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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 ∼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.

A Critical Analysis of Atoh7 (Math5) mRNA Splicing in the Developing Mouse Retina