Polypyrimidine tract binding protein functions as a repressor to regulate alternative splicing of alpha-actinin mutally exclusive exons

Abstract

The smooth muscle (SM) and nonmuscle (NM) isoforms of alpha-actinin are produced by mutually exclusive splicing of an upstream NM exon and a downstream SM-specific exon. A rat alpha-actinin genomic clone encompassing the mutually exclusive exons was isolated and sequenced. The SM exon was found to utilize two branch points located 382 and 386 nucleotides (nt) upstream of the 3' splice site, while the NM exon used a single branch point 191 nt upstream. Mutually exclusive splicing arises from the proximity of the SM branch points to the NM 5' splice site, and this steric repression could be relieved in part by the insertion of spacer elements. In addition, the SM exon is repressed in non-SM cells and extracts. In vitro splicing of spacer-containing transcripts could be activated by (i) truncation of the transcript between the SM polypyrimidine tract and exon, (ii) addition of competitor RNAs containing the 3' end of the actinin intron or regulatory sequences from alpha-tropomyosin (TM), and (iii) depletion of the splicing extract by using biotinylated alpha-TM RNAs. A number of lines of evidence point to polypyrimidine tract binding protein (PTB) as the trans-acting factor responsible for repression. PTB was the only nuclear protein observed to cross-link to the actinin RNA, and the ability of various competitor RNAs to activate splicing correlated with their ability to bind PTB. Furthermore, repression of alpha-actinin splicing in the nuclear extracts depleted of PTB by using biotinylated RNA could be specifically restored by the addition of recombinant PTB. Thus, alpha-actinin mutually exclusive splicing is enforced by the unusual location of the SM branch point, while constitutive repression of the SM exon is conferred by regulatory elements between the branch point and 3' splice site and by PTB.

FIG. 1

Organization of the mutually exclusive exons of α-actinin and α-TM. Exons are shown as boxes, and introns are shown as lines. The sizes (in nucleotides) of the exons and introns are indicated. The α-TM exon 1 has a variable size according to the transcription start site used (72). The nonmuscle splicing pattern is shown above the gene, and the smooth muscle pattern is shown below. The relative arrangement of the mutually exclusive exons differs; in α-actinin, the SM-specific exon is the downstream exon of the pair, while in α-TM, it is the upstream exon. The C-terminal domain of α-actinin contains two EF hand calcium-binding motifs. In both the NM and SM α-actinin isoforms, the first part of the first EF hand is encoded by the EF1a exon, while the second part is encoded by the NM exon in the NM isoform and the SM exon in the SM isoform. Inclusion of the SM exon produces a nonfunctional Ca²⁺-binding domain. The second EF hand, which is common to the two isoforms, is encoded by the EF2 exon.

FIG. 2

Sequence of the rat α-actinin gene in the region encoding the two EF hand calcium-binding motifs. The exons are shown in uppercase, and the introns are in lowercase. The putative branch point adenosines utilized in splicing of the NM and SM exons are shown in boldface uppercase and underlined (nt 911 for the NM exon and nt 1237 and 1241 for the SM exon). The adjacent polypyrimidine tracts are shown in italics. Clusters of TGC motifs, similar to those of the URE and DRE of α-TM (19, 20), are shown in italic boldface (nt 701 to 725 and 1319 to 1335). Optimal PTB binding TCTT motifs (50) in the vicinity of the two alternative exons are underlined, and GCATG motifs (30) are shown in boldface.

FIG. 3

Increasing the distance between the 5′ splice site and the branch point and the length of truncation activate α-actinin NM-SM splicing. Full-length α-actinin transcripts (BamHI [B]) or transcripts truncated at the AflIII (A), NcoI (N), or EcoRI (E) site, with (+) or without (−) a 30-nt spacer between the NM 5′ splice site and putative branch point, were incubated with HeLa nuclear extract for 3 h, and the RNA species were resolved on an 8% polyacrylamide gel. The initial transcripts and splicing intermediates are indicated to the sides of the gel. The lariats for the BamHI and AflIII transcripts are not well resolved on gels with this percentage of polyacrylamide; the 5′ exon is the clearest diagnostic band for processing. The insertion of the spacer between the 5′ splice site and branch point activates splicing, particularly for transcripts truncated at the NcoI and EcoRI sites.

FIG. 4

Mapping the SM and NM branch points. (A) NcoI truncated α-actinin NM-SM transcripts, containing the spacer, were not processed or were spliced for the times indicated over the gels, followed by primer extension with an oligonucleotide complementary to nt 1267 to 1282. In addition, RNA from a 2-h splicing reaction was debranched (2D) prior to primer extension. The samples were run alongside a sequencing reaction with the same primer. The extended products arising from arrest of the RT at the branch points at A₁₂₃₇ and A₁₂₄₁ are indicated by the arrows, and the corresponding branch points in the sequence are underlined. Use of the minor branch point at nt 1241 is indicated by the increased proportion of the lower of the two bands that are present as background in the unprocessed RNA sample. The 1-h timepoint in this experiment was loaded incorrectly, resulting in the anomalously low signal. (B) Splicing of transcripts containing EF1a and NM exons and intron between these two exons. Transcripts were not processed or were incubated in HeLa nuclear extract for the times indicated. The upper gel has a lower percentage of polyacrylamide to facilitate resolution of the lariats. (C) Transcripts containing the EF1a and NM exons were not processed, spliced for 2 h, or spliced for 2 h and debranched (2D [lane 3]), followed by primer extension with an oligonucleotide complementary to nt 934 to 951. The extended product is indicated by an arrow, and the corresponding branch point at A₉₁₁ is underlined.

FIG. 5

Increasing the distance between the 5′ splice site and the branch point activates α-actinin NM-SM splicing in vivo. (A) HeLa cells were transiently transfected with α-actinin constructs. An α-actinin construct extending from the EF1a exon to the EF2 exon (lane 1), a construct containing a 30-bp spacer element between the NM 5′ splice site and SM branch point (lane 2), a construct containing an EcoRI-AflIII (E-A) deletion in the intron between the NM and SM exons (lane 3), or a construct with both the spacer element and deletion (lane 4). (B) PhosphorImager analysis was performed, the intensity of each of the three products was expressed as a percentage of the total signal, and the mean and standard deviation of the results of five experiments were calculated. Increasing the distance between the 5′ splice site and the branch point activates α-actinin NM-SM splicing in vivo.

FIG. 6

Derepression of the α-actinin SM exon by RNA competitors. Full-length α-actinin transcripts, containing the spacer, were not processed (lane 1) or were spliced for 3 h in the absence (−) (lane 2) or presence of a 10-, 25-, 50-, or 100-fold molar excess over the pre-mRNA (22.5, 56, 113, or 225 nM) of α-actinin competitor RNA (lanes 3 to 6, respectively); 10-, 25-, 50-, or 100-fold molar excess of α-TM DY competitor RNA (lanes 7 to 10); 50- or 100-fold molar excess of α-TM DYΔPC competitor RNA (lanes 11 and 12); or 50- or 100-fold molar excess of the α-actinin ClaI competitor RNA (lanes 13 and 14). The RNA species were resolved on a 12% polyacrylamide gel so that the lariats migrated above the pre-mRNA. α-Actinin NM-SM splicing was activated by both the α-actinin and α-TM DY competitors but not the truncated actinin or mutant TM competitors which lack UCUU motifs.

FIG. 7

UV cross-linking of HeLa extract proteins with α-actinin transcripts. Cross-linking reactions of α-actinin transcripts containing the spacer i.e., full-length (BamHI [B]), or truncated at the AflIII (A), NcoI (N), or EcoRI (E) site or at the ClaI (C) site in the spacer were done in HeLa nuclear extract (lanes 1 to 5). Cross-linking reactions of the full-length and AflIII and NcoI truncated transcripts were immunoprecipitated with anti-PTB antiserum (+) or preimmune serum (−) (lanes 6 to 11). PTB cross-links to α-actinin sequences, and this cross-link decreases with progressive truncations of the α-actinin intron.

FIG. 8

Depletion of PTB from HeLa nuclear extract activates α-actinin splicing. (A) PTB was depleted from HeLa nuclear extract by using biotinylated α-TM DY RNA bound to streptavidin magnetic beads. Western blot analysis with an anti-PTB antibody was performed on recombinant His-tagged PTB (His-PTB) (25 ng) (lane 1), serial dilutions of undepleted HeLa nuclear extract (NE) (0.25 to 2 μl) (lanes 2 to 6), equal protein loads (18 μg) of complete nuclear extract (NE) and depleted extract (DEP) (lanes 7 & 8), and on an equivalent volume of the proteins eluted from the beads (EP) (1 μl) (lane 9). The majority of the PTB was removed from the extract (80 to 90% as estimated by comparison with serial dilutions of undepleted extract). (B) Full-length α-actinin transcripts, containing the spacer, were not processed (lane 1) or were spliced for 3 h in the complete nuclear extract (NE) (lane 2), mock-depleted (MOCK DEP) extract (lane 3), complete extract in the presence of 240 nM of α-TM DY competitor RNA (lane 4) or PTB-depleted extract (−) (lane 5). The proteins eluted from the streptavidin beads (1 and 2 μl) were included in splicing reactions with the depleted extract (lanes 6 and 7, respectively), or recombinant His-tagged PTB (lanes 8 to 10), sxl (lanes 11 to 13), or unr (lanes 14 to 16) was added to the reaction mixtures to a final concentration of 86, 172, or 345 nM, respectively. α-Actinin splicing is activated in PTB-depleted HeLa nuclear extract. This activation can be reversed by the addition of rPTB but not by the RNA binding proteins sxl and unr. (C) β-Globin transcripts were not processed (lane 1) or were incubated with complete HeLa nuclear extract (NE) (lane 2), depleted extract (lane 3), or depleted extract with rPTB at a concentration of 86, 172, or 345 nM (lanes 4 to 6, respectively).