原创力文档a critical analysis of atoh7 (math5) mrna splicing in the developing mouse retina的关键分析atoh7(math5)信使rna剪接在发展中老鼠的视网膜.pdfVIP
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a critical analysis of atoh7 (math5) mrna splicing in the developing mouse retina的关键分析atoh7(math5)信使rna剪接在发展中老鼠的视网膜
A Critical Analysis of Atoh7 (Math5) mRNA Splicing in the
Developing Mouse Retina
1 2 1
Lev Prasov , Nadean L. Brown , Tom Glaser *
1 Departments of Human Genetics and Internal Medicine, University of Michigan, Ann Arbor, Michigan, United States of America, 2 Division of Developmental Biology,
Department of Pediatrics and Ophthalmology, Cincinnati Children’s Research Foundation, University of Cincinnati School of Medicine, Cincinnati, Ohio, 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, 39RACE, 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 h
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