TIA1 prevents skipping of a critical exon associated with spinal muscular atrophy

Abstract

Prevention of skipping of exon 7 during pre-mRNA splicing of Survival Motor Neuron 2 (SMN2) holds the promise for cure of spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. Here, we report T-cell-restricted intracellular antigen 1 (TIA1) and TIA1-related (TIAR) proteins as intron-associated positive regulators of SMN2 exon 7 splicing. We show that TIA1/TIAR stimulate exon recognition in an entirely novel context in which intronic U-rich motifs are separated from the 5' splice site by overlapping inhibitory elements. TIA1 and TIAR are modular proteins with three N-terminal RNA recognition motifs (RRMs) and a C-terminal glutamine-rich (Q-rich) domain. Our results reveal that any one RRM in combination with a Q domain is necessary and sufficient for TIA1-associated regulation of SMN2 exon 7 splicing in vivo. We also show that increased expression of TIA1 counteracts the inhibitory effect of polypyrimidine tract binding protein, a ubiquitously expressed factor recently implicated in regulation of SMN exon 7 splicing. Our findings expand the scope of TIA1/TIAR in genome-wide regulation of alternative splicing under normal and pathological conditions.

FIG. 1.

Effect of TIA1 on SMN exon 7 splicing. (A) Schematic diagram of the SMN gene. Exons are shown as gray boxes and introns are shown as broken lines. Sizes of exons and introns are indicated. Locations of the start and stop codons as well as the SMN2-specific DdeI restriction site are indicated by bold vertical arrows. The annealing positions of primers used for amplifications of endogenous SMN transcripts are shown by horizontal arrows. (B) In vivo splicing pattern of the SMN2 minigene in the presence of different nuclear proteins from the TIA1 family. A schematic diagram of the SMN minigenes is shown in the left panel. SMN exons are represented by gray boxes, and intervening introns are shown by broken lines. Sizes of exons and introns are indicated. Of note, intron 6 has been shortened to improve transfection efficiency (49). Annealing positions of primers for amplification of minigene-derived transcripts are shown. Results of in vivo splicing of the SMN2 minigene are shown in the right panel. Data were generated employing HeLa cells grown in 24-well plates. Cells were cotransfected with 0.04 μg of SMN2 and 0.46 μg of a 3XFLAG protein expression vector. Total RNA for the splicing assay was prepared from cells harvested 24 h posttransfection. Exon 7-included (+) and exon 7-skipped (−) spliced products are indicated. Results were analyzed as described previously (49). (C) In vivo splicing pattern of different minigenes in the presence of the overexpressed human full-length TIA1. HeLa cells plated in 24-well plates were cotransfected with 0.04 μg of a given minigene and 0.46 μg of either pCI-neo (empty vector) or 3XFLAG-hTIA1, and total RNA was prepared 24 h later. In every lane, upper and lower bands represent exon-included and exon-skipped products, respectively. In CFTR, ApoA-II, Casp3Avr, and Casp3ISS-N1, the major bands represent the exon-included products while in Casp3-ex7-ISS-N1 the single band represents an exon-skipped product. Numbers on the left and right are sizes in base pairs. The percentage of exon skipping was calculated from the total value of exon-skipped and exon-included products. (D) Effect of TIA1 overexpression on endogenous SMN exon 7 splicing. HeLa cells plated in 100-mm dishes were transfected with increasing amounts of 3XFLAG-hTIA1 plasmid (2.0, 4.5, 9.0, and 18 μg). After 24 h cells were collected for total RNA preparation/protein fractionation. Splicing patterns of endogenous SMN1 and SMN2 are shown in the left panel. Spliced products were analyzed by RT-PCR using primers located in exon 6 (N-24 in panel A) and exon 8 (P25 in panel A). Following RT-PCR, DdeI digestion was performed to distinguish spliced products from SMN2 (35). SMN1 and SMN2 spliced products with exon 7 included or skipped are indicated. The percentage of SMN2 exon 7 skipping was calculated from the total value of SMN2 exon-skipped and exon-included products. Control refers to untransfected cells. The results of Western blotting are shown in the right panel. Nuclear and cytoplasmic fractions are marked. A total of 15 μg of protein was used for nuclear and cytoplasmic samples. Primary antibodies used for probing are indicated on the left. Bands corresponding to FLAG-tagged (*) and endogenous (**) TIA1 are marked on the right. TATA binding protein (TBP) served as a nuclear marker, whereas GAPDH served as a cytoplasmic marker. Cytoplasmic fraction from untransfected cells was used as a negative and positive control for nuclear and cytoplasmic markers, respectively.

FIG. 2.

Effect of TIA1 knockout on splicing of SMN exon 7. (A) Western blot showing levels of TIA1 and its homolog TIAR in wild-type and in TIA1^−/− knockout mouse embryonic fibroblasts. Cellular proteins were fractionated, and 15 μg of protein of the nuclear/cytoplasmic fractions was used for SDS-PAGE. Primary antibodies used for probing are indicated on the left. GAPDH and histone 3 (H3) were used as markers for cytoplasmic and nuclear fractions, respectively. (B) Splicing pattern of mouse endogenous Smn exon 7 in TIA1^−/− knockout mouse embryonic fibroblasts. Total RNA was prepared from wild-type or TIA1^−/− knockout mouse embryonic fibroblasts, DNase treated and used for RT-PCR. One microgram of total RNA was used per 20 μl of reverse transcriptase reaction mixture. For the PCR step, mouse-specific primers annealing to exons 6 and 8 were employed. PCR amplification was done for 22 cycles; PCR products were ethanol precipitated and resolved on a native 5% PAGE gel. (C) In vivo splicing patterns of SMN1 and SMN2 minigenes in mouse embryonic fibroblasts. Mouse embryonic fibroblasts (wild type or TIA1 knockout) were transfected with 1 μg of SMN1 or SMN2 minigene and collected for total RNA preparation 24 h later. Spliced products were analyzed by RT-PCR as described in the legend of Fig. 1B. (D) Comparison of introns 6 and 7 of human (SMN) and mouse (Smn) genes. Exons are shown as colored boxes, and introns are shown as broken lines. Sizes of introns are given. ¹⁰C, a human intronic nucleotide involved in long-distance interactions, is shown (53). (E) Alignment of the 5′ end of human and mouse intron 7. Numbering starts from the beginning of the intron. Nucleotides identical between human and mouse are highlighted in gray. Nonconserved mouse residues are shown in lowercase letters. The 10th intronic position is circled. ISS-N1, GC-rich sequence, and ¹⁰C are specific to humans (48, 52, 53).

FIG. 3.

Effect of depletion of TIA1 and other stimulatory proteins on SMN exon 7 splicing. (A) Western blot showing the effect of the indicated siRNAs on the level of corresponding proteins. HeLa cells were cotransfected with 1 μg of the SMN2 minigene, and either 100 nM individual siRNA or all siRNAs simultaneously (25 nM each); whole-cell lysates were prepared 48 h after transfection. Primary antibodies used for probing are indicated on the left. (B) Time course of siRNA effect on splicing of SMN2 minigene-derived exon 7. Cotransfections were done as described in panel A. Spliced products were analyzed by RT-PCR using minigene-specific primers, and total RNA was isolated from cells collected 24, 48, and 72 h after transfection. (C) Time course of siRNA effect on splicing of endogenous SMN exon 7. Cotransfections were done as described in panel A. Spliced products were analyzed by RT-PCR as described in panel B except that different primers, one located in exon 5 (N-23) and the other in exon 8 (P25), were used. Following RT-PCR, DdeI digestion was performed to distinguish spliced products from SMN2 (35). The percentage of SMN2 exon 7 skipping was calculated as in Fig. 1D.

FIG. 4.

Identification of intronic sequences responsible for TIA1-associated stimulatory effect on SMN2 exon 7 splicing. (A) Effect of deletions in intron 7 on the ability of TIA1 to promote inclusion of SMN2 exon 7. Diagrammatic representation of deletions within intron 7 of the SMN2 minigene is given. Numbering of nucleotides starts from the first position of intron 7. Deletions are represented by dotted lines. Names of mutants are given on the left. Numbers in the names represent positions of the first and the last deleted nucleotides. In vivo splicing patterns of the wild-type SMN2 minigene and intron 7 deletion mutants in the absence and presence of overexpressed TIA1 are shown in the lower panel. HeLa cells grown in 24-well plates were cotransfected with 0.04 μg of a given minigene and 0.118 μg of either empty vector or 3XFLAG-hTIA1. This amount of 3XFLAG-hTIA1 plasmid is similar to the lowest concentration used in the experiment shown in Fig. 1D downscaled to a 24-well plate. Results were analyzed as described in the legend of Fig. 1B. (B) Splicing pattern of the SMN2 minigene cotransfected with different ASOs targeting sequences between ISS-N1 and element 2. Diagrammatic representation of ASOs and their annealing positions are given. Numbering of nucleotides starts from the first position of intron 7. ISS-N1 (gray) and element 2 (black) sequences are demarcated. ASOs are shown as horizontal bars. The lower panel shows the in vivo splicing pattern of the SMN2 minigene in the presence of different ASOs with (+) or without (−) overexpressed TIA1. Experiments were performed with HeLa cells cotransfected with 0.04 μg of minigene, a 100 nM concentration of a given ASO, and 0.2 μg of either an empty vector or 3XFLAG-hTIA1. Results were analyzed as described in the legend of Fig. 1B.

FIG. 5.

Role of URC1 and URC2 on the TIA1-associated stimulatory effect on SMN2 exon 7 splicing. (A) Diagrammatic representation of mutations within intron 7 of the SMN2 minigene. Numbering of nucleotides starts from the first position of intron 7. Deletions are represented by dotted lines. Site-specific mutations are highlighted in black. Names of mutants are given on the left. Numbers in the names of deletion mutants represent positions of the first and the last deleted nucleotides. ISS-N1, element 2, and URCs are highlighted in red, green, and chartreuse yellow, respectively. (B) In vivo splicing pattern of the SMN2 mutants shown in panel A. Cotransfection and splicing analysis were done similar to the method described in the legend of Fig. 4A. The bar diagram shown in the bottom panel represents residual exon 7 skipping for different mutants in the presence of 3XFLAG-hTIA1. Results in the bar diagram are expressed as per the following equation: 100 × [(exon 7 skipped in the presence of TIA1)/(exon 7 skipped in the absence of TIA1)]. Error bars based on standard deviation have been indicated. Standard deviation was determined based on three independent experiments performed in duplicates.

FIG. 6.

Portability of TIA1-associated stimulatory response of URC1/URC2 in a heterologous context. (A) Diagrammatic representation of the Casp3SMN2 hybrid minigene and its mutants. Casp3 and SMN2 sequences in the minigene are highlighted in dark and light red, respectively. Numbering of nucleotides starts from the first position of SMN2 intron 7. ISS-N1, element 2, and URCs are highlighted in red, green, and yellow, respectively. Deletions are represented by dotted lines. Numbers in the names of the hybrid minigenes represent positions of the first and the last deleted nucleotides. ASO 35Dn15 is indicated by a blue horizontal bar, and its annealing position is shown. (B) In vivo splicing pattern of hybrid minigenes shown in panel A in the presence of overexpressed TIA1. HeLa cells grown in 24-well plates were cotransfected with 0.04 μg of a given hybrid minigene and 0.36 μg of either an empty vector or 3XFLAG-hTIA1. Splicing analysis was done similarly as described in the legend of Fig. 4A. The bar diagram shown in the bottom panel represents residual exon 7 skipping for different mutants in the presence of 3XFLAG-hTIA1. Results in the bar diagram were expressed as described in the legend of Fig. 5B. Error bars based on standard deviation have been indicated. (C) In vivo splicing patterns of Casp3SMN2 and Casp3HYBΔ29-41 in the presence of TIA1 and a URC2-targeting ASO. HeLa cells grown in 24-well plates were cotransfected with 0.04 μg of minigene, a 100 nM concentration of a given ASO, and 0.36 μg of either an empty vector or 3XFLAG-hTIA1. Results were analyzed as described in the legend of Fig. 1B. Error bars were generated from standard deviations similar to the method described in the legend of Fig. 5B.

FIG. 7.

In vitro binding of purified TIA1 with RNA substrates harboring URC1 and/or URC2. (A) Diagrammatic representation of RNA transcripts used for in vitro binding. Deletions in transcripts are represented by dotted lines. Site-specific mutations are highlighted in black. Names of transcripts are given on the left, and their lengths are shown on the right. URC1, URC2, and URC3 are highlighted in gray. Sequence 1-1 (seq 1-1) is a high-affinity ligand reported earlier (16). (B) Relative affinity of TIA1 for different RNA substrates described in panel A. Employing a nitrocellulose filter assay, binding strengths of different RNA substrates were compared with either wild-type sequence (upper panel) or sequence 1-1 (lower panel). Results were expressed by computing the fraction of input RNA substrate bound to the membrane against a common competing RNA (55). The bound molar ratio of each mutant was compared with the wild-type construct, the value for which was assigned as 1.

FIG. 8.

Effect of different domains of TIA1 on SMN2 exon 7 splicing. (A) Diagrammatic representation of the structure of full-length TIA1 and its derivative constructs. RRMs and the Q-rich domain are indicated. Numbers represent amino acid positions. Numbers in the full-length TIA1 were assigned based on Gilks et al. and Tian et al. (20, 57). (B) In vivo splicing pattern of the SMN2 minigene in the presence of different TIA1 variants shown in panel A. All expressed TIA1 variants carried 3XFLAG tags at their N termini. HeLa cells were cotransfected with 0.04 μg of SMN2 and either an empty vector or a TIA1 variant of interest. Amounts of expression vectors were adjusted to produce comparable levels of protein expression (see panel C) and varied from 0.4 μg to 1.96 μg. The total amount of DNA used per transfection was maintained constant by adding an empty vector when needed. Cells were collected 24 h after transfection. Results were analyzed as described in the legend of Fig. 1B. (C) Western blot confirming expression levels of TIA1 variants shown in panel A. Twenty micrograms of total protein was used for each sample. Equal protein loading was confirmed by reprobing the blot for α-tubulin. Primary antibodies used for probing are indicated on the left.

FIG. 9.

Effect of Q-rich domain shortening on SMN2 exon 7 splicing. (A) Diagrammatic representation of deletions/substitutions in the Q-rich domain of TIA1. RRMs and the Q-rich domain are indicated. Numbers represent amino acid positions. For the purposes of comparison, amino acid sequences of different deleted variants of the Q domain are given in the lower panel. Red letters represent C-terminal residues of RRM3. Black and blue letters denote the first 54 residues of the Q domain and the last 43 residues of TIA1, respectively. Green letters represent unrelated amino acid sequences added to the C terminus of a truncated TIA1 lacking the last 87 residues. Glutamine residues of TIA1 are shown in white letters and highlighted in black. (B) In vivo splicing pattern of the SMN2 minigene in the presence of TIA1 proteins shown in panel A. All expressed TIA1 variants carried 3XFLAG tags at their N termini. HeLa cells were cotransfected with 0.04 μg of SMN2 and either an empty vector or the TIA1 protein expression vector of interest. The amounts of TIA1 expression vectors were adjusted to produce comparable levels of protein expression (Western blot panel) and varied from 0.4 μg to 0.96 μg. The total amount of DNA used per transfection was maintained constant by the addition of empty vector when needed. Cells were collected for analysis 24 h after transfection as described in the legend of Fig. 8B and C.

FIG. 10.

(A) In vivo splicing pattern of the SMN2 minigene in the presence of overexpressed PTB1 and TIA1. HeLa cells grown in six-well plates were cotransfected with 0.04 μg of SMN2 (lanes 1 to 8), 0.3 μg of myc-PTB expression vector (lanes 2 to 5), and various amounts of 3XFLAG-hTIA1 (0. 36 μg in lanes 3 and 6; 0. 73 μg in lanes 4 and 7; and 1.46 μg in lanes 5 and 8). Lane 9 served as an untransfected control. The total amount of DNA used per transfection was maintained constant by the addition of empty vector when needed. Cells were collected 24 h after transfection. Results were analyzed as described in the legend of Fig. 1B. (B) Effect of overexpressed PTB and TIA1 on splicing of endogenous SMN exon 7. Cotransfections were done as described in panel A. Spliced products were analyzed by RT-PCR as described in panel A, except that different primers, one located in exon 6 (N-24) and the other in the portion of exon 8 that is absent in the minigene (P26), were used. Following RT-PCR, DdeI digestion was performed to distinguish spliced products from SMN2 (35). The percentage of SMN2 exon 7 skipping was calculated as described in the legend of Fig. 1D. (C) Western blot showing the levels of endogenous and recombinant myc-tagged PTB as well as endogenous and recombinant FLAG-tagged TIA1. Twenty micrograms of total protein was used for each sample. Equal protein loading was confirmed by reprobing the blot for β-actin. Primary antibodies used for probing are indicated on the left. Bands corresponding to FLAG-tagged (*) and endogenous (**) TIA1 are marked on the right. (D) Effect of URC1 and URC2 deletions on the ability of PTB to promote SMN2 exon 7 skipping. HeLa cells grown in 24-well plates were cotransfected with 0.04 μg of a given minigene and 0.30 μg of either empty vector or myc-PTB. Mutants are described the legend of Fig. 5A. Total RNA was isolated 24 h posttransfection. Results were analyzed as described in the legend of Fig. 1B.

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 cis elements ISS-N1, GC-rich, element 2, and hnRNP A1 and PTB motifs have been described previously (4, 24, 41, 48, 52). Plus and minus signs indicate that a given splicing factor promotes exon 7 inclusion and skipping, respectively. The nature of the stimulatory factor(s) (represented by “?”) interacting with element 2 is not known. Binding of TIA1 to URC1/URC2 brings a change in the context leading to recruitment of U1 snRNP at the 5′ ss of exon 7. Also, a TIA1-associated change in the context creates an unfavorable condition for the recruitment of negative splicing factors including hnRNP A1 and PTB.