The human CYP2C locus: a prototype for intergenic and exon repetition splicing events

In human there are four known CYP2C genes that have been mapped to chromosome 10q24 with the order Cen-2C18-2C19-2C9-2C8-Tel. Previously we have shown that splicing events joining exons from the neighboring 2C18 and 2C19 genes occur in human liver and epidermis. Here evidence is presented that the terminal genes of this cluster, 2C18 and 2C8, are also involved in intergenic splicing. Most interestingly, several of these 2C18/2C8 RNAs were composed of all nine exons, thus conceivably having the potential for coding functional proteins. Moreover, chimeric RNA species consisting of exons originating not only from the CYP2C8 and CYP2C18 genes, but also from the CYP2C19 gene were detected. In all cases the exons from the different CYP2C genes were joined at the correct canonical splice sites. However, the closely linked RBP4 gene is not participating in intergenic splicing with the CYP2C genes. In addition, CYP2C8 gene expression was found to generate a variety of scrambled RNA molecules including species that contained repetitions of certain exons.     

The Human CYP2C Locus: A Prototype for Intergenic and Exon Repetition Splicing Events

In human there are four known CYP2C genes that have been mapped to chromosome 10q24 with the order Cen–2C18–2C19–2C9–2C8–Tel. Previously we have shown that splicing events joining exons from the neighboring 2C18 and 2C19 genes occur in human liver and epidermis. Here evidence is presented that the terminal genes of this cluster, 2C18 and 2C8, are also involved in intergenic splicing. Most interestingly, several of these 2C18/2C8 RNAs were composed of all nine exons, thus conceivably having the potential for coding functional proteins. Moreover, chimeric RNA species consisting of exons originating not only from the CYP2C8 and CYP2C18 genes, but also from the CYP2C19 gene were detected. In all cases the exons from the different CYP2C genes were joined at the correct canonical splice sites. However, the closely linked RBP4 gene is not participating in intergenic splicing with the CYP2C genes. In addition, CYP2C8 gene expression was found to generate a variety of scrambled RNA molecules including species that contained repetitions of certain exons.     

Intergenic transcripts containing a novel human cytochrome P450 2C exon 1 spliced to sequences from the CYP2C9 gene

The cytochrome P450 2C (CYP2C) gene locus was found to includea novel exon 1 sequence with high similarity to the canonicalexon 1 of CYP2C18. Rapid amplification of cDNA ends (RACE) andPCR amplifications of human liver cDNA revealed the presenceof several intergenic species containing the CYP2C18 exon 1–likesequence spliced to different combinations of exonic and intronicsequences from the CYP2C9 gene. One splice variant was foundto have an open reading frame starting at the canonical translationinitiation codon of the CYP2C18 exon 1–like sequence.Another variant consisted of the nine typical CYP2C9 exons splicedafter the CYP2C18 exon 1–like sequence through a segmentof CYP2C9 5' flanking sequences. Moreover, analysis of bacterialartificial chromosome (BAC) clones revealed that the CYP2C18exon 1–like sequence was located in the intergenic regionbetween the CYP2C19 and CYP2C9 genes. The finding that a solitaryexon is spliced with sequences from a neighboring gene may beinterpreted as representing a general evolutionary mechanismaimed at using the full expression potential of a cell's genomicinformational content.     

Trans splicing of mRNA precursors in vitro

Two exon segments from two separate RNA molecules can be joined in a trans splicing process. In trans splicing reactions, an RNA molecule containing an exon, a 5' splice site, and adjacent intron sequences was mixed with an RNA molecule containing an exon, a 3' splice site, and adjacent intron sequences. The efficiency of trans splicing of these two RNAs increased if the two termini of the intervening sequences were paired in a short RNA duplex. However, trans splicing of two RNA molecules with no significant complementarity was also observed. These results strongly suggest that significant secondary structures within intervening sequences could affect the splicing of flanking exons. Similarly, RNAs that are complementary to segments within the intervening sequences could potentially regulate the selection of splice sites. Finally, some organisms might use trans splicing to distribute a single exon to many different mRNAs.     

Muscleblind-like 1 knockout mice reveal novel splicing defects in the myotonic dystrophy brain

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by a CTG trinucleotide repeat expansion (CTG(exp)) in the DMPK gene. In skeletal muscle, nuclear sequestration of the alternative splicing factor muscleblind-like 1 (MBNL1) explains the majority of the alternative splicing defects observed in the HSA(LR) transgenic mouse model which expresses a pathogenic range CTG(exp). In the present study, we addressed the possibility that MBNL1 sequestration by CUG(exp) RNA also contributes to splicing defects in the mammalian brain. We examined RNA from the brains of homozygous Mbnl1(ΔE3/ΔE3) knockout mice using splicing-sensitive microarrays. We used RT-PCR to validate a subset of alternative cassette exons identified by microarray analysis with brain tissues from Mbnl1(ΔE3/ΔE3) knockout mice and post-mortem DM1 patients. Surprisingly, splicing-sensitive microarray analysis of Mbnl1(ΔE3/ΔE3) brains yielded only 14 candidates for mis-spliced exons. While we confirmed that several of these splicing events are perturbed in both Mbnl1 knockout and DM1 brains, the extent of splicing mis-regulation in the mouse model was significantly less than observed in DM1. Additionally, several alternative exons, including Grin1 exon 4, App exon 7 and Mapt exons 3 and 9, which have previously been reported to be aberrantly spliced in human DM1 brain, were spliced normally in the Mbnl1 knockout brain. The sequestration of MBNL1 by CUG(exp) RNA results in some of the aberrant splicing events in the DM1 brain. However, we conclude that other factors, possibly other MBNL proteins, likely contribute to splicing mis-regulation in the DM1 brain.     

Differential alternative splicing activity of isoforms of polypyrimidine tract binding protein (PTB).

Polypyrimidine tract binding protein (PTB) is an RNA-binding protein that regulates splicing by repressing specific splicing events. It also has roles in 3'-end processing, internal initiation of translation, and RNA localization. PTB exists in three alternatively spliced isoforms, PTB1, PTB2, and PTB4, which differ by the insertion of 19 or 26 amino acids, respectively, between the second and third RNA recognition motif domains. Here we show that the PTB isoforms have distinct activities upon alpha-tropomyosin (TM) alternative splicing. PTB1 reduced the repression of TM exon 3 in transfected smooth muscle cells, whereas PTB4 enhanced TM exon 3 skipping in vivo and in vitro. PTB2 had an intermediate effect. The PTB4 > PTB2 > PTB1 repressive hierarchy was observed in all in vivo and in vitro assays with TM, but the isoforms were equally active in inducing skipping of alpha-actinin exons and showed the opposite hierarchy of activity when tested for activation of IRES-driven translation. These findings establish that the ratio of PTB isoforms could form part of a cellular code that in turn controls the splicing of various other pre-mRNAs.