript and abundant levels of an aberrantly spliced transcript lacking exon 7, referred to as SMN7 (Fig. 2B, lanes 1, 5, 9, and Table 1). Like Htra2-ß1, hnRNP-G and RBM increased FL-SMN2 production and concomitantly decreased SMN7 (Fig. 2B, lanes 2–4, 6–8 and 10–12). Numerous repetitions of these experiments confirmed 3-fold or a 2-fold increase of FL-SMN2 transcript at the highest concentration of hnRNP-G and RBM, respectively. However, this increase was less efficient than that obtained with 5 µg of Htra2-ß1 (Fig. 2B, lane 4), which does induce a 4-fold increase of FL-SMN2 (see also Table 1).
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Similar in vivo splicing assays were performed in murine NIH3T3 cells to determine whether the splicing regulation observed in human cells was species-specific. This is important because: (1) Htra2-ß1 was shown to include SMN2 exon 7 even on a mouse background (25) and therefore to have a conserved function in mouse and human; and (2) an SMN2 transgenic mouse exists in which the murine Smn gene has been replaced by the human SMN2 gene (12,13). Factors that stimulate FL-SMN2 expression could serve as therapeutic agents and could first be tested in SMN2 transgenic mice regarding the alteration of the phenotypic severity and appearance of side-effects. Co-transfection of increasing amounts of either hnRNP-G or RBM with the pSMN2 minigene into murine NIH3T3 fibroblasts conferred high levels of FL-SMN2 expression and lower levels of SMN
7, comparable to the splicing pattern observed in human HEK293 cells (data not shown). In order to see if these splicing factors support each other's function or whether there is competition for binding sites on RNA between hnRNP-G and Htra2-ß1 or RBM and Htra2-ß1, we performed similar in vivo splicing assays with a combination of Htra2-ß1 and hnRNP-G, or Htra2-ß1 and RBM, respectively at equal rates (Fig. 2C). Combined co-transfection of these splicing factors converted the splicing pattern as follows: 5 µg of hnRNP-G together with 5 µg of Htra2-ß1 led to accumulation of some 83±4% FL-SMN2 transcript (lane 4), which did not significantly exceed the amount of FL-SMN2 transcript (78±3%) exclusively produced by Htra2-ß1 (Table 1). Similar results were observed for the combination of 5 µg of RBM and Htra2-ß1, which restored 77±1% FL-SMN transcript (Fig. 2C, lane 8). However, it is difficult to determine the contributions of the respective splicing factors to FL-SMN2 restoration by this means. Therefore, functional splicing assays were carried out on the basis of constant low amounts of Htra2-ß1 (1 µg) and increasing amounts (0–5 µg) of hnRNP-G or RBM (Fig. 2D). By exclusive use of 1 µg of Htra2-ß1, the inclusion of SMN2 exon 7 into FL-SMN2 transcript is increased from an initial 15±3% to some 40% (Fig. 2D, lanes 2 and 8). Interestingly, addition of increasing amounts of hnRNP-G restored the splicing pattern of FL-SMN2 to 81±1% (Fig. 2D, lanes 3–5), indicating a synergistic effect of these two proteins. Remarkably, 5 µg of hnRNP-G, when tested exclusively, restored only 54% full-length transcript (Table 1 and Fig. 2B, lane 8). Similar, although weaker, effects were observed for RBM (Fig. 2D, lanes 9–11 and Table 2).
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In order to show that the effect observed for SMN2 is exon 7 specific, we performed a similar experiment in which the SMN2 minigene was replaced by the SRp20/X16 minigene (Fig. 2A) (40). pSRp20/X16 contains genomic mouse DNA from exon 3–5 (Fig. 2A) (40), and was chosen because exon 4 of SRp20/X16 was recently shown to be spliced alternatively (41), and Htra2-ß1 has already been shown to have no effect on SRp20/X16 pre-mRNA processing. Both hnRNP-G and RBM failed to show any influence on the regulation of alternative splicing of SRp20/X16 exon 4 (Fig. 2E), indicating that the effects observed for hnRNP-G and RBM on SMN2 pre-mRNA are SMN exon 7 specific.
hnRNP-G and RBM do not need a specific motif to bind SMN exon 7 RNA
To verify the interaction of hnRNP-G and RBM proteins with SMN pre-mRNA and to determine which sequence motif is required, in vitro RNA–protein binding studies were carried out (Fig. 3). 35S-radiolabeled proteins of hnRNP-G, RBM, Fyn and Htra2-ß1 were produced by in vitro translation. The ability to express full-length fusion protein from the corresponding expression constructs has been demonstrated before by western blot analysis of cell lysates obtained after transient transfection of HEK293 cells (Fig. 1A, lanes 1–5). Htra2-ß1 was used as a positive control for in vitro RNA–protein interaction studies, whereas Fyn (p59), proven not to interact with RNA (42), served as negative control. Radiolabeled proteins were subsequently reacted with in vitro transcribed and biotin-conjugated RNA corresponding to SMN exon 6, SMN1 exon 7 and SMN2 exon 7 as well as to SMN1 exon 7 containing mutated subdomains SE1, SE2 or SE3, or three wild-type copies of SE2 (3xSE2). Both hnRNP-G and RBM bind non-specifically to all SMN RNAs (Fig. 3). Neither protein distinguishs between SMN1 and SMN2 exon 7 RNA, and nor do they bind specifically better to three wild-type copies of the SE2 domain (Fig. 3, lanes 4, 5 and 6), as Htra2-ß1 does (Fig. 3, lane 6). They also bind SMN exon 6 very well (Fig. 3, lane 3), whereas Htra2-ß1 does not bind to exon 6 at all, indicating that while Htra2-ß1 interacts specifically with SMN exon 7, hnRNP-G and RBM bind RNA in general and do not require a particular motif to bind SMN exon 7 RNA. The non-specific character of hnRNP-G and RBM binding to RNA is further supported by the use of exon 7 mutated RNA (in SE1, SE2 and SE3) in comparison to wild-type RNA, which are all equally well bound (Fig. 3, lanes 7–9). Similar protein–RNA binding assays were performed for exon 4 of murine SRp20/X16, which does not share any similarity with the SMN exon 7 nucleotide sequence. As indicated in Figure 3, lane 11, hnRNP-G and RMB bind murine SRp20/X16 exon 4 RNA, whereas Htra2-ß1 does not. This further supports the finding that, in contrast to Htra2-ß1, which requires a specific binding motif (GAA-rich), hnRNP-G and RBM bind RNA in general.
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Further characterization of the binding capacity of hnRNP-G for RNA showed that stable interaction is not sequence specific but depends on the length of the substrate. Studies of binding between hnRNP-G and RNAs of a length of
100 nt using buffers with increased ionic strength (100 mM, 150 mM, 180 mM and 200 mM NaCl) did not show a higher affinity for SMN exon 7 RNA compared to RNAs of the same length but of other origin. HnRNP-G was always released from the RNA substrate at NaCl concentrations higher than 100 mM (data not shown). A 5-fold increase of the RNA length led to a delayed dissociation of the protein from the RNA substrate at 180 mM salt (data not shown).
Direct interaction of hnRNP-G with Htra2-ß1 facilitates SMN exon 7 inclusion
From the observations that (1) hnRNP-G does not require a specific motif in order to bind SMN2 exon 7 pre-mRNA and to promote exon inclusion during pre-mRNA processing, (2) hnRNP-G directly interacts with Htra2-ß1 in vivo, (3) Htra2-ß1 binds specifically the AG-rich SE2 domain in SMN2 exon 7 RNA, and (4) low levels of Htra2-ß1 combined with high levels of hnRNP-G lead to abundant accumulation of full-length SMN2 transcript that cannot be achieved by any of these two splicing factors on its own at the amounts used, we concluded that only the direct protein–protein interaction of hnRNP-G with Htra2-ß1 bound to the SE2 domain is responsible for the effects noticed for hnRNP-G during SMN2 pre-mRNA processing. Furthermore, production of full-length transcript seems to be most efficient when both proteins act in concert by stabilizing each other's function on exon 7 RNA. If this hypothesis is correct, a mutated hnRNP-G protein which, on the one hand, is still able to bind RNA, but on the other hand shows strongly reduced affinity towards Htra2-ß1 protein, should fail to restore the full-length SMN2 transcript in an in vivo splicing assay. To prove this hypothesis, we generated mutated hnRNP-G proteins with successively larger deletions of the C-terminus designated hnRNP-Gc41, hnRNP-Gc102 and hnRNP-Gc141 (Fig. 4A). By in vitro co-immunoprecipitation of radiolabeled wild-type and deleted hnRNP-G with endogenous Htra2-ß1 from HEK293 cells, and through protein–RNA binding studies, we identified the hnRNP-Gc41 protein as fulfilling these criteria. Compared to the wild-type hnRNP-G (Fig. 4C, lanes 1 and 2 and Fig. 4D), the affinity for hnRNP-Gc41 for Htra2-ß1 (Fig. 4C, lanes 3 and 4 and Fig. 4D) is almost 60% decreased, whereas the ability of this mutant to bind RNA is not diminished (Fig. 5). Further truncation of the C-terminus of hnRNP-G leads to complete lack of Htra2-ß1 binding capacity (Fig. 4C, lanes 6 and 8; Fig. 4D) and drastically reduces RNA affinity (Fig. 5).
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Therefore, the hnRNP-G
c41 mutant was used to perform in vivo splicing assays of the SMN2 minigene (Fig. 6) in HEK293 cells. 1 µg of the SMN2 minigene was co-transfected with 5 µg of the deletion mutant (Fig. 6, lane 5) and the ability to restore full-length SMN2 transcript was compared with the ability of wild-type hnRNP-G (Fig. 6, lane 3 and Fig. 2B, lane 8) and Htra2-ß1 (Fig. 6, lane 2 and Fig. 2B, lane 4).
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Remarkably, the hnRNP-G
c41 mutant was not capable of stimulating exon 7 inclusion at all (Fig. 6, lane 5), whereas Htra2-ß1 (Fig. 6, lane 2) and wild-type hnRNP-G (Fig. 6, lane 3) yielded abundant accumulation of FL-SMN2, consistent with the results of previous splicing assays. Again, combining high amounts of wild-type hnRNP-G with low amounts of Htra2-ß1 restored some 80% of FL-SMN2 (Fig. 6, lane 4; Fig. 2D, lane 5; and Table 2), but the combination of 5 µg of the mutant hnRNP-Gc41 (which does not interact with Htra2-ß1) with only 1 µg of Htra2-ß1 revealed a weak upregulation of FL-SMN2 typical for low levels of Htra2-ß1 (compare Fig. 6, lane 1 and 6, and lane 5 and 6). These data give firm evidence that at least hnRNP-G requires the direct protein interaction to promote SMN2 exon 7 inclusion. Taking these results together, hnRNP-G must be considered a trans-acting factor of exon 7 alternative splicing via interaction with Htra2-ß1 which is simultaneously bound to the AG-rich sequence in SMN exon 7 pre-mRNA.
Recombinant Htra2-ß1 and hnRNP-G can also alter endogenous SMN protein level
The amount of stable SMN protein in alpha-motor neurons derived from the remaining SMN2 copies is crucial for the phenotypic severity of an SMA patient. Factors promoting the inclusion of exon 7 in SMN2 transcripts could be of great therapeutic value. However, the accumulation of full-length SMN2 mRNA from an SMN2 minigene facilitated by splicing factors such as Htra2-ß1 and hnRNP-G as demonstrated by in vivo splicing assays still does not answer the question of whether these effects ever influence the endogenous protein level. In order to demonstrate this, recombinant hnRNP-G and Htra2-ß1 expression constructs were transiently co-transfected into HEK293 cells. These cells were chosen because: (1) they are of human origin; (2) they can easily be transfected, yielding high transfection efficiencies; and (3) the use of primary fibroblast cultures from SMA patients resulted in a very low transfection efficiency (data not shown). Previous determination of the SMN copy number based on real-time LightCycler PCR (14) revealed two SMN1 and two SMN2 copies in HEK293 cells. Proteins were isolated 48 h post-transfection and subjected to western-blot analysis by using an anti-SMN antibody. The use of 5 µg Htra2-ß1 led to a 1.8-fold increase of endogenous SMN in HEK293 cells, compared to mock-treated cells (Fig. 7, lanes 1 and 2), whereas the use of 5 µg hnRNP-G gives only a 1.4-fold increase of endogenous SMN protein (Fig. 7, lane 3). Consistent with in vivo splicing assays, the combination of high amounts of hnRNP-G with a low level of Htra2-ß1 led to a further increase (2.2-fold) of endogenous SMN protein (Fig. 7, lane 4), an amount that none of the two proteins can achieve on its own. This suggests again that the two splicing factors support each other's function even on endogenous SMN pre-mRNA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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SMN2 and SMN1 encode identical proteins. However, a translationally silent C to T transition in SMN2 exon 7 disrupts an exonic splicing enhancer, causing the production of mainly alternatively spliced transcripts lacking exon 7 that encode a truncated, unstable protein (7,43,44). In SMA patients, the low level of full-length SMN2 protein is not sufficient to compensate for the loss of SMN1 and thus cannot maintain viability of alpha-motor neurons, which finally leads to disease development.
In this report, we identified hnRNP-G and its paralogue RBM as new trans-acting factors capable of compensating for the C–T transition by stimulating abundant accumulation of full-length SMN2 transcript. HnRNP-G is a member of a heterogeneous population of hnRNP proteins that are thought to be involved in the packaging, splicing and post-transcriptional processing of all pre-mRNAs (30,31). We showed that hnRNP-G interacts in vitro and in vivo with recombinant and endogenous Htra2-ß1, a member of the SR family of splicing regulatory factors, that binds specifically to the AG-rich enhancer sequence located in the center of SMN exon 7, and by this means facilitates the inclusion of exon 7 in SMN2 full-length transcripts (25). Since hnRNP-G was also found to bind very efficiently although non-specifically to RNA, we can postulate, that SMN2 exon 7 RNA, Htra2-ß1 and hnRNP-G form a stable complex that is active during SMN2 pre-mRNA processing. Transient co-expression of high levels of hnRNP-G and low levels of Htra2-ß1 revealed the highest amount of restored FL-SMN2 mRNA (>80%) and demonstrated the synergistic effect of both splicing factors during exon 7 processing. Neither of the two trans-acting factors transfected exclusively at the given amounts (5 µg hnRNP-G or 1 µg Htra2-ß1) can restore FL-SMN2 mRNA to >80%. Furthermore, we have proven that it is not the binding of hnRNP-G to a specific RNA motif of exon 7 SMN2 RNA—since hnRNP-G binds non-specifically to RNA in general—but the specific protein–protein interaction of hnRNP-G with Htra2-ß1 that promotes the specific inclusion of SMN2 exon 7, giving rise to high amounts of FL-SMN2 mRNA.
Taken together, these results clearly demonstrate that exon 7 inclusion facilitated by hnRNP-G depends, on the one hand, on the direct interaction with Htra2-ß1, which simultaneously must be bound to the AG-rich SE2 element within the exonic splicing enhancer of SMN exon 7, and on the other hand, on direct binding to SMN RNA hnRNP-G, which enhances Htra2-ß1 function by stabilizing Htra2-ß1 binding to the SE2 element. These findings provide insight into the molecular network of various factors that are required to form a stable and functional complex on exon 7 during SMN2 pre-mRNA processing in order to overcome the C–T transition in SMN2 and finally the loss of SMN1. Additionally, for the first time, hnRNP-G was found to play a specific role as a trans-acting splicing factor during pre-mRNA splicing. So far, hnRNP-G has only been reported to play a secondary role within the subset of hnRNP proteins that maintain pre-mRNA processing in general. Moreover, SMN pre-mRNA is the first target described that is affected by hnRNP-G, even though direct interaction with Htra2-ß1 is necessary. Other promising hnRNP proteins, in particular hnRNP-A1 (26), hnRNP-K and hnRNP-L, did not show any influence on restoration of full-length SMN2 transcript (data not shown). These proteins were partly identified as not interacting with Htra2-ß1 (39), which might be the reason why no effect on SMN pre-mRNA processing was observed. Even kinases (T-STAR and Sam68) that directly interact with hnRNP-G (39) and were thought to have a regulatory influence on hnRNP-G and thus on exon 7 processing failed to show any influence (data not shown). During the preparation of this manuscript, it has been reported that SRp30c, a member of the SR family of splicing factors, converts the SMN2 splicing pattern and forms a trimeric complex with SMN exon 7 RNA via direct interaction with Htra2-ß1 through the SE2 element in the exonic splicing enhancer (45). SRp30c was also demonstrated to interact with hnRNP-G, and this interaction is even stronger than that of SRp30c with Htra2-ß1 (39).
Finally, we would like to propose a model for mRNA processing of SMN exon 7 (Fig. 8). SMN1 contains within the SE1 domain a heptamer motif which is recognized and specifically bound by the SR-splicing factor SF2/ASF and is mainly responsible for correct splicing of exon 7 SMN mRNA (Fig. 8A). The C–T transition in SMN2 exon 7 disrupts the SF2/ASF-dependent heptamer motif. Consequently, SF2/ASF cannot bind this motif and fails to promote correct inclusion of exon 7 (8). This loss of efficient recognition of the heptamer motif by SF2/ASF can be partly overcome by the interplay of the AG-rich exonic splicing enhancer within the SE2 element and the recruitment of several other trans-acting splicing factors which remain despite the disruption of the heptamer in SE1. This might be one of the reasons why low amounts of full-length SMN2 transcripts are still produced in SMA patients and SMN2 transgenic mice. The overexpression of these SE2-dependent splicing factors can efficiently convert the SMN2 splicing pattern (25), although SF2/ASF is not present in this SMN2-specific spliceosomal complex. Moreover, functional splicing assays on an SMN1 minigene construct with a mutated SE2 domain highlight that this region in the center of exon 7 is essential for exon 7 inclusion (43). So far, Htra2-ß1 is the most important SE2-dependent factor of those identified, since it binds directly and specifically to the SE2 element within the ESE on SMN RNA and facilitates exon 7 inclusion. This protein–RNA interaction is most likely stabilized by direct protein–protein interaction with hnRNP-G and simultaneous protein–RNA interaction of hnRNP-G and SMN RNA—thus enhancing the function of Htra2-ß1. SRp30c, for its part, binds very efficiently to Htra2-ß1 but barely to RNA (45). However, since hnRNP-G and SRp30c are attached to each another very efficiently (39), the whole complex is further stabilized (Fig. 8).
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Although we could not use alpha-motor neurons for our assays, the use of mammalian cells was still a great advantage, since in contrast to a prokaryotic expression system, post-translational modification could be guaranteed.
Further experiments are needed to identify additional splicing factors that are components of this SMN exon 7-specific spliceosomal complex which forms during pre-mRNA processing, in order to understand how this assembly is regulated. It is also very important to take the unique tissue distribution, abundances or developmentally regulated manner of splicing factors into account, as these result in corresponding changes in the ratio of individual SR and hnRNP proteins. Post-translational modification such as phosphorylation of SR proteins also plays an important role and needs to be studied in more detail. Moreover, as drugs are identified that convert the SMN2 splicing pattern, it will be critical to have an idea of the molecular network and the signals required to stimulate those targets. In summary, for the first time we demonstrate here that the negative effect of the C–T transition in SMN2 on the correct splicing can be compensated by overexpression of hnRNP-G and Htra2-ß1, not only in a more or less artificial system such as the SMN2 minigene, but also on endogenous SMN2-derived full-length protein. It remains speculative, and further experiments are needed to address the issue of whether accumulation of SMN2 protein leads to abundant oligomerization of SMN and to simultaneous increase of further proteins that are part of the SMN complex (46). At least for Gemin2, a tight co-regulation with SMN has been determined in SMA patients and heterozygous mice (47). However, since each SMA patient retains at least one SMN2 copy, stimulation of exon 7 inclusion and accumulation of endogenous full-length SMN2 by overexpression of splicing factors or use of stimulating compounds is of great therapeutic importance.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Construction of plasmids and expression of recombinant proteins
Wild-type SMN minigene pSMN2 contains the human genomic SMN2 (GenBank accession no. U80017) sequence from exon 6–8 (7). The wild-type SRp20/X16 minigene contains murine genomic DNA from exon 3 to exon 5 (GenBank accession no. X91656) (40). The cDNA of wild-type hnRNP-G/RBMX (GenBank accession no. NM_002139 and Z23064), RBM/RBMY1A1 (GenBank accession no. NM_005058), Htra2-ß1 (GenBank accession no. U61267) and Fyn-p59 (GenBank accession no. Z97989) was subcloned into the CMV-driven mammalian expression vector pcDNA3.1/V5-His-TOPO (Invitrogen, Groningen, The Netherlands) to generate V5-tagged fusion proteins. Moreover, shorter cDNA fragments of hnRNP-G were subcloned into pcDNA3.1/V5-His-TOPO in order to generate mutant recombinant proteins that lack the C-terminal 41, 102 and 141 amino acids. Correct sequences were verified by sequencing the recombinant plasmid DNA on an Applied Biosystems Inc. 377 DNA sequencer (PE Applied Biosystems, Foster City, CA, USA). Protein expression was confirmed by western blot analysis after harvest of 3x105 transiently transfected HEK293 cells. The lysates were resolved by 12% SDS–PAGE and transferred to nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany). Proteins were detected with anti-V5 antibody (1 : 5000 dilution, Invitrogen, Groningen, The Netherlands) by subsequently using a goat–anti-mouse IgG polyclonal antibody conjugated to horseradish peroxidase (1 : 2500 dilution) (Calbiochem, Bad Soden, Germany) and chemiluminescence (Super Signal West Pico, Pierce, Rockford, IL, USA).
For in vitro RNA expression, SMN1 or SMN2 exon 7, exon 6 or different site-directed mutated fragments of SMN exon 7 (3xSE3, SE1, SE2, SE3) (43) were subcloned into either the pCR 2.1-TOPO vector (Invitrogen, Groningen, The Netherlands) or the pSP72 vector (43). In the case of SRp20/X16, the complete exon 4 was also subcloned into the pCR 2.1-TOPO vector. All PCR reactions were carried out with Pfu proofreading polymerase (Promega, Madison, WI, USA), and all constructs were sequenced to confirm correct sequences.
GFP-tagged Htra2-ß1 was derived from a pEGFP-C2 mammalian expression construct (a kind gift from S. Stamm) (25).
In vivo splicing
Recombinant plasmid DNA of wild-type pSMN2 minigene and increasing amounts of the indicated mammalian expression construct were transiently transfected into either HEK293 or NIH3T3 cells. Subsequently, total RNA was isolated and RT–PCR performed as previously described (25). In the case of the SRp20/X16 minigene construct, universal T7 and Sp6 primers derived from vector sequences flanking the insert were used. To ensure quantitative measurements within the linear range, 22 cycles (16 cycles for radioactive PCR of SRp20/X16 exon 4) with denaturing at 95°C for 20 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, were performed for all PCR reactions. PCR products were resolved on 10% polyacrylamide gels (PAA 49 : 1) and visualized by ethidium bromide staining or by autoradiography. The ratio of FL-SMN and alternatively spliced SMN7 was densitometrically quantified with ONE-DScan software (MWG Biotech, Ebersberg, Germany). Representative results are shown from multiple separate experiments.
Anti-Htra2-ß1 antibody and immunoblotting
A polyclonal antibody was raised against Htra2-ß1 by immunization of two New Zealand rabbits with a synthetic peptide corresponding to the N-terminus of the protein (amino acids 1–15: MSDSGEQNYGERESR) conjugated to keyhole limpet hemocyanin (KLH) (Eurogentec, Herstal, Belgium). Since human Htra2-ß1 and murine tra2-ß1 (SIG41) bear a 100% identical amino acid sequence, this antibody is useful for recognizing both the human and murine proteins. Specificity of the antibody was demonstrated by ELISA and preabsorption assays (Eurogentec) and subsequently confirmed by western blot analysis. To detect endogenous and recombinant Htra2-ß1 (SIG41 in mouse), 30 µg of total protein extract from cell lysates of human HEK293 and NIH3T3 murine fibroblasts, both mock and transiently transfected with pEGFP-Htra2-ß1, was resolved by 12% SDS–PAGE and transferred to nitrocellulose. The membrane was blocked overnight in PBS–Tween containing 6% non-fat dry milk, and this was followed by incubation with anti-Htra2-ß1 antibody (1 : 1000 dilution) in PBS–Tween containing 2% non-fat dry milk at room temperature for 1 h, washing three times in PBS–Tween, and detection with goat–anti-rabbit polyclonal antibody conjugated to horseradish peroxidase (Pierce; 1 : 10000 dilution) using chemiluminescence (SuperSignal West Pico).
In vivo co-immunoprecipitation
HEK293 cells, 2x106, cells were transiently transfected with CMV-driven Htra2-ß1-V5, RBM-V5 and hnRNP-G-V5 plasmid DNA by using standard calcium phosphate transfection. Forty-eight hours post-transfection, cells were harvested in IP buffer (25 mM HEPES, pH 8.0, 150 mM KCl, 2 mM EDTA, 20 mM NaF, 1 mM DTT, 0.5% NP-40). To exclude nucleic acid-mediated interactions, all lysates were treated with 5 units of DNase I and RNase A (100 µg/ml) for 20 min at 37°C, prior to binding. After preclearing the lysates, equivalent amounts of cellular extracts were incubated with polyclonal anti-Htra2-ß1 antibody for 2 h at 4°C, and bound with protein G–Sepharose (pre-blocked in 0.5% bovine serum albumin (BSA) Amersham, Uppsala, Sweden) for 60 min. Bound fractions were washed in 800 µl IP buffer five times, resolved by 12% SDS–PAGE, transferred to nitrocellulose, and detected with monoclonal anti-V5 monoclonal antibodies using chemiluminescence.
In vitro transcription
Depending on the in vitro RNA expression construct, recombinant plasmid DNA was linearized either with BamHI, HindIII or XbaI to achieve 5'-overhanging ends. Then, biotinylated sense strand RNA products corresponding to SMN1, SMN2 and SRp20/X16 fragments of discrete sequences were generated by in vitro transcription of 4–5 µg of plasmid DNA for 6 h at 37°C using T7 RNA polymerase (T7-Megashortscript, Ambion, Austin, TX, USA) and Bio-11-CTP (NEN, Boston, MA, USA). Following DNase digestion, the RNA transcripts were gel purified and subsequently quantified by TCA precipitation.
RNA–protein binding
[35S]Methionine radiolabeled recombinant Fyn-V5, Htra2-ß1-V5, RBM-V5, hnRNP-G-V5, hnRNP-Gc41-V5, hnRNP-Gc102-V5 and hnRNP-Gc141-V5 fusion proteins were expressed in reticulocyte lysates (T7 Quick Coupled TnT System, Promega) and reacted with 4 µg of biotin-conjugated RNA corresponding to wild-type exon 6, to exon 7 of SMN1 or SMN2, to SMN1 mutated in the subdomains SE1, SE2 or SE3, to three wild-type copies of SE2 (3xSE2) (43), or to exon 4 of SRp20/X16 for 30 min at 4°C, before the addition of streptavidin beads (pre-blocked; High Performance Streptavidin Sepharose, Amersham) for 30 min at 4°C. Bound fractions were washed five times in 800 µl of pre-chilled phosphate buffered saline (PBS) with 100 mM NaCl and 0.05% NP-40 and analyzed by SDS–PAGE and autoradiography. Experiments were repeated twice, with similar results.
In vitro protein–protein binding
Total protein lysate, 800 µg, from HEK293 cells containing sufficient amounts of endogenous Htra2-ß1, previously confirmed by western blot analysis, were nuclease treated, pre-cleared and incubated with anti-Htra2-ß1 antibody for 2 h at 4°C. Then, lysates were reacted in IP buffer with equivalent amounts of [35S]-methionine-radiolabeled recombinant wild-type hnRNP-G-V5, hnRNP-Gc41-V5, hnRNP-Gc102-V5 and hnRNP-Gc141-V5 fusion proteins, expressed in reticulocyte lysates (T7 Quick Coupled TnT System). Following incubation with protein-G–Sepharose (Amersham) and after extensive washing, bound fractions were analyzed by 12% SDS–PAGE, autoradiography and ONE-DScan software.
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ACKNOWLEDGEMENTS |
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The pSP72 constructs containing SMN
SE1, SE2, SE3 or 3xSE3 were a kind gift from C. L. Lorson at Arizona State University and E. J. Androphy at Massachusetts University, USA. We thank P. J. Nielsen at the Max-Planck-Institute of Immunobiology in Freiburg, Germany, for providing the SRp20/X16 minigene construct. We are grateful to S. Raeder and J. Herchenbach for quantitative analyses of SMN1 and SMN2 copy number in HEK293 cells based on real-time LightCycler PCR. We thank W. Friedl and C. Helmken for critical reading of the manuscript. This study was funded by the Deutsche Forschungsgemeinschaft (Grant SFB400-A6, Graduiertenkolleg 246), Families of SMA and BONFOR.
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FOOTNOTES |
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* To whom correspondence should be addressed at: University of Bonn, Institute of Human Genetics, Wilhelmstr. 31, 53111 Bonn, Germany. Tel: +49 2282872344; Fax: +49 2282872380; Email: bwirth{at}uni-bonn.de
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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