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

• Published in: PloS one Vol. 5; no. 8; p. e12315
• Main Authors: Prasov, Lev, Brown, Nadean L, Glaser, Tom
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 24.08.2010; Public Library of Science (PLoS)
• Subjects: Alternative splicing; Analysis; Animals; Base Sequence; Basic Helix-Loop-Helix Transcription Factors - genetics; Betaine; Blotting, Northern; Cerebellum; Cerebellum - metabolism; Cloning; Cloning, Molecular; Deoxyribonucleic acid; Developmental Biology/Molecular Development; Developmental Biology/Neurodevelopment; Developmental Biology/Organogenesis; DNA; DNA, Complementary - genetics; Embryos; Eye (anatomy); Gene expression; Genetics and Genomics/Gene Expression; Genomics; Hybridization; Internal medicine; Laboratories; Messenger RNA; Mice; Mitosis; Molecular Biology/RNA Splicing; Mutants; Nerve Tissue Proteins - genetics; Nerves; Neurogenesis; Neuroscience/Neurodevelopment; Optic nerve; Polymerase Chain Reaction; Protein structure; Retina; Retina - growth & development; Retina - metabolism; Retinal ganglion cells; Retinogenesis; Ribonuclease; Ribonucleases - metabolism; Ribonucleic acid; RNA; RNA Splicing; RNA, Messenger - genetics; RNA, Messenger - metabolism; Secondary structure; Switching; Vertebrates; Ann Arbor Michigan; United States > US; Michigan
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0012315

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.