Circular RNAs from transcripts of the rat cytochrome P450 2C24 gene: correlation with exon skipping.

The cytochrome P450 2C24 gene is characterized by the capability to generate, in rat kidney, a transcript containing exons 2 and 4 spliced at correct sites but having the donor site of exon 4 directly joined to the acceptor site of exon 2 (exon scrambling). By reverse transcriptase-PCR analysis, it is now shown that the only exons present in the scrambled transcript are exons 2, 3, and 4 and that this molecule lacks a poly(A)+ tail. Furthermore, the use of PCR primers in both orientations of either exon 2 or exon 4 revealed that the orders of the exons in the scrambled transcript are 2-3-4-2 and 4-2-3-4, respectively. These results, combined with the observation that P450 2C24 is a single-copy gene, with no duplication of the exon 2 to exon 4 segment, suggest that the scrambled transcript has properties consistent with that of a circular molecule. In line with this is the observation of an increased resistance of the transcript to phosphodiesterase I, a 3'-exonuclease. Moreover, an alternatively processed cytochrome P450 2C24 mRNA, lacking the three scrambled exons and having exon 1 directly joined to exon 5, has been identified in kidney and liver, tissues that express the scrambled transcript. This complete identity of the exons that are absent in the alternatively processed mRNA but present in the scrambled transcript is interpreted as indicative of the possibility that exon scrambling and exon skipping might be interrelated phenomena. It is therefore proposed that alternative pre-mRNA processing has the potential to generate not only mRNAs lacking one or more exons but also circular RNA molecules.

Proviral insertions induce the expression of bone-specific isoforms of PEBP2αA (CBFA1): Evidence for a new myc collaborating oncogene

The til-1 locus was identified as a common retroviral integration site in virus-accelerated lymphomas of CD2-myc transgenic mice. We now show that viral insertions at til-1 lead to transcriptional activation of PEBP2αA (CBFA1), a transcription factor related to the Drosophila segmentation gene product, Runt. Insertions are upstream and in the opposite orientation to the gene and appear to activate a variant promoter that is normally silent in T cells. Activity of this promoter was detected in rodent osteogenic sarcoma cells and primary osteoblasts, implicating bone as the normal site of promoter activity. The isoforms encoded by the activated gene all encompass the conserved runt DNA-binding domain and share a novel N terminus different from the previously reported PEBP2αA products. Minor products include isoforms with internal deletions due to exon skipping and a novel C-terminal domain unrelated to known runt domain factors. The major isoform expressed from the activated til-1 locus (G1) was found to account for virtually all of the core binding factor activity in nuclear extracts from its corresponding lymphoma cell line. Another member of this gene family, AML1(CBFA2), is well known for its involvement in human hemopoietic tumors. These results provide evidence of a direct oncogenic role for PEBP2αA and indicate that the Myc and Runt family genes can cooperate in oncogenesis.

Intron self-complementarity enforces exon inclusion in a yeast pre-mRNA

Skipping of internal exons during removal of introns from pre-mRNA must be avoided for proper expression of most eukaryotic genes. Despite significant understanding of the mechanics of intron removal, mechanisms that ensure inclusion of internal exons in multi-intron pre-mRNAs remain mysterious. Using a natural two-intron yeast gene, we have identified distinct RNA–RNA complementarities within each intron that prevent exon skipping and ensure inclusion of internal exons. We show that these complementarities are positioned to act as intron identity elements, bringing together only the appropriate 5′ splice sites and branchpoints. Destroying either intron self-complementarity allows exon skipping to occur, and restoring the complementarity using compensatory mutations rescues exon inclusion, indicating that the elements act through formation of RNA secondary structure. Introducing new pairing potential between regions near the 5′ splice site of intron 1 and the branchpoint of intron 2 dramatically enhances exon skipping. Similar elements identified in single intron yeast genes contribute to splicing efficiency. Our results illustrate how intron secondary structure serves to coordinate splice site pairing and enforce exon inclusion. We suggest that similar elements in vertebrate genes could assist in the splicing of very large introns and in the evolution of alternative splicing.

A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy

SMN1 and SMN2 (survival motor neuron) encode identical proteins. A critical question is why only the homozygous loss of SMN1, and not SMN2, results in spinal muscular atrophy (SMA). Analysis of transcripts from SMN1/SMN2 hybrid genes and a new SMN1 mutation showed a direct relationship between presence of disease and exon 7 skipping. We have reported previously that the exon-skipped product SMNΔ7 is partially defective for self-association and SMN self-oligomerization correlated with clinical severity. To evaluate systematically which of the five nucleotides that differ between SMN1 and SMN2 effect alternative splicing of exon 7, a series of SMN minigenes was engineered and transfected into cultured cells, and their transcripts were characterized. Of these nucleotide differences, the exon 7 C-to-T transition at codon 280, a translationally silent variance, was necessary and sufficient to dictate exon 7 alternative splicing. Thus, the failure of SMN2 to fully compensate for SMN1 and protect from SMA is due to a nucleotide exchange (C/T) that attenuates activity of an exonic enhancer. These findings demonstrate the molecular genetic basis for the nature and pathogenesis of SMA and illustrate a novel disease mechanism. Because individuals with SMA retain the SMN2 allele, therapy targeted at preventing exon 7 skipping could modify clinical outcome.

Complex mtDNA constitutes an approximate 620-kb insertion on Arabidopsis thaliana chromosome 2: Implication of potential sequencing errors caused by large-unit repeats

Previously conducted sequence analysis of Arabidopsis thaliana (ecotype Columbia-0) reported an insertion of 270-kb mtDNA into the pericentric region on the short arm of chromosome 2. DNA fiber-based fluorescence in situ hybridization analyses reveal that the mtDNA insert is 618 ± 42 kb, ≈2.3 times greater than that determined by contig assembly and sequencing analysis. Portions of the mitochondrial genome previously believed to be absent were identified within the insert. Sections of the mtDNA are repeated throughout the insert. The cytological data illustrate that DNA contig assembly by using bacterial artificial chromosomes tends to produce a minimal clone path by skipping over duplicated regions, thereby resulting in sequencing errors. We demonstrate that fiber-fluorescence in situ hybridization is a powerful technique to analyze large repetitive regions in the higher eukaryotic genomes and is a valuable complement to ongoing large genome sequencing projects.

Ullrich scleroatonic muscular dystrophy is caused by recessive mutations in collagen type VI

Ullrich syndrome is a recessive congenital muscular dystrophy affecting connective tissue and muscle. The molecular basis is unknown. Reverse transcription–PCR amplification performed on RNA extracted from fibroblasts or muscle of three Ullrich patients followed by heteroduplex analysis displayed heteroduplexes in one of the three genes coding for collagen type VI (COL6). In patient A, we detected a homozygous insertion of a C leading to a premature termination codon in the triple-helical domain of COL6A2 mRNA. Both healthy consanguineous parents were carriers. In patient B, we found a deletion of 28 nucleotides because of an A → G substitution at nucleotide −2 of intron 17 causing the activation of a cryptic acceptor site inside exon 18. The second mutation was an exon skipping because of a G → A substitution at nucleotide −1 of intron 23. Both mutations are present in an affected brother. The first mutation is also present in the healthy mother, whereas the second mutation is carried by their healthy father. In patient C, we found only one mutation so far—the same deletion of 28 nucleotides found in patient B. In this case, it was a de novo mutation, as it is absent in her parents. mRNA and protein analysis of patient B showed very low amounts of COL6A2 mRNA and of COL6. A near total absence of COL6 was demonstrated by immunofluorescence in fibroblasts and muscle. Our results demonstrate that Ullrich syndrome is caused by recessive mutations leading to a severe reduction of COL6.

Molecular basis of human mitochondrial very-long-chain acyl-CoA dehydrogenase deficiency causing cardiomyopathy and sudden death in childhood.

beta-Oxidation of long-chain fatty acids provides the major source of energy in the heart. Defects in enzymes of the beta-oxidation pathway cause sudden, unexplained death in childhood, acute hepatic encephalopathy or liver failure, skeletal myopathy, and cardiomyopathy. Very-long-chain acyl-CoA dehydrogenase [VLCAD; very-long-chain-acyl-CoA:(acceptor) 2,3-oxidoreductase, EC 1.3.99.13] catalyzes the first step in beta-oxidation. We have isolated the human VLCAD cDNA and gene and determined the complete nucleotide sequences. Polymerase chain reaction amplification of VLCAD mRNA and genomic exons defined the molecular defects in two patients with VLCAD deficiency who presented with unexplained cardiac arrest and cardiomyopathy. In one, a homozygous mutation in the consensus dinucleotide of the donor splice site (g+1-->a) was associated with universal skipping of the prior exon (exon 11). The second patient was a compound heterozygote, with a missense mutation, C1837-->T, changing the arginine at residue 613 to tryptophan on one allele and a single base deletion at the intron-exon 6 boundary as the second mutation. This initial delineation of human mutations in VLCAD suggests that VLCAD deficiency reduces myocardial fatty acid beta-oxidation and energy production and is associated with cardiomyopathy and sudden death in childhood.