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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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Ribonuclease protection assays.A. Diagram showing RPA strategy, with Math5 cDNA, two different antisense cRNA probes, protected fragments expected for FL (full length, unspliced) and ECO (spliced) transcripts, and positive control RNAs generated by sense IVT reactions. B. Autoradiogram, showing undigested probes A and B (366 nt and 632 nt) and exclusively unspliced fragments protected by E14.5 eye RNA (567 nt and 301 nt). No fragment corresponding to the presumptive ECO transcript (212 nt) was protected by eye RNA using either cRNA probe, although a doublet of this size was protected by the ECO IVT positive control. Background fragments observed with probe B (arrowheads) are caused by intrinsic sensitivity of the cRNA-mRNA duplex to RNase cleavage at particular sites and were also present in the full length IVT positive control. The probe (no RNase) and IVT controls were diluted 20- and 10-fold respectively, compared to the E14.5 eye RNA hybridization lanes.
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pone-0012315-g006: Ribonuclease protection assays.A. Diagram showing RPA strategy, with Math5 cDNA, two different antisense cRNA probes, protected fragments expected for FL (full length, unspliced) and ECO (spliced) transcripts, and positive control RNAs generated by sense IVT reactions. B. Autoradiogram, showing undigested probes A and B (366 nt and 632 nt) and exclusively unspliced fragments protected by E14.5 eye RNA (567 nt and 301 nt). No fragment corresponding to the presumptive ECO transcript (212 nt) was protected by eye RNA using either cRNA probe, although a doublet of this size was protected by the ECO IVT positive control. Background fragments observed with probe B (arrowheads) are caused by intrinsic sensitivity of the cRNA-mRNA duplex to RNase cleavage at particular sites and were also present in the full length IVT positive control. The probe (no RNase) and IVT controls were diluted 20- and 10-fold respectively, compared to the E14.5 eye RNA hybridization lanes.

Mentions: To evaluate the ratio of spliced and unspliced Math5 transcripts, we hybridized total eye RNA from E14.5 embryos, in parallel, with a molar excess of two 32P-labeled antisense RNAs (Figure 6a). These cRNAs were prepared by in vitro transcription of two cDNA clones derived from unspliced 301 bp (A) and 567 bp (B) competitive RT-PCR products (Figure 5b). After hybridization and RNase digestion, surviving probe RNA molecules were resolved by polyacrylamide gel electrophoresis (Figure 6b). Probes A and B were protected by full-length Math5 mRNA in the embryonic eye, giving 301 nt and 567 nt digestion products. No hybridizing fragments were detected at the size predicted for ECO mRNA (212 nt). The absence of smaller protected fragments in this sensitive assay further indicates that the Math5 coding segment is not significantly spliced in the embryonic eye.


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

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

Ribonuclease protection assays.A. Diagram showing RPA strategy, with Math5 cDNA, two different antisense cRNA probes, protected fragments expected for FL (full length, unspliced) and ECO (spliced) transcripts, and positive control RNAs generated by sense IVT reactions. B. Autoradiogram, showing undigested probes A and B (366 nt and 632 nt) and exclusively unspliced fragments protected by E14.5 eye RNA (567 nt and 301 nt). No fragment corresponding to the presumptive ECO transcript (212 nt) was protected by eye RNA using either cRNA probe, although a doublet of this size was protected by the ECO IVT positive control. Background fragments observed with probe B (arrowheads) are caused by intrinsic sensitivity of the cRNA-mRNA duplex to RNase cleavage at particular sites and were also present in the full length IVT positive control. The probe (no RNase) and IVT controls were diluted 20- and 10-fold respectively, compared to the E14.5 eye RNA hybridization lanes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2927423&req=5

pone-0012315-g006: Ribonuclease protection assays.A. Diagram showing RPA strategy, with Math5 cDNA, two different antisense cRNA probes, protected fragments expected for FL (full length, unspliced) and ECO (spliced) transcripts, and positive control RNAs generated by sense IVT reactions. B. Autoradiogram, showing undigested probes A and B (366 nt and 632 nt) and exclusively unspliced fragments protected by E14.5 eye RNA (567 nt and 301 nt). No fragment corresponding to the presumptive ECO transcript (212 nt) was protected by eye RNA using either cRNA probe, although a doublet of this size was protected by the ECO IVT positive control. Background fragments observed with probe B (arrowheads) are caused by intrinsic sensitivity of the cRNA-mRNA duplex to RNase cleavage at particular sites and were also present in the full length IVT positive control. The probe (no RNase) and IVT controls were diluted 20- and 10-fold respectively, compared to the E14.5 eye RNA hybridization lanes.
Mentions: To evaluate the ratio of spliced and unspliced Math5 transcripts, we hybridized total eye RNA from E14.5 embryos, in parallel, with a molar excess of two 32P-labeled antisense RNAs (Figure 6a). These cRNAs were prepared by in vitro transcription of two cDNA clones derived from unspliced 301 bp (A) and 567 bp (B) competitive RT-PCR products (Figure 5b). After hybridization and RNase digestion, surviving probe RNA molecules were resolved by polyacrylamide gel electrophoresis (Figure 6b). Probes A and B were protected by full-length Math5 mRNA in the embryonic eye, giving 301 nt and 567 nt digestion products. No hybridizing fragments were detected at the size predicted for ECO mRNA (212 nt). The absence of smaller protected fragments in this sensitive assay further indicates that the Math5 coding segment is not significantly spliced in the embryonic eye.

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