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Identification of splicing silencers and enhancers in sense Alus: a role for pseudoacceptors in splice site repression - PubMed

Identification of splicing silencers and enhancers in sense Alus: a role for pseudoacceptors in splice site repression

Haixin Lei et al. Mol Cell Biol. 2005 Aug.

Abstract

Auxiliary splicing signals in introns play an important role in splice site selection, but these elements are poorly understood. We show that a subset of serine/arginine (SR)-rich proteins activate a cryptic 3' splice site in a sense Alu repeat located in intron 4 of the human LST1 gene. Utilization of this cryptic splice site is controlled by juxtaposed Alu-derived splicing silencers and enhancers between closely linked short tandem repeats TNFd and TNFe. Systematic mutagenesis of these elements showed that AG dinucleotides that were not preceded by purine residues were critical for repressing exon inclusion of a chimeric splicing reporter. Since the splice acceptor-like sequences are present in excess in exonic splicing silencers, these signals may contribute to inhibition of a large number of pseudosites in primate genomes.

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Figures

FIG. 1.
FIG. 1.

SR-dependent activation of a cryptic 3′ splice site in LST1. (A) Schematic representation of the LST1 minigene (to scale; scale units are kilobases). Exons are shown as shadowed boxes; introns are shown as lines. TNFe and TNFd are indicated by small open boxes; segment A is denoted by a closed rectangle. Arrows indicate primers. For a full genomic sequence of this region, see Fig. S1 in the supplemental material. Naturally occurring LST1 isoforms arising by alternative 3′ ss and designated LST1/C, LST1/K (53), and LST1/O (this study) are shown above the minigene, whereas cryptic 3′ ss activation generating LST1/n is shown below. The location of the alternative 3′ ss of intron 4 was as described in reference (and see Fig. S1 in the supplemental material). (B) Cryptic 3′ ss activation in cells overexpressing SR proteins in constructs lacking TNFd. LST1 reporters are shown below each panel, and LST1 RNA products are shown to the right. LST1/K is a predominant isoform in peripheral blood mononuclear cells generated by alternative 3′ ss of intron 4 (53). WT, wild-type; NC and VO; no-cotransfection and vector-only controls, respec-tively. No-template controls are not shown. (C) SR-dependent upregulation of LST1/n and LST1/C. We used 0.5 μg of each reporter plasmid together with 0.1, 0.5, 1.0, and 2.0 μg of plasmids expressing SRp40. Hd, heteroduplexes. (D) Binding of ASF/SF2 to the pre-mRNA is essential for the LST1/n formation. d, deletion of ASF/SF2 domains RRM1, RRM2, and RS or a heptaglycine repeat (Gly7). LST1 isoforms described above are shown on the right side.

FIG. 2.
FIG. 2.

Identification of Alu-derived splicing signals that inhibit LST1/n. (A) Cryptic 3′ ss and segment A sequences are derived from Alu repeats. Sequence alignment was generated by the RepeatMasker (

http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker

). The cryptic 3′ ss (position 33/34 in the Alu consensus sequence) (2, 37) is boxed. Deleted segments A1 (positions 40 to 51 in the Alu consensus), A2 (positions 51 to 66), A3 (positions 66 to 77), and A4 (positions 78 to 96) are underlined. The telomeric end of TNFd is shown as a gray bar. Mismatches are indicated by the letter i (transition) or v (transversion). (B) Activation of cryptic 3′ ss upon removal of segment A3 or A2. The amount of plasmid expressing ASF/SF2 is in micrograms. Mutated constructs (bottom) are described in the text. d, deletion; >, replacement. PL refers to a polylinker sequence. Expression of LST1/C and LST1/O was negligible and is not shown. Reporter constructs containing the TNFd repeat that lacked segment A were not obtained because of a detection bias against RNA isoforms with this STR (see Fig. 1B and Discussion). (C) A3 and A2 in the sense orientation promote skipping of XPC exon 4. (Upper panel) Mutated minigenes transfected into 293T cells are shown at the top. F and R, forward (sense) and reverse (antisense) orientation, respectively. ES, exon skipping as a ratio of transcripts lacking exon 4 (E4−) to the sum of E4− and E4+ transcripts. SD, standard deviation as calculated from two transfection experiments. (Lower panel) ES following transfection of the truncated (T) XPC minigene carrying identical insertions.

FIG. 3.
FIG. 3.

Characterization of Alu-derived splicing silencer A3. (A) Exon inclusion levels following transfection of mutated A3 in XPC exon 4. Point mutations above each panel are numbered according to the A3 position as shown in panels B and C. ES, exon skipping as a ratio of transcripts lacking exon 4 (E4−) to the sum of E4− and E4+ transcripts. SD, standard deviation of two transfection experiments. (B) The influence of AG-AG distance in segment A3 on inclusion of a heterologous exon. (Left panel) Two-, 4-, and 6-nt insertions in the XPC-T-A3F construct. The XPC exon 4 sequences flanking A3 were GTGCTGGGTG (upstream) and ACGTGAGAGA (downstream). (Right panel) Exon inclusion levels from a single transfection experiment in duplicate. (C) Alignment of LST1 A3 segment and consensus sequences of Alu subfamilies. Alu sequences were derived from the left arm (2, 37), except for those denoted by -R (the right arm of Alu). The designation of Alu subfamilies was as described previously (2, 37). Mutations are shown in bold. (D) The influence of minigene mutations in A3 on exon inclusion. Constructs A to L shown at the top correspond to mutations listed in Fig. 3C.

FIG. 4.
FIG. 4.

Characterization of splicing silencer A2. (A) The influence of A2 mutations in four AGs on exon inclusion. The splicing reporter constructs are shown at the top. Guanine-to-cytosine mutations are in positions shown in panel B. ES, exon skipping as a ratio of transcripts lacking exon 4 (E4−) to the sum of E4− and E4+ transcripts. SD, standard deviation of two transfection experiments. (B) Alignment of LST1 segment A2 with consensus sequences of Alu subfamilies. Mutations (in bold) that corresponded to sequence variations in the subfamilies were introduced in segment A2 inserted in the XPC-T construct in the sense orientation. The designation of Alu subfamilies was as described previously (2, 37). (C) Influence of A2 variants representing Alu subfamilies on inclusion of XPC exon 4 in mRNA. Constructs A to M shown at the top correspond to mutations listed in Fig. 4B. A-R, constructs with segment A2 inserted in the antisense orientation.

FIG. 5.
FIG. 5.

Identification and characterization of Alu-derived splicing enhancer A4. (A) Deletion of A4 in TNFd-(AG)0 constructs eliminated the expression of SRp40- and ASF/SF2-induced LST1/n. We used 1 μg of plasmids expressing the two SR proteins and 0.5 μg of the reporter plasmid. NC, no-cotransfection controls. No-template controls are not shown. (B) Insertion of A4 into the TH minigene in both sense and antisense orientations resulted in full exon inclusion. The insertions were made in TH exon 12. ES, exon skipping as a ratio of transcripts lacking exon 12 (E12−) to the sum of E12− and E12+ transcripts. WT, wild type. SD, standard deviation as calculated from two transfection experiments. (C) Alignment of LST1 segment A4 with consensus sequences of Alu subfamilies. Mutations (in bold) that corresponded to sequence variations in the subfamilies were introduced in segment A4 inserted in the XPC-T construct in the sense orientation. The designation of Alu subfamilies was as described previously (2, 37). (D) Splicing of the XPC-T minigene containing the branchpoint A→G mutation could be rescued by A4F but not by A4 sequences representing most Alu subfamilies. BP-G, constructs containing the adenine-to-guanine mutation in the predicted branchpoint of XPC exon 4. Constructs A to H shown at the top correspond to mutations listed in panel C. A-R, construct with segment A4 inserted in the antisense orientation.

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