Alternative splicing and bioinformatic analysis of human U12-type introns

Abstract

U12-type introns exist, albeit rarely, in a variety of multicellular organisms. Splicing of U12 intron-containing precursor mRNAs takes place in the U12-type spliceosome that is distinct from the major U2-type spliceosome. Due to incompatibility of these two spliceosomes, alternative splicing involving a U12-type intron may give rise to a relatively complicated impact on gene expression. We studied alternative U12-type intron splicing in an attempt to gain more mechanistic insights. First, we characterized mutually exclusive exon selection of the human JNK2 gene, which involves an unusual intron possessing the U12-type 5' splice site and the U2-type 3' splice site. We demonstrated that the long and evolutionary conserved polypyrimidine tract of this hybrid intron provides important signals for inclusion of its downstream alternative exon. In addition, we examined the effects of single nucleotide polymorphisms in the human WDFY1 U12-type intron on pre-mRNA splicing. These results provide mechanistic implications on splice-site selection of U12-type intron splicing. We finally discuss the potential effects of splicing of a U12-type intron with genetic defects or within a set of genes encoding RNA processing factors on global gene expression.

Figure 1.

The role of the U2–U12 hybrid intron in alternative splicing of human JNK2 pre-mRNA. (A) Schematic diagram shows exons 5 to 7 of JNK2. Between alternative exon 6a and 6b is the U2–U12 hybrid intron (heavy line); mutually exclusive selection of either exon generates mRNA isoforms. The numbers indicate the exon or intron length in nucleotides. U2 and U12 splice sites are indicated by hatched and filled boxes, respectively. The triangle indicates an EcoRV restriction site in exon 6b. Below the diagram is the sequence of the Py tract of the hybrid intron. (B) The minigene contains exons 5 to 8 of human JNK2, in which all introns except for the hybrid one are internally truncated (length indicated by the numbers). Transcription of the minigenes is driven by the CMV promoter. Translation start codon was in frame introduced in exon 5. Within the hybrid intron, the vertical line and grey boxes indicate the site for exon 6a/b swap and CU-rich sequences, respectively. The E6a and E6b control PCR products were amplified from the minigene, whereas the E6a and E6b transcripts represent the RT-PCR products that were amplified from the JNK transcripts. EcoRV digestion of the exon 6b-containing DNA fragment (472 bp) yielded two bands (294 and 178 bp). The modified minigenes are also depicted; the numbers of total and C/U residues of their polypyrimidine (Py) tract are listed. (C) Analysis of the JNK transcripts of four cell lines. The first four lanes show the E6a and E6b control fragments with or without EcoRV digestion. RT-PCR DNA fragments amplified from the endogenous JNK2 transcripts were of 316 bp; EcoRV digestion of the exon 6b-containing product generates two nearly comigrating fragments (cut) of 171 and 145 bp. Percentage of E6a-containing transcripts [cut/(cut + uncut) × 100%] in four cell lines was measured from three independent experiments; average with standard deviation is indicated. (D) HEK 293 cells were transfected with a minigene reporter as indicted. Total RNA was collected 24-h post-transfection and analyzed as in panel B. Shown on the gel are uncut PCR products (634 bp) and the larger digested fragment (463 bp). (E) The graph represents absolute sequence complexity (Y-axis) of a JNK2 genomic segment containing the alternative exon 6a/b and the hybrid intron (X-axis) from eight vertebrates (human, chimp, dog, cow, rat, mouse, opossum and chicken). Conservation of the Py tracts is indicated as percentage. (F) Model shows that mutually exclusive exon (hatched and squared boxes) selection of the JNK2 gene is driven by the U2–U12 intron (heavy line). The Py tract (grey box) of the hybrid intron dominates over that upstream of exon 6a, thus leading to exon 6b inclusion as the default pathway, particularly in non-neuronal cells. In neuronal cells, specific splicing activators, such as Nova, induce the use of exon 6a (21,22). On the other hand, since several YCAY elements (vertical lines) exist in the Py tract of the hybrid intron, YCAY-binding factors (such as Nova) may antagonize the activity of the ubiquitous CU-rich element-binding factors to reduce the utilization of exon 6b.

Figure 2.

Effects of single nucleotide polymorphisms of the human WDFY1 U12-type intron. (A) Schematic diagram shows exons 4 to 7 of the human WDFY1 gene. Nucleotide variations were found at residue 6 (+6) of the 5′SS consensus sequence and residue 14 (−14) upstream of the branch site. The minigenes used for the splicing assay contain a single mutation at either the 5′ + 6 or the B-4 position or dual mutations (5′Bdm) at both sites. The numbers indicate the exon or intron length in nucleotides. (B) The minigene is composed of exons 4 to 7 with internally truncated introns (length indicated by the numbers). Translation start and stop codons are in frame at the 5′ end of exon 4 and 3′ end of exon 7, respectively. The use of the cryptic splice sites b1, b2, d, c yielded aberrant transcripts (see panel C). (C) HEK 293 cells were transfected with a minigene reporter as indicted. Total RNA was collected 24-h post-transfection and subjected to RT-PCR analysis using ³²P-labeled primers. The identity of the cDNA products is shown at the right; hatched boxes represent truncated exons. The bar graph shows the relative abundance of the major splicing products as a percentage; the data were obtained from two to three independent experiments. The ∼330-bp band (asterisk) included a variety of aberrantly spliced JNK products, none of which expressed dominantly, and therefore was tentatively ignored in the graph. (D) A U12 snRNA expression vector was co-transfected with the wild-type or B-4CA WDFY1 minigene into HEK 293 cells. Splicing of the WDFY1 transcripts was examined as in panel C. The bar graph is as in panel C. (E) Model shows alternative splicing of the WDFY1 U12-type intron induced by genetic mutations. A 5′SS mutation activates cryptic U2-type 5′SSs in exon 5 (upper panel). A branch-site consensus mutation promotes the use of cryptic 5′SSs in exon 5 by the U2-type spliceosome or cryptic 3′SSs in exon 6 by the U2-type spliceosome (middle panel). Bottom: A double mutant is spliced only by the U2-type spliceosome, yielding aberrant mRNA products (lower panel).

Figure 3.

U12-type introns present in splicing factor genes may impact on gene expression. When the level or activity of a splicing factor encoded by a U12-type intron-containing gene is altered due to defective U12-type intron splicing (grey lines), aberrant splicing of its target pre-mRNAs may thus be observed.