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A critical analysis of Atoh7 (Math5) mRNA splicing in the developing mouse retina.

Prasov L, Brown NL, Glaser T - PLoS ONE (2010)

Bottom Line: Although approximately 10% of cerebellar Math5 RNAs are spliced, truncating the polypeptide, our results show few, if any, spliced Math5 transcripts exist in the developing retina (<1%).These data differ starkly from a recent study (Kanadia and Cepko 2010), which concluded that the vast majority of Math5 and other bHLH transcripts are spliced to generate noncoding RNAs.These results have implications for all members of the bHLH gene family, for any gene that is alternatively spliced, and for the interpretation of all RT-PCR experiments.

View Article: PubMed Central - PubMed

Affiliation: Departments of Human Genetics and Internal Medicine, University of Michigan, Ann Arbor, Michigan, United States of America.

ABSTRACT
The Math5 (Atoh7) gene is transiently expressed during retinogenesis by progenitors exiting mitosis, and is essential for ganglion cell (RGC) development. Math5 contains a single exon, and its 1.7 kb mRNA encodes a 149-aa polypeptide. Mouse Math5 mutants have essentially no RGCs or optic nerves. Given the importance of this gene in retinal development, we thoroughly investigated the possibility of Math5 mRNA splicing by Northern blot, 3'RACE, RNase protection assays, and RT-PCR, using RNAs extracted from embryonic eyes and adult cerebellum, or transcribed in vitro from cDNA clones. Because Math5 mRNA contains an elevated G+C content, we used graded concentrations of betaine, an isostabilizing agent that disrupts secondary structure. Although approximately 10% of cerebellar Math5 RNAs are spliced, truncating the polypeptide, our results show few, if any, spliced Math5 transcripts exist in the developing retina (<1%). Rare deleted cDNAs do arise via RT-mediated RNA template switching in vitro, and are selectively amplified during PCR. These data differ starkly from a recent study (Kanadia and Cepko 2010), which concluded that the vast majority of Math5 and other bHLH transcripts are spliced to generate noncoding RNAs. Our findings clarify the architecture of the Math5 gene and its mechanism of action. These results have implications for all members of the bHLH gene family, for any gene that is alternatively spliced, and for the interpretation of all RT-PCR experiments.

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Math5 messenger RNAs.A. Northern blot probed with 1.2 kb Math5 (JN4C) and 1.1 kb β-actin cDNAs. Two Math5 mRNAs are visible (left arrowheads), but no hybridizing RNA species is present in the 0.8–1.0 kb size range. The RNA size ladder cross-hybridized to vector DNA in the plasmid probes. B. Map of the 3′UTR and flanking genomic DNA (6 kb), showing eight potential polyA signals ATTAAA (blue) and AATAAA (green); the internal A14 priming site in the UTR (ψpA); interspersed repeats (gray); and the nested 3′RACE primers (dark red) for pA1 and pA6 sites, which have the most favorable sequence context. Clones JN2 and BC092234 terminate at pA1, whereas cDNAs JN1, JN4 and JN6 terminate at ψpA [7], [13]. ψpA2 marks an A-rich genomic site captured in the pA6 assay. C. polyADQ scores for all potential pA sites, calculated using human genome parameters [22]. Only pA1 and pA6 have scores above threshold. D. Embryonic eye RT-PCRs with 260 bp and 365 bp 3′RACE products (arrowheads) showing utilization of pA1 and pA6 sites. The 900 bp product was primed from ψpA2 (open arrowhead). m, marker (1 kb-plus ladder); RT, reverse transcriptase. E. Sequence of pA1 RACE products originating from the 1.7 kb Math5 mRNA. F. Sequence of pA6 RACE products originating from the 4.4 kb Math5 mRNA.
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pone-0012315-g002: Math5 messenger RNAs.A. Northern blot probed with 1.2 kb Math5 (JN4C) and 1.1 kb β-actin cDNAs. Two Math5 mRNAs are visible (left arrowheads), but no hybridizing RNA species is present in the 0.8–1.0 kb size range. The RNA size ladder cross-hybridized to vector DNA in the plasmid probes. B. Map of the 3′UTR and flanking genomic DNA (6 kb), showing eight potential polyA signals ATTAAA (blue) and AATAAA (green); the internal A14 priming site in the UTR (ψpA); interspersed repeats (gray); and the nested 3′RACE primers (dark red) for pA1 and pA6 sites, which have the most favorable sequence context. Clones JN2 and BC092234 terminate at pA1, whereas cDNAs JN1, JN4 and JN6 terminate at ψpA [7], [13]. ψpA2 marks an A-rich genomic site captured in the pA6 assay. C. polyADQ scores for all potential pA sites, calculated using human genome parameters [22]. Only pA1 and pA6 have scores above threshold. D. Embryonic eye RT-PCRs with 260 bp and 365 bp 3′RACE products (arrowheads) showing utilization of pA1 and pA6 sites. The 900 bp product was primed from ψpA2 (open arrowhead). m, marker (1 kb-plus ladder); RT, reverse transcriptase. E. Sequence of pA1 RACE products originating from the 1.7 kb Math5 mRNA. F. Sequence of pA6 RACE products originating from the 4.4 kb Math5 mRNA.

Mentions: During our initial characterization of Math5 [7], we identified four independent retinal cDNA clones, which were colinear with mouse genomic DNA (Genbank accession no. AF418923). The 5′ and 3′ termini, and internal sequences were consistent with RNA hybridization data suggesting a single-exon transcription unit, with an initiation site 23 bp downstream from a TATAAA box and a polyadenylation (pA) site 669 bp downstream from the TAA stop codon, giving 1.7 kb as the predicted size for polyA+ Math5 mRNA (Figure 1a,d). This major Math5 transcript was detected by Northern blot analysis of E15.5 mRNA with an 1155 bp radiolabeled cDNA probe (JN4C) that includes 318 bp 5′UTR, 447 bp coding sequence (CDS) and 390 bp 3′ UTR (Figure 2a). A second, less abundant 4.4 kb transcript was also detected at this age, which is close to the peak time-point for Math5 expression during embryogenesis [15]. Careful inspection of the autoradiogram, in relation to the RNA size markers, revealed no smaller Math5 transcripts, particularly in the 0.8–1.0 kb size range expected for spliced isoforms lacking the coding region. This pattern resembles Northern data obtained by Kanadia and Cepko with UTR probes (cf. Figure 1f and 1f'), but appears inverted compared to the unsized blot hybridized with a CDS probe in their report (cf. Figure 1f). We cannot explain this discrepancy.


A critical analysis of Atoh7 (Math5) mRNA splicing in the developing mouse retina.

Prasov L, Brown NL, Glaser T - PLoS ONE (2010)

Math5 messenger RNAs.A. Northern blot probed with 1.2 kb Math5 (JN4C) and 1.1 kb β-actin cDNAs. Two Math5 mRNAs are visible (left arrowheads), but no hybridizing RNA species is present in the 0.8–1.0 kb size range. The RNA size ladder cross-hybridized to vector DNA in the plasmid probes. B. Map of the 3′UTR and flanking genomic DNA (6 kb), showing eight potential polyA signals ATTAAA (blue) and AATAAA (green); the internal A14 priming site in the UTR (ψpA); interspersed repeats (gray); and the nested 3′RACE primers (dark red) for pA1 and pA6 sites, which have the most favorable sequence context. Clones JN2 and BC092234 terminate at pA1, whereas cDNAs JN1, JN4 and JN6 terminate at ψpA [7], [13]. ψpA2 marks an A-rich genomic site captured in the pA6 assay. C. polyADQ scores for all potential pA sites, calculated using human genome parameters [22]. Only pA1 and pA6 have scores above threshold. D. Embryonic eye RT-PCRs with 260 bp and 365 bp 3′RACE products (arrowheads) showing utilization of pA1 and pA6 sites. The 900 bp product was primed from ψpA2 (open arrowhead). m, marker (1 kb-plus ladder); RT, reverse transcriptase. E. Sequence of pA1 RACE products originating from the 1.7 kb Math5 mRNA. F. Sequence of pA6 RACE products originating from the 4.4 kb Math5 mRNA.
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pone-0012315-g002: Math5 messenger RNAs.A. Northern blot probed with 1.2 kb Math5 (JN4C) and 1.1 kb β-actin cDNAs. Two Math5 mRNAs are visible (left arrowheads), but no hybridizing RNA species is present in the 0.8–1.0 kb size range. The RNA size ladder cross-hybridized to vector DNA in the plasmid probes. B. Map of the 3′UTR and flanking genomic DNA (6 kb), showing eight potential polyA signals ATTAAA (blue) and AATAAA (green); the internal A14 priming site in the UTR (ψpA); interspersed repeats (gray); and the nested 3′RACE primers (dark red) for pA1 and pA6 sites, which have the most favorable sequence context. Clones JN2 and BC092234 terminate at pA1, whereas cDNAs JN1, JN4 and JN6 terminate at ψpA [7], [13]. ψpA2 marks an A-rich genomic site captured in the pA6 assay. C. polyADQ scores for all potential pA sites, calculated using human genome parameters [22]. Only pA1 and pA6 have scores above threshold. D. Embryonic eye RT-PCRs with 260 bp and 365 bp 3′RACE products (arrowheads) showing utilization of pA1 and pA6 sites. The 900 bp product was primed from ψpA2 (open arrowhead). m, marker (1 kb-plus ladder); RT, reverse transcriptase. E. Sequence of pA1 RACE products originating from the 1.7 kb Math5 mRNA. F. Sequence of pA6 RACE products originating from the 4.4 kb Math5 mRNA.
Mentions: During our initial characterization of Math5 [7], we identified four independent retinal cDNA clones, which were colinear with mouse genomic DNA (Genbank accession no. AF418923). The 5′ and 3′ termini, and internal sequences were consistent with RNA hybridization data suggesting a single-exon transcription unit, with an initiation site 23 bp downstream from a TATAAA box and a polyadenylation (pA) site 669 bp downstream from the TAA stop codon, giving 1.7 kb as the predicted size for polyA+ Math5 mRNA (Figure 1a,d). This major Math5 transcript was detected by Northern blot analysis of E15.5 mRNA with an 1155 bp radiolabeled cDNA probe (JN4C) that includes 318 bp 5′UTR, 447 bp coding sequence (CDS) and 390 bp 3′ UTR (Figure 2a). A second, less abundant 4.4 kb transcript was also detected at this age, which is close to the peak time-point for Math5 expression during embryogenesis [15]. Careful inspection of the autoradiogram, in relation to the RNA size markers, revealed no smaller Math5 transcripts, particularly in the 0.8–1.0 kb size range expected for spliced isoforms lacking the coding region. This pattern resembles Northern data obtained by Kanadia and Cepko with UTR probes (cf. Figure 1f and 1f'), but appears inverted compared to the unsized blot hybridized with a CDS probe in their report (cf. Figure 1f). We cannot explain this discrepancy.

Bottom Line: Although approximately 10% of cerebellar Math5 RNAs are spliced, truncating the polypeptide, our results show few, if any, spliced Math5 transcripts exist in the developing retina (<1%).These data differ starkly from a recent study (Kanadia and Cepko 2010), which concluded that the vast majority of Math5 and other bHLH transcripts are spliced to generate noncoding RNAs.These results have implications for all members of the bHLH gene family, for any gene that is alternatively spliced, and for the interpretation of all RT-PCR experiments.

View Article: PubMed Central - PubMed

Affiliation: Departments of Human Genetics and Internal Medicine, University of Michigan, Ann Arbor, Michigan, United States of America.

ABSTRACT
The Math5 (Atoh7) gene is transiently expressed during retinogenesis by progenitors exiting mitosis, and is essential for ganglion cell (RGC) development. Math5 contains a single exon, and its 1.7 kb mRNA encodes a 149-aa polypeptide. Mouse Math5 mutants have essentially no RGCs or optic nerves. Given the importance of this gene in retinal development, we thoroughly investigated the possibility of Math5 mRNA splicing by Northern blot, 3'RACE, RNase protection assays, and RT-PCR, using RNAs extracted from embryonic eyes and adult cerebellum, or transcribed in vitro from cDNA clones. Because Math5 mRNA contains an elevated G+C content, we used graded concentrations of betaine, an isostabilizing agent that disrupts secondary structure. Although approximately 10% of cerebellar Math5 RNAs are spliced, truncating the polypeptide, our results show few, if any, spliced Math5 transcripts exist in the developing retina (<1%). Rare deleted cDNAs do arise via RT-mediated RNA template switching in vitro, and are selectively amplified during PCR. These data differ starkly from a recent study (Kanadia and Cepko 2010), which concluded that the vast majority of Math5 and other bHLH transcripts are spliced to generate noncoding RNAs. Our findings clarify the architecture of the Math5 gene and its mechanism of action. These results have implications for all members of the bHLH gene family, for any gene that is alternatively spliced, and for the interpretation of all RT-PCR experiments.

Show MeSH
Related in: MedlinePlus