Recent reports reveal critical roles for TIA1 and TIAR in genome-wide positive regulation of pre-mRNA splicing through specific interactions involving U-rich intronic motifs adjacent to the 5′ ss of exons (3
). The close proximity of intronic U-rich sequences to the 5′ ss allows TIA1 to facilitate 5′ ss recognition by recruitment of U1 snRNP through direct interactions (9
). Such a mechanism may exclude a role for TIA1 in different contexts in which U-rich intronic motifs are separated from the 5′ ss by intervening negative intronic cis
elements. Moreover, it is not known if the stimulatory role of TIA1 is at all applicable to contexts in which U-rich intronic motifs are located more than 30 nucleotides away from the 5′ ss. To explore the role of TIA1 in such a context, we took advantage of SMN2
exon 7, skipping of which is associated with SMA, a leading neurodegenerative disease. The intronic sequences immediately downstream of the 5′ ss of exon 7 are marked by the absence of U-rich motifs characteristic of TIA1 binding sites. In addition, the first 24 residues of intron 7 harbor overlapping inhibitory cis
) (Fig. ). Here, we report that TIA1 plays a prominent stimulatory role in SMN2
exon 7 splicing through novel intronic motifs located away from the 5′ ss.
FIG. 11. Model of TIA1-mediated splicing regulation of SMN2 exon 7 splicing. The 5′ portion of SMN intron 7 sequence containing various splicing cis elements is shown. Numbering of nucleotides starts from the first position of intron 7. Roles of intronic (more ...)
A significant effect of TIA1 on SMN2 exon 7 splicing regulation emerged from a series of complementary experiments that we performed in vivo. First, overexpression of recombinant TIA1 or its closely related analog, TIAR, led to a substantial increase in exon 7 inclusion in transcripts derived from the SMN2 minigene. A stimulatory effect of TIA1 was also observed on exon 7 splicing from endogenous SMN2. Supporting the result of TIA1 overexpression, SMN minigenes transfected in a TIA1 knockout mouse cell line (TIA1−/− cells) showed increased skipping of exon 7. The negative consequence of the absence of TIA1 was specific to human SMN as splicing of mouse Smn exon 7 appeared unaffected in TIA1−/− cells. Further validating the positive role of TIA1 and TIAR on SMN2 exon 7 splicing, depletion of these factors using an siRNA-based approach led to an increase in SMN2 exon 7 skipping.
To determine the role of a specific sequence(s) responsible for a TIA1-associated stimulatory effect on SMN2 exon 7 splicing, we adopted an open-ended approach in which we first scanned the entire intron 7 through large deletions. With our largest deletion (I7Δ190-406), which eliminated numerous URCs, including the longest U-rich motif comprised of eight U residues in the 3′-half of intron 7, the stimulatory effect of TIA1 was fully retained. These results ruled out the possibility that the TIA1 binding site is located in the 3′ half of intron 7. Thus, we focused on sequences in the first half of intron 7. Overlapping deletions in this region revealed URC1 (UCUUACUUUUGU) and URC2 (UUUAUGGUUUGU) as possible sites of TIA1 interaction. URC1 and URC2 fall within 29th and 57th positions of intron 7. Of note, simultaneous deletion of URC1 and URC2 led to a complete loss of the TIA1-associated stimulatory effect despite the fact that this deletion brought URC3 (UGUUUUUGAACAUUUA) closer to the 5′ ss. Complementing the results of deletion mutations, ASOs blocking URC1 and URC2 led to increased SMN2 exon 7 skipping, even in the presence of overexpressed TIA1. The results of ASO-based experiments were significant in ruling out the possibility of an indirect effect of inhibitory elements that may have been inadvertently created by deletion mutations. Further supporting the URC1/URC2-dependent stimulatory role of TIA1, overexpression of TIA1 led to an increase in exon inclusion in a heterologous context. Portability of URC1/URC2 in a heterologous context underscores that TIA1/TIAR may have an expanded role in splicing regulation through URC1/URC2-like motifs away from the 5′ ss.
To accurately define the TIA1 binding site, we used small overlapping deletions. Several of these short deletions that either fully or partially eliminated URC1 and/or URC2 reduced the stimulatory effect of TIA1. These results imply roles for both URC1 and URC2 in TIA1 binding. Complementing the results of deletion mutations, substitutions within URC1 and URC2 also led to a decrease in/loss of the stimulatory effect of TIA1. To confirm that in vivo splicing results of SMN2 minigenes containing deletion and substitution mutations are in concert with the loss of a direct interaction of TIA1 with URC1 and URC2, we performed in vitro binding using purified TIA1 and transcripts harboring identical mutations. The results of in vitro binding fully supported a direct interaction of TIA1 with URC1 and URC2. Consistently, substitutions within URC1 or URC2 led to a reduction in affinity of TIA1 for RNA. Results of in vitro binding also revealed that the interaction between URC1/URC2 and TIA1 is tighter than the interaction of TIA1 with a high-affinity TIA1 binder isolated using in vitro selection (Fig. ). These results underscore that very low levels of TIA1 in the nucleus should be able to modulate SMN2 exon 7 splicing.
Having determined the sequence-specific effect of TIA1 in a novel context, we next examined the impact of individual TIA1 domains on SMN2
exon 7 splicing. Our results revealed the essential role of the Q domain and confirmed that any RRM in combination with the Q domain is necessary and sufficient to stimulate SMN2
exon 7 splicing in vivo
. This is not totally unexpected since the RRMs of TIA1 share a high degree of homology (57
). Of note, a positive effect of a given TIA1 domain combination on pre-mRNA splicing in vivo
is dependent upon several limiting events, including a relatively moderate expression, an appreciable level of nuclear import, a reasonable affinity for the target, and the ability of this domain combination to favorably interact with components of spliceosome. Our finding that any RRM in combination with the Q domain is able to stimulate SMN2
exon 7 splicing is distinct from an in vitro
study showing a specific requirement for RRM1 in addition to the Q domain for the formation of a tight interaction with U1 snRNP (18
). The discrepancy between in vivo
and in vitro
results could be due to several factors, including a change in the context of the 5′ ss and coupling of pre-mRNA splicing with transcription and polyadenylation in vivo
. A previous localization study had suggested a requirement for RRM2 and RRM3 in nuclear import and export, respectively (68
). However, since the study did not employ a nuclear function in a sufficiently sensitive assay, the significance of reduced nuclear import or export of a particular domain combination could not be evaluated. Our finding that the lack of RRM2 has no adverse effect on SMN2
exon 7 splicing suggests that RRM2 might be dispensable for nuclear import. Our results argue that even a suboptimal level of nuclear import of a critical splicing factor may have a substantial impact on pre-mRNA splicing. In agreement with the results of different domain combinations of TIA1, overexpression of TIA1Δ5 and TIARΔ3 increased SMN2
exon 7 inclusion. These two proteins are the major spliced variants of TIA1 and TIAR, respectively. Based on our findings, we believe that even shorter and less frequent spliced variants of TIA1 and TIAR would be able to stimulate SMN2
exon 7 inclusion.
The essential role of the Q domain in SMN2 exon 7 splicing prompted us to examine this domain in further detail. Unlike the characteristic two RNP motifs within each RRM, the Q domain does not contain a signature motif. Twenty-one glutamine residues are randomly distributed within the Q domain. With a deletion of the last half of the Q domain, most of the splicing activity was retained, and some splicing activity was retained with deletion of the first half of the Q domain. These results suggest a cumulative effect in which a large number of glutamine residues in the first half of the Q domain contributed toward the maximum activity. Consistently, the first 19 amino acids of the Q domain contain the highest density of glutamine residues (~32%) as well as the longest Q-rich motif (QQQNQ), and this region was able to retain about one-third of the total activity. These results, however, do not rule out the possibility that amino acids other than glutamine residues in the Q domain might play a supportive role in the splicing-associated function of TIA1.
Our results support the bipartite nature of an interaction in which TIA1 contacts two motifs (URC1 and URC2) within a span of 29 nucleotides between the 29th and 57th positions of intron 7. Such an interaction is consistent with the structural studies in which the interface between RNA and an interacting RRM domain is generally occupied by less than seven nucleotides (11
). In the case of TIA1, the bipartite nature of interaction is possible through two RRMs of either the same or different TIA1 molecules. Our finding that a single RRM in combination with the Q domain is able to stimulate exon 7 inclusion suggests that URC1/URC2 is bound by a TIA1 dimer. In addition, the high affinity of TIA1 to URC1/URC2 fits well with the dimeric mode of interactions in which protein binding to RNA is stabilized by protein-protein interactions and vice versa.
Different mechanisms may account for the stimulation of SMN2
exon 7 inclusion by TIA1. The most plausible among those is the “prevention of negative recruitment.” According to this model, TIA1 interaction with URC1/URC2 prevents recruitment of negative factors, including hnRNP A1, upstream of the TIA1 binding site (Fig. ). This, in turn, would lead to enhanced recruitment of U1 snRNP due to a now accessible 5′ ss. This hypothesis is consistent with complementary findings in which ASO-mediated blocking of a GC-rich motif or ISS-N1 promotes SMN2
exon 7 inclusion (24
). In addition, our recent report revealed a long-distance negative interaction involving the 10th intronic position (53
). Binding of TIA1 to URC1/URC2 has a potential to prevent/abrogate this negative interaction through hindering and/or sterically impacting the accessibility of the 10th intronic position. The hypothesis of the prevention of negative recruitment does not exclude another possibility, i.e., that URC1/URC2-bound TIA1 directly recruits U1 snRNP through looping out sequences between the 5′ ss and the TIA1 binding site. This hypothesis is consistent with the ability of RRMs and the Q domain to participate in protein-protein interactions (11
Notable finding that TIA1 is able to counteract the inhibitory effect of PTB suggests additional mechanisms in which recruitment of TIA1 to URC1/URC2 brings dramatic changes in the context of pre-mRNA. PTB promotes exon skipping through different mechanisms (2
). The most common among them is the looping out of the skipped exon through binding to flanking intronic sequences (30
). A recent genome-wide analysis revealed subsequence UYUYU as the top scored consensus motif for PTB-RNA interactions in vivo
). Intronic sequences flanking SMN
exon 7 are replete with such sequences. Also, PTB has been shown to interact with element 1, which is located within intron 6 of SMN
). Element 1 harbors subsequence UUUUU as one of the top-scored PTB binding motifs. Consistent with the absence of a UYUYU motif, our functional study ruled out the presence of a PTB binding site within URC1/URC2, which is the site of interaction with TIA1. However, there are additional UYUYU motifs downstream of URC1/URC2. Interaction of PTB with element 1 within SMN
intron 6 and possible interaction within intron 7 may support the looping-out mechanism. Another mechanism of PTB-associated splicing regulation supports the formation of a zone of silencing around a skipped exon (60
). In addition to PTB, other inhibitory factors such as hnRNP A1 and Sam68 have been implicated in promotion of SMN2
exon 7 skipping. It is possible that PTB forms a zone of silencing around SMN2
exon 7 through interactions involving additional negative factors including, but not limited to, hnRNP A1 and Sam68. Our results suggest that the TIA1-induced contextual change within SMN
pre-mRNA is sufficient to prevent the inhibitory effect of PTB on SMN
exon 7 splicing.
SMN is a multifunctional protein with more than 20 reported interacting partners. Through regulation of snRNP biogenesis in the nucleus, SMN controls the rate of pre-mRNA splicing. Consistently, depletion of SMN leads to genome-wide perturbations of pre-mRNA splicing (70
). SMN is a component of stress granules, a dynamic cytoplasmic structure, formation of which requires TIA1 and hnRNP A1 (22
). hnRNP A1 is a negative regulator of SMN2
exon 7 splicing. Two out of four putative hnRNP A1 binding sites are located immediately upstream of the TIA1 binding site within intron 7. Our discovery of TIA1 as a regulator of SMN
exon 7 splicing provides a unique precedence in which two splicing factors (TIA1 and hnRNP A1) that cooperate in response to cellular stress play opposing roles in regulation of SMN2
exon 7 splicing through adjacent intronic motifs located in the vicinity of the 5′ ss. Thus far, studies on TIA1-assisted pre-mRNA splicing regulation have focused on U-rich motifs close to the 5′ ss. The finding that TIA1 could modulate exon 7 inclusion through U-rich intronic motifs separated from the 5′ ss by negative cis
elements brings new insight into our understanding of pre-mRNA splicing of a critical exon associated with SMA, a leading genetic disease of children and infants. Reduced levels of SMN in SMA primarily affect motor neurons that generally maintain high levels of SMN. It is also known that TIA1, TIAR, and PTB are abundantly expressed in the brain (5
). Hence, regulation SMN2
exon 7 splicing by TIA1/TIAR has significance in maintaining the high levels of SMN in the brain. Skipping of SMN2
exon 7 has been also associated with conditions of oxidative stress that causes Parkinson's and Alzheimer's disease (37
). Thus, our findings provide a broader role of TIA1/TIAR in alleviating the severity of a large number of diseases associated with low levels of SMN.