Journal of Heredity Advance Access originally published online on May 19, 2006
Journal of Heredity 2006 97(3):253-260; doi:10.1093/jhered/esj037
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Noncanonical RNAs From Transcripts of the Drosophila muscleblind Gene
From the Department of Genetics, University of Valencia, Doctor Moliner 50, 46100 Burjasot, Valencia, Spain (Garcia-Casado, Pascual, Paricio, and Artero); and Institute of Biomedical and Life Sciences, University of Glasgow, Anderson College Complex, 56 Dumbarton Road, Glasgow G11 6NU, UK (Houseley, O'Dell, and Monckton). Jonathan M. Houseley is now at The Wellcome Trust Centre for Cell Biology, Michael Swann Building, Mayfield Road, Edinburgh EH9 3JR, UK; and Zaida Garcia-Casado is now at the Department of Haematology, Hospital Universitario La Fe, Valencia, Spain
Address correspondence to R. D. Artero at the address above, or e-mail: ruben.artero{at}uv.es.
It has become increasingly evident that eukaryotic cells produce RNA molecules from coding genes with constitutions other than those of typically spliced mRNA transcripts. Here we describe new cDNAs from the Drosophila melanogaster muscleblind (mbl) locus that identify two such atypical RNA molecules: RNAs containing an incomplete exon 2 tandem repetition (mblE2E2') or having exons with a different order compared to the corresponding genomic DNA (mblE2E3'E2'; exon scrambling). The existence of exon duplications and rearrangements in the genomic locus that might explain such cDNAs was ruled out by genomic Southern blotting and in silico analysis of the Drosophila genome sequence. The incomplete exon 2 tandem repetition was confirmed by sequencing reverse transcriptase-polymerase chain reaction (RT-PCR) products, rapid amplification of cDNA ends, and detection of a band consistent with cDNA sizes in total RNA northern blots. RT-PCRs with exon-specific primers downstream of exon 2 were unable to amplify products other than those expected from canonical mbl isoforms, thus indicating that no other exons were efficiently spliced downstream of exon 2. Moreover, mblE2E2' transcripts seem to be poorly polyadenylated, if at all, and behave aberrantly in a polyacrylamide gel electrophoresis (PAGE) mobility assay. Taken together, lack of polyadenylation, lack of downstream splicing events, small size of mblE2E2', and PAGE behavior all suggest that these noncanonical transcripts may be circular RNAs. The functional implications for these noncanonical transcripts are unclear. A developmental expression profile of mblE2E2' revealed an almost constant expression except during early embryogenesis and early adulthood. The protein putatively encoded is unlikely to be functional because an in-frame stop codon occurs almost immediately after the splice site. Such noncanonical transcripts have previously been observed in vertebrates, and these data provide the first experimental evidence for similar phenomena in invertebrates.
Transcript heterogeneity is a feature of many genes and one that serves in many cases to facilitate additional functionality. Several mechanisms, which occur at both the DNA and RNA levels, have been shown to contribute to this heterogeneity, all of which involve either the rearrangement of sequences within a genome or the use of alternative signals in contiguous RNA. However, the basic requirement to maintain the linear order of gene sequences in RNA transcripts has been challenged in recent years by the description of nontypical RNA molecules. Some of these molecules differ from canonical mRNAs in having tandem duplications of one or more exons in the context of a typical mRNA in the absence of duplications or rearrangements in the DNA. Such exon repetition events have been proposed to constitute a new pathway for transcript heterogeneity and not to arise from trans-splicing as previously assumed (Rigatti et al. 2004). Through serendipity, only a small number of human and rat genes have been found to contain this specific pattern of repetition, the best-studied examples being the rat COT and Sa genes (Caudevilla et al. 1998; Frantz et al. 1999; Rigatti et al. 2004). Exon repetition has been shown to be allele specific, thus suggesting that it is a cis-acting property of the allele rather than a by-product of the splicing machinery (Rigatti et al. 2004). The detection of a larger protein product from the COT gene suggests that in some cases the repetition might have a biological role (Caudevilla et al. 1998).
The linear layout of genomic sequences is also altered in transcripts showing exon scrambling, in which exons are accurately joined at consensus splice sites but in a different order compared to the corresponding genomic DNA. Exon scrambling was initially correlated with the presence of large introns (Cocquerelle et al. 1992) and exon skipping (Zaphiropoulos 1996, 1997), and the first transcripts described were neither polyadenylated nor expressed at their normal level (Nigro et al. 1991; Zaphiropoulos 1996, 1997). However, later studies reported scrambled transcripts that were polyadenylated, adjacent to small introns, and joined at nonconsensus sites (Caldas et al. 1998; Crawford et al. 1999). Scrambled exons have properties consistent with a circular RNA (Megonigal et al. 2000; Nigro et al. 1991), and RNA molecules composed of circularized single exons, or circons, have indeed been observed at low levels (Schindewolf et al. 1996; Zaphiropoulos 1996, 1997). The functional and evolutionary implications of these nontypical RNAs are largely unknown, so they are typically regarded as erroneous by-products of the splicing machinery. For the most part, scrambled transcripts do not seem to have the potential for encoding functional proteins. Recent computational searches, however, showed that exon scrambling and repetition events may be relatively widespread affecting up to approximately 1% of mammalian genes. In addition, conservation of the phenomenon in one gene in humans and mice suggests a biological function (Dixon et al. 2005).
Primary transcripts arising from the Drosophila muscleblind (mbl) locus have been described to follow a canonical alternative-splicing pathway, giving rise to four transcript isoforms known as mblA, mblB, mblC, and mblD. Each transcript encodes for a nuclear protein isoform with a constant N-terminus encoding one or two Cys3His zinc fingers (Begemann et al. 1997). mbl is an essential gene during Drosophila embryogenesis, and homozygous mutant mbl embryos die inside the chorion as fully formed first-instar larvae, with severe defects in the terminal differentiation of embryonic muscles (Artero et al. 1998). Mosaic analysis has also revealed that mbl is required later in development for photoreceptor differentiation (Begemann et al. 1997). Recent studies with one of the human Mbl orthologs the muscle blind-like 1 protein indicate that Mbl proteins are regulators of alternative splicing of pre-mRNAs (Ho et al. 2004). Human Mbl proteins, in addition, play a pivotal role in the pathogenesis of the myotonic dystrophies (for recent reviews, see Pascual et al. 2006; Ranum and Day 2004).
Here we report that mbl primary transcripts, in addition to their canonical splicing pathway, give rise to noncanonical RNA molecules. Noncanonical transcripts are small, poorly polyadenylated, strongly retarded in high-percentage polyacrylamide gel electrophoreses (PAGEs), and characterized by a partial exon 2 tandem repetition in the absence of additional exonic sequences. All these properties are consistent with circular RNA molecules. We also describe an example of exon scrambling at nonconsensus splice sites within exon 2 and exon 3. To our knowledge, experimental characterization of such noncanonical RNAs has been limited to vertebrate genes (including humans), making mbl the first example of a nontypical Drosophila RNA that is verified in vivo.
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Materials and Methods |
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cDNA Isolation and Characterization
A 12- to 24-h embryonic cDNA library from an isogenic second chromosome stock dp cn bw (Brown and Kafatos 1988) was screened using a 0.6-kb HindIII genomic fragment, containing most of the mbl exon 2, by following standard procedures adapted to the use of nonradioactive probes (Ausubel et al. 1992; Roche Molecular Biochemicals, Mannheim, Germany). Six cDNAs were isolated, and their ends were manually sequenced with the use of Sequenase (United States Biochemical, Cleveland, OH). Sequence analyses were performed with programs from the GCG bioinformatics package (Genetics Computer Group, University of Wisconsin).
Reverse Transcriptase-Polymerase Chain Reaction Amplification and Rapid Amplification of cDNA Ends
Total RNA from Oregon-R wild-type strain was prepared by the guanidinium isothiocyanate method (TriReagent, Sigma, St. Louis, MO), and contaminating genomic DNA was eliminated by incubating with RNase-free DNaseI for 15 min at 37°C. cDNA was made from 1 µg RNA using the AMV reverse transcriptase (RT) kit (Roche Molecular Biochemicals) following the manufacturer's recommendations for oligo(dT)-primed synthesis. Of the 20 µl cDNA synthesis mixture, 2 µl was directly used for polymerase chain reaction (PCR) amplification. Primers used for PCR amplification were as follows: B, 5'-CCCACAAGCTTTGTAATCC-3'; A, 5'-GCTGATAGCTTGGGCTAT-3'; E3, 5'-TTCCCCTCGGCGCTTTTC-3'; E4, 5'-GCACGGCGGTTTATCACG-3'; and E5a, 5'-GCTGTGCCACCTGCACTG. For the primer combination A and B, cycling conditions were denaturation for 30 s at 94°C, annealing for 30 s at 55°C, and extension for 1 min at 72°C. Thirty amplification cycles were performed in a total volume of 20 µl. For all other primer combinations, the annealing temperature was 50°C.
For rapid amplification of cDNA ends (3' RACE), RNA isolated as above was reverse transcribed from ADAPT-dT (5'-CGAGGGGGATGGTCGACGGAAGCGACCTTTTTTTTTTTTTTT-3') using SuperScript II (Invitrogen, Carlsbad, CA) and amplified using mblF2 (5'-CTATAACCCCAAAGATCTATGGCC-3') and X45 (5'-CGAGGGGGATGGTCGACGG-3'), with 35 cycles of 94°C for 45 s, 60°C for 45 s, and 72°C for 3 min. Products were gel purified and cloned into pGEM T-Easy (Promega, Madison, WI) for sequencing. Existence of rearranged exon 2 was assessed in developmentally staged cDNA reverse transcribed from random hexamers with SuperScript II using primers mblF5 (5'-GGATTCGCGCTGGCTGCAATTGG-3') and mblXR (5'-GCTTGTGGGGGTAGCTGATAGCTTGGG-3'); PCR conditions were as above, except the extension time was reduced to 1 min.
Northern Blot Analysis
RNA samples (1 µg total Oregon-R RNA or 200 ng poly(A)+ RNA isolated with a Dynabeads® mRNA DirectTM kit) were denatured at 85°C for 10 min in 0.2% sodium dodecyl sulfate, snap chilled, and separated through a 1.5% tris-acetate EDTA agarose gel with 20 mM guanidine thiocyanate and 200 ng/ml ethidium bromide. The RNA was transferred onto a nylon membrane in 20x standard saline citrate and hybridized with digoxigenin-11-dUTP-labeled antisense riboprobes to mbl and rp49 at 68°C in EasyHyb (Roche Molecular Biochemicals). For PAGE analysis of mblE2E2', 10 µg total Oregon-R RNA and 1 µg New England Biolabs (Beverly, MA) 50-bp ladder were separated in 4%–8% polyacrylamide/7 M urea gels containing 1x tris-borate-EDTA. Gels were briefly stained with approximately 150 ng/ml ethidium bromide and imaged before transfer overnight to HyBond N+ membrane in 0.5x TBE at 8 V. Blots were probed with 32P-labeled, random-primed probes according to standard protocols.
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Isolation of Noncanonical cDNA Clones From the mbl Locus
The alternative use of the 11 exons described in the mbl transcription unit gives rise to four transcript isoforms (Figure 1A; Begemann et al. 1997; Garcia-Casado Z, unpublished observations). To further characterize the alternative splicing of the gene, an additional embryonic cDNA library was screened with a genomic probe containing most of exon 2, which includes the putative start codon of the Mbl protein (see Materials and Methods). Surprisingly, all six cDNAs isolated were small, ranging in size from 0.9 to 1.2 kb, and showed an unusual structure. Restriction analysis and sequencing of cDNA ends revealed that clones 2.3, 2.4, and 2.8 had an incomplete tandem repetition of exon 2. The tandem repetition consisted of a complete exon 2 followed by the acceptor site of another exon 2 to an interrupted adenine-rich region lying approximately 100 bp 5' of the start codon. We refer to this noncanonical transcript structure as mblE2E2', where E2' denotes exon 2 sequence from the start of exon 2 to the adenine-rich region. Similar analyses with cDNA 2.6 revealed a structure in which a partial exon 3 and a partial exon 2 (to the adenine-rich region) followed a complete exon 2, an exonic organization we refer to as mblE2E3'E2' (Figure 1B). Rearrangements of exonic sequences such as the one present in cDNA 2.6 have been defined as exon scrambling (Nigro et al. 1991). While the incomplete exon repetition event in mblE2E2' used genuine splice sites, we infer that scrambled exons in mblE2E3'E2' used a cryptic donor splice site in E3 because exon E3 is larger (2.6 kb) than the size of cDNA 2.6 (<1.2 kb).
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mbl Transcripts With Duplicated Exons Exist In Vivo
A number of possibilities might explain the isolation of atypical cDNA clones from the mbl locus, most notably the existence of a duplicated and rearranged extra copy of the gene, a possible trans-splicing event that would result in exon repetition and exon scrambling, and the artifactual joining of partial cDNAs. Homology searches of the Drosophila genome sequence for near-exact matches of exon 2 returned a single hit, thus indicating that no extra copies of exon 2 exist. This result was confirmed by genomic Southern blot analysis, which detected only a single copy of exon 2 in the genome of Drosophila strains Oregon-R and Canton-S (data not shown). Intermolecular splicing of two mbl pre-mRNAs is also an unlikely explanation because trans-splicing of intact genes has been reported very rarely (Horiuchi et al. 2003). If exon repetition and scrambling resulted from a trans-splicing event, it is likely that this would generate two products: a long mRNA having a duplicated exon 2 and a short mRNA lacking that exon and having exon 1 directly joined to exon 3 (or exon 4). No cDNAs corresponding to the short transcript have been isolated. Trans-splicing, in addition, would require the use of cryptic splice sites inside exons 2 and 3 in order to explain structures such as mblE2E2' and mblE2E3'E2' and would include the remaining exonic sequences to the end of the transcript.
Noncanonical mbl transcripts were detected in vivo at several developmental stages by RT-PCR and 3'RACE. PCR primers in exon 2 that are oriented outward gave a strong product from cDNA but not from genomic DNA, confirming the absence of this arrangement at the DNA level and indicating that the exon 2 repetition takes place during primary mbl transcript maturation. The PCR product was purified and sequenced, confirming the repeated structure found in cDNAs 2.3, 2.4, and 2.8 as well as the use of genuine donor and acceptor splice sites (Figure 2A,B,D). Similarly, 3'RACE with an oligo(dT) primer gave a product whose sequence also confirms both the exon 2 repetition and the use of correct splice sites (Figure 2C). Like cDNAs 2.3, 2.4, and 2.8, the 3' end of the RACE product is an interrupted adenine-rich region of exon 2 that presumably binds the oligo(dT) cDNA synthesis primer. Transcripts with the structure mblE2E2' could encode a small protein with a partial Cys3His zinc finger motif (Figure 2E). However, the presence of an in-frame stop codon near the splice site makes it unlikely that a functional protein is actually translated.
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Noncanonical Transcripts From the mbl Locus Are Abundant but Poorly Polyadenylated
The abundance of noncanonical mbl transcripts, such as mblE2E2', was investigated by northern blot hybridization of total RNA using an antisense mblA probe. Unexpectedly, although no known mbl transcript isoforms were observed in this blot, a strong signal was detected at approximately 0.65 kb, which indicates that noncanonical mbl transcripts are very common (Figure 3A). Northern blot analysis of poly(A)+ RNA under similar conditions, in contrast, detected a faint band around 0.65 kb, while canonical mbl transcripts such as mblA, mblB, and mblC were clearly detected (Figure 3B). The difference in intensity of the 0.65-kb band in total and poly(A)+ RNA demonstrates that this product is poorly polyadenylated. Densitometric analysis of the poly(A)+ RNA northern revealed that the 0.65-kb species comprises less than 10% of the total polyadenylated mbl transcript pool (data not shown). Conversely, the absence of a detectable signal from the known transcripts in total RNA (even after overexposing the 0.65-kb band) suggests that the 0.65 kb is present at steady-state levels at least 10-fold those of the canonical transcripts.
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The developmental expression profile of mblE2E2', as an example of noncanonical RNA, was generated using RT-PCR amplification of developmentally staged cDNAs. To specifically amplify cDNA sequences containing a repetition of exon 2, we used the same approach described in Figure 2A, that is, primers oriented outward from within exon 2 so that a product should be amplified from cDNA but not from the genomic DNA. This experiment showed the expression of mblE2E2' at approximately constant levels, except that no early embryonic and early adulthood expression was detected (Figure 3C).
mblE2E2' Transcripts Do Not Include Downstream Exonic Sequences
In order to detect genuine or aberrant splicing of exon 2 sequences to downstream exons in noncanonical transcripts, the B primer was used in combination with exon E3-, E4-, and E5a-specific primers in RT-PCR amplifications. In these experiments, the B oligonucleotide could prime DNA synthesis from annealing sites located either in E2, thus detecting canonical splice events, or in E2', which would detect additional splicing events downstream of E2' (note that because the exon 2 repetition is incomplete, the product size arising from both possibilities would be different; see Figure 2B). In these experiments, only the amplification product from canonical transcripts was detected (Figure 4A), indicating that no additional known exonic sequences are spliced 3' to exon 2 sequences in noncanonical transcripts.
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mblE2E2' Represents Two RNA Populations
To assess whether mblE2E2' transcripts are in fact circular, total Drosophila RNA was separated through various percentages of denaturing polyacrylamide gel (Figure 4B). The mobility of linear RNA species in polyacrylamide is invariant with gel percentage relative to linear DNA molecular weight markers; however, circular species are strongly retarded by increasing acrylamide concentration. Two species are highlighted by the mbl probe: a faster migrating species that is the correct size for mbl exon 2 and a slower migrating species that fails to leave the wells in the 6% polyacrylamide (and in 8%, data not shown). The migration of the faster species is invariant with gel percentage compared to the DNA ladder and an rp49 mRNA control, showing that this species is linear. The slower species migrates differently in the 4% and 6% gels, and the separation between this species and the apparently smaller rRNA is dramatically decreased in the 4% relative to the 6% acrylamide; the opposite would be expected if this band represented a high–molecular weight linear species. We also note the sharpness of the mbl band compared to the blurry rp49 signal, which is consistent with mblE2E2' being poorly polyadenylated. In summary, the aberrant behavior of this species in various gel concentrations strongly suggests it is the circular species detected by RT-PCR.
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Discussion |
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In this paper, we describe new noncanonical transcripts from the mbl locus characterized by an incomplete repetition of exon 2 and scrambling of exons 2 and 3. We demonstrate that these transcripts are small and abundant, show some degree of developmental regulation, are poorly polyadenylated, and are strongly retarded in high-percentage acrylamide gels. Moreover, we show that no additional exonic sequences are spliced 3' to the duplicated exon 2. Taken together, the properties of these RNA molecules are consistent with a circular RNA, as similarly proposed for the ets-1, Sry, or cytochrome P450 2C24 gene transcripts (Capel et al. 1993; Cocquerelle et al. 1993; Zaphiropoulos 1996). Circular RNAs seem rather stable molecules due to the absence of exonuclease-sensitive 5' and 3' ends (Cocquerelle et al. 1993). Therefore, even low levels of RNA processing into noncanonical transcripts might lead to the observed accumulation in the Drosophila cells (Figure 3).
In a number of cases, exon scrambling and exon skipping have been shown to be reciprocal events, thus suggesting a mechanistic relationship (Zaphiropoulos 1996, 1997). However, the study of other gene transcripts such as MLL (Caldas et al. 1998) and our own results with mbl transcripts do not support a correlation. In particular, all mbl transcript isoforms described to date contain exon 2, which also harbors the start codon of the encoded protein, making it unlikely that an exon 2–skipping event was the origin of the noncanonical transcripts we describe. Large introns adjacent to the exons that are scrambled, as well as the presence of other structural characteristics in the pre-mRNA such as large inverted repeats that would facilitate or stabilize the formation of an intermediary stem-loop, have been proposed to influence exon scrambling (Capel et al. 1993; Cocquerelle et al. 1992). Both requirements are fulfilled in mbl primary transcripts: First, the introns between exons 2 and 3 and between exons 3 and 4 are approximately 15 and 60 kb long, respectively, which is relatively large for a Drosophila gene. Second, the intronic sequence between exons 2 and 3 contains the non-LTR retrotransposon jockey J-1 immediately upstream of exon 2, while another non-LTR retrotransposon, an F element, is found downstream of exon 3. The presence of partially homologous sequences between both elements could facilitate the hairpin formation in primary transcripts, which is an intermediary potentially implicated in the mechanism of exon scrambling.
Exon 2 of mbl transcripts contains a number of oligo(A) tracts upstream of the start codon. In a recent study, the molecular basis for the allele-specific exon repetition event associated with the Sa gene (Rigatti et al. 2004) was concluded to involve cis-acting elements. Among the potentially involved sequences were six single nucleotide polymorphisms and a difference of five nucleotides in the length of an oligo(A) tract. It is thus temping to propose that tracts of oligo(A) may participate in the processing or regulation of noncanonical transcripts.
Evidence presented herein indicates unusual features of the processing of mbl primary transcripts, possibly leading to the generation of circular RNA molecules. A basic question about this phenomenon is whether it is unique to mbl or a general occurrence to Drosophila genes. Recent computational searches identified 245 genes in mammals, and five Drosophila genes, showing nonlinear exon splicing, which include exon repetition and exon-scrambling events (Dixon et al. 2005). Interestingly, the same exon 2 repetition event we describe was found in four expressed sequence tags from the mbl gene, thus providing independent confirmation of our results. Additionally, nonlinear exon splicing was identified in another four Drosophila genes: capulet, G protein 
30A, centaurin gamma1A, and CG32306. These observations suggest that the exon repetition and exon-scrambling phenomena is not limited to mbl but is nonetheless limited to a relatively small subset of specific genes. However, it should be considered that the libraries employed to date to search for such transcripts effectively select against circular (poorly polyadenylated) transcripts, and nonlinear exon splicing is thus likely to be more common than currently appreciated (Dixon et al. 2005).
The splicing mechanism yielding circular RNAs may simply represent errors in the normal splicing process, although a possible role of the scrambled transcripts as a feedback regulator of alternative pre-mRNA splicing cannot be excluded. This is especially tempting in the case of Mbl, which itself is involved in the regulation of alternative splicing (Ho et al. 2004). Whatever the answer, occasional errors may have profound impact in cells, such as mutations generated during DNA replication or oncogene transduction by retrovirus, and potential applications can be derived from them.
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Acknowledgments |
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The authors thank the Dynamic Mutation Group and the Drosophila Fly Neurobiology Groups of the University of Glasgow for helpful discussion throughout the course of this work. This work was financially supported in part from research grants SAF2003-03536 (Spanish Ministry of Science and Technology) and GV2004-B-164 (Valencian Community research agency CCEyD) to R.A. and by awards from the Lister Institute and the Wellcome Trust to D.G.M. R.A. was supported by a contract from the Ramon y Cajal program of the Ministerio de Educacion y Ciencia.
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* These authors contributed equally to this work.
Corresponding Editor: Ross MacIntyre
Received August 30, 2005
Accepted March 28, 2006
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