Regulation of SMN
exon 7 splicing attracted attention due to the realization that SMA patients that lack SMN1
always carry at least one copy of SMN2
; therefore, redirecting SMN2
exon 7 splicing represents a potential SMA therapy. Manipulation of splicing has been proposed for a number of diseases, including Duchenne muscular dystrophy, beta-thalassemia and certain cancers (81
). Although many cis-elements and transacting factors are known to modulate SMN
exon 7 splicing, the role of local RNA structure in regulation of exon 7 splicing has not been studied. Our interest in a potential role of RNA structure began with the results of in vivo
selection that identified a number of highly mutable (inhibitory for exon 7 inclusion) residues that overlap the predicted stem–loop structure, TSL2 [(62
) and ]. To demonstrate that the presence of TSL2 negatively affects exon 7 inclusion we used extended mutational analyses combined with enzymatic structure probing.
Recently, we have shown that a weak 5′ ss aids in SMN2
exon 7 skipping (53
). Accordingly, improvement of the 5′ ss by 54G mutation or by deletion of the inhibitory element (ISS-N1) located in the vicinity of the 5′ ss promoted exon 7 inclusion in SMN2
). Interestingly, as mentioned in the recent review by Baralle and co-authors, several computational programs failed to detect a major improvement in the strength of the 5′ ss caused by 54G mutation indicating the importance of the ‘local context’ (82
). Our results show that RNA structure (TSL2) provides this local context and is a new addition to the existing repertoire of cis-elements that contribute toward the weak 5′ ss of exon 7. Based on its location, size and base composition, TSL2 presents a unique structure locked within a short exon. Its triloop and 8 bp-long stem are formed almost exclusively by exonic sequences; only two intronic residues are involved in TSL2 folding (). In contrast, a well studied stem–loop structure at the junction of exon 10 and intron 10 of tau
contains a 6 nt loop entirely comprised of intronic sequences, and a bulged stem >70% of which are formed by intronic nucleotides (27
). Compared to the tau
stem with its high GC content, TSL2, in which the number of GC base pairs is only 25%, appears to be relatively weak.
Crucial evidence of the inhibitory function of TSL2 came from the observation that compensatory mutations ‘rescued’ skipping of exon 7 by restoring the predicted stem. The most convincing among these were 40G and 54C, separated from each other by thirteen nucleotides. When these two substitutions were combined, the effect of the compensatory mutation was fully realized (). This result cannot be explained based on the concept of traditional cis-elements that are generally comprised of short sequences (63
). An RNA structure, on the other hand, would provide the most plausible interpretation for the effect of the long-distance compensatory mutations, such as 40G54C, on exon 7 splicing. In fact, a single compensatory mutation involving long-distance interactions is sufficient to implicate the role of RNA structure (83
To demonstrate the inhibitory impact of TSL2 on SMN
exon 7 splicing we were confronted with the usual challenge of delineating the role of structure from the role of overlapping cis-elements. For example, the top portion of the stem and the entire loop of TSL2 coincide with 3′-Cluster, an inhibitory cis-element predicted by the results of in vivo
selection of the entire exon (53
). It is possible that several mutations in the region of TSL2 improved SMN2
exon 7 inclusion due to both, abrogations of 3′-Cluster and disruption of TSL2. This is illustrated by comparing the effect of 51C and 43G mutations located on the complementary sides of the TSL2 stem. The former mutation fully restored exon 7 inclusion in SMN2
, whereas the latter one had only partial stimulatory effect (). According to our structure probing results, both mutations have similar structure-destabilizing consequences (). However, in contrast to position 43, position 51 is also the part of 3′-Cluster. Therefore, 51C mutation has an additional advantage of disrupting this negative cis-element. Our results imply that a potential transacting factor that recognizes and binds to 3′-Cluster may require both, nucleotide sequence and RNA secondary structure.
The inhibitory impact of the distal portion of TSL2 may be distinct from its proximal part that partially sequesters the 5′ ss of exon 7. Strengthening of the TSL2 stem by three additional base pairs, which results in increase of the sequestered portion of the 5′ ss from two to five nucleotides, causes complete skipping of exon 7 even in SMN1
(). Of note, we believe the inhibitory effect of a stem–loop structure that sequesters the 5′ ss could not be over-generalized. For example, a 9 bp-long stem that sequestered the entire 5′ ss produced no in vivo
effect on donor site selection in the reporter construct containing RP51A
intron inserted in the lacZ
). A 15 bp-long stem was necessary to achieve complete inhibition of splicing in the above system (84
). In contrast, an 8 bp-long stem in TSL2 of SMN1/39C resulted in greater than 80% exon skipping (). Interestingly, the inhibitory effect of TSL2 in SMN1/39C was gained due to a single substitution that changed a Wobble base pair to a stronger Watson–Crick base pair, causing stem strengthening without changing its size. This result highlights that the impact of RNA structure is not merely a matter of stem size but also stem composition. Further, presence of other negative cis-elements in the vicinity of TSL2 may contribute to the inhibitory role of this structure. For example, it is possible that transacting factors that interact with Exinct (52
) and/or ISS-N1 (60
) make secondary contacts with TSL2. In this case, the role of TSL2 goes beyond sequestering the 5′ ss of exon 7.
It has been shown that loop size rather than loop composition contributes to stability of a hairpin (70
). Consistent with this observation, only two out of nine mutations in the loop caused some improvement in SMN2
exon 7 inclusions. However, when the TSL2 triloop was substituted with several known stable tetraloops, exon 7 skipping was promoted even in SMN1
(). In this regard it should be noted that the above tetraloops would also disrupt 3′-Cluster. We believe that the created tetraloop hairpins form a rigid structure that is stable enough to cause skipping of exon 7.
The inhibitory effect of a relatively long stem in TSL2 correlated with the small size of an RNA duplex formed between the 5′ ss of exon 7 and U1 snRNA. Increasing the size of the 5′ ss:U1 duplex from 6 to 11 bp restored exon 7 inclusion even in mutants that had an 11 bp-long stem (). Furthermore, a single intronic mutation at position 61 that extended the size of 5′ ss:U1 duplex from six to eight base pairs completely restored SMN2
exon 7 inclusion in the context of wild-type TSL2 (data not shown). In contrast, a 7 bp-long duplex formed between the 5′ ss of tau
exon 10 and U1 snRNA did not overcome the inhibitory impact of a larger stem that sequesters this 5′ ss (27
). These results further support the exon-specific relationship between the size of the 5′ ss:U1 duplex and the length of the stem that sequesters the 5′ ss of exon 7.
Our previous study indicated that 54G restores exon 7 inclusion in SMN2
). Here we show that 54G was able to restore inclusion of exon 7 in mutants that combined inhibitory C6U and strengthened TSL2. Several possibilities could account for such a strong positive impact of 54G on exon 7 inclusion. First, 54G improves complementarity between the 5′ ss of exon 7 and U1 snRNA. Second, it decreases the stability of TSL2 stem by changing a canonical (Watson–Crick) base pair to a Wobble base pair. In addition, 54G may help recruit U1C protein, which prefers a G residue at the last position of the exon (85
). Importantly, 54G creates a 4 nt G run GGG/gu at the exon7/intron 7 junction. Experimental results accumulated by now indicate that G repeats play an important role in splice site recognition. Their effect on splicing appears to be position dependent. For example, it has been suggested that intronic G repeats increase the content of information used by the spliceosome to distinguish between authentic and cryptic splice sites (86
). It has also been shown that intronic G triplets located immediately downstream of 5′ ss directly interact with U1 snRNA facilitating splice site recognition and functioning as ISE (87
). On the other hand, G quadruplets adjacent to the 5′ ss have been shown to promote silencing of a brain-region-specific exon 19 of the GRIN1
) and inhibit the splicing of intron 3 in DQB1
reporter construct (88
). Interestingly, the GGG/gu sequence similar to the one created by 54G was found to be inhibitory for NF1
exon 3 inclusion (89
). This differential effect of GGG/gu on splicing stresses the importance of ‘local context’ and highlights the differences in the regulation of splicing at the level of individual exons. The stimulatory effect of 54G on splicing of SMN2
mutants with strengthened TSL2 was fully neutralized by an 11 bp stem with four consecutive C-G pairs located in the middle of this structure. Weakening of this strong stem by a single substitution that changed a Watson–Crick base pair to a Wobble base pair restored exon 7 inclusion in SMN2
Interestingly, 48G mutation created a cryptic splice site A/gtaagg (). It is located only seven nucleotides upstream of the wild-type 5′ ss. Despite limited complementarity with U1 snRNA (only 5 bp), this cryptic 5′ ss was not discriminated in favor of the wild-type 5′ ss as judged by the amount of exon 7-included products generated from both splice sites. Due to the fact that both cryptic and wild-type donor sites were partially sequestered in TSL2, none was efficiently recognized. Once again, our results highlight the difficulty in evaluating the role of RNA structure using substitution mutations. In addition to creating and/or abrogating cis-elements, substitution mutations are also capable of creating cryptic splice sites. It is worth mentioning that a cryptic splice site is also created when the wild-type triloop is substituted with the tetraloop sequences UUCG or CUUG. However, as indicated by the results in , this cryptic splice site is not activated in ‘tetraloop’ constructs. This is in line with our hypothesis that UUCG and CUUG stabilize the structure and as a consequence suppress the usage of the cryptic splice site.
As final demonstration of the role of RNA structure in SMN exon 7 splicing, we confirmed the existence of TSL2 by enzymatic structure probing. Our probing results were able to capture structural changes caused by mutations that either destabilized or reinstated TSL2. As expected, these mutations affected only the TSL2 region, while the remainder of exon 7 structure was unchanged. We also probed the structure of SMN2/36U37U39C40C54G, which was predicted to have a very strong stem. Consistently, this structure was not disrupted even at 50°C in 7 M urea. In agreement with the inhibitory effect of the strong stem, mutant U1 snRNA with increased base pairing to the 5′ ss failed to stimulate exon 7 inclusion in SMN2/36U37U39C40C54G (data not shown).
In comparison to other mammalian SMN
exon 7, regulation of human SMN
exon 7 splicing is distinct and unique (53
). Some of these differences arise in part due to presence of an additional codon UUA (codes for 293
Leu), which corresponds to positions 43, 44 and 45 of exon 7. Since 293
Leu is the second last aminoacid in human SMN, it is unlikely that SMN protein containing or lacking 293
Leu would have different functions. In fact, only the first eight (out of total 16) amino acids coded by exon 7, in particular QNQKE sequence, have been shown to be sufficient enough to restore SMN function (90
). The presence of remaining coding sequences may have been necessary to increase the exon size to facilitate its recognition. Furthermore, 293
Leu codon participates in constitution of both 3′-Cluster and TSL2, and may have contributed to human specific regulation of SMN
exon 7 splicing. Of note, deletion of the codon that precedes 293
Leu would also break TSL2 but showed no stimulatory effect on SMN2
exon 7 inclusion (53
). It is likely that this deletion created alternative structure and/or abrogated the Conserved tract, a stimulatory cis-element (53
). The conflicting effects of coding sequences on TSL2 formation reveal complexity of evolution in which only three positions (43rd, 44th and 45th) were available for UUA-codon insertion that produced the specific effect by constituting TSL2. The inhibitory effect of TSL2 must be viewed as complementary to other inhibitory cis-elements that participate in promoting SMN
exon 7 skipping. Abrogation or deletion of these elements leads to SMN2
exon 7 inclusion despite the presence of TSL2 (53
Pending further examination of additional factors that interact with 3′-Cluster and flanking inhibitory cis-elements, such as Exinct and ISS-N1, we can only infer that TSL2 takes advantage of a short 5′ ss:U1 duplex and facilitates SMN2 exon 7 skipping through a concerted effort of direct and indirect interactions that sequester the 5′ ss of exon 7. Since local RNA structures (or secondary structures) are formed instantaneously, TSL2 may provide the very first regulatory element aimed at weakening the 5′ ss of exon 7. To a larger significance, our results highlight TSL2 as a unique and essential component of the combinatorial control that modulates alternative splicing of SMN exon 7. Considering structure-specific compounds can be synthesized or selected from the available libraries, TSL2 provides an additional target for correcting the aberrant splicing of SMN2 exon 7 in SMA.
In summary, we have demonstrated that the 5′ ss of SMN exon 7 is sequestered in a stem–loop structure, TSL2. Disruption of TSL2 promoted exon 7 inclusion in SMN2 mRNA. Discovery of TSL2 as a regulatory element reveals for the first time the critical role of a RNA structure in alternative splicing of human SMN genes. It also provides a novel therapeutic target for correction of aberrant splicing of SMN2 in SMA.