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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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RT-PCRs of Math5 RNA transcribed in vitro.A. Diagram and agarose gel showing linearized pJN4C and Math5 sense RNA generated by T3 polymerase and treated with DNaseI. B. cDNA products amplified by RT-PCR from IVT-derived RNA with UTR primers LP8 and LP4. Only the full-length 1087 bp Math5 cDNA product was amplified in the presence of 3X Masteramp (MA, indicated above brackets). In the absence of betaine, a variety of weak products were observed, with a heterogeneous deletion profile, reflecting a low level of RT template-switching. This background could be increased by using suboptimal PCR conditions or omitting the mouse liver RNA carrier. IVT, in vitro transcribed Math5 RNA (10 ng); ML, mouse liver RNA (3 µg). C. Similar RT-PCRs performed using internal primers LP6 and LP7. Only the expected 486 bp cDNA was amplified in 3X MA, while spurious products were amplified at lower MA concentrations. The right three panels in B and C represent adjacent lanes in the same gels, displayed separately for clarity. D. Alignment of lacunar cDNAs generated from IVT or E14.5 eye RNA templates. The deletion profile is comparable to the distribution reported by Kanadia and Cepko [18] (cf. Table S1 and Figure 1), using the same primer pairs with no precautions for GC secondary structure. The sequence of breakpoints is given in Table S3, with microhomology at the inferred sites of RT template-switching.
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pone-0012315-g004: RT-PCRs of Math5 RNA transcribed in vitro.A. Diagram and agarose gel showing linearized pJN4C and Math5 sense RNA generated by T3 polymerase and treated with DNaseI. B. cDNA products amplified by RT-PCR from IVT-derived RNA with UTR primers LP8 and LP4. Only the full-length 1087 bp Math5 cDNA product was amplified in the presence of 3X Masteramp (MA, indicated above brackets). In the absence of betaine, a variety of weak products were observed, with a heterogeneous deletion profile, reflecting a low level of RT template-switching. This background could be increased by using suboptimal PCR conditions or omitting the mouse liver RNA carrier. IVT, in vitro transcribed Math5 RNA (10 ng); ML, mouse liver RNA (3 µg). C. Similar RT-PCRs performed using internal primers LP6 and LP7. Only the expected 486 bp cDNA was amplified in 3X MA, while spurious products were amplified at lower MA concentrations. The right three panels in B and C represent adjacent lanes in the same gels, displayed separately for clarity. D. Alignment of lacunar cDNAs generated from IVT or E14.5 eye RNA templates. The deletion profile is comparable to the distribution reported by Kanadia and Cepko [18] (cf. Table S1 and Figure 1), using the same primer pairs with no precautions for GC secondary structure. The sequence of breakpoints is given in Table S3, with microhomology at the inferred sites of RT template-switching.

Mentions: Similar experiments were performed with a second pair of primers (LP6 and LP7), which are separated by 486 bp in genomic DNA and flank the GC-rich segment (Figure 3c,d). In the absence of betaine, these primers did not amplify any product. However, when 2–3X Masteramp™ was included in the PCR, only the expected 486 bp amplimer was observed. When we extended the PCR beyond 35 cycles, preincubated the reaction at 25°C (“cold start”) or used crude Taq polymerase preparations in the absence of betaine, a heterogeneous group of deleted (lacunar) products was observed (not shown), with a size and sequence distribution (Figure 4d, Table S3) similar to that reported by Kanadia and Cepko.


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

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

RT-PCRs of Math5 RNA transcribed in vitro.A. Diagram and agarose gel showing linearized pJN4C and Math5 sense RNA generated by T3 polymerase and treated with DNaseI. B. cDNA products amplified by RT-PCR from IVT-derived RNA with UTR primers LP8 and LP4. Only the full-length 1087 bp Math5 cDNA product was amplified in the presence of 3X Masteramp (MA, indicated above brackets). In the absence of betaine, a variety of weak products were observed, with a heterogeneous deletion profile, reflecting a low level of RT template-switching. This background could be increased by using suboptimal PCR conditions or omitting the mouse liver RNA carrier. IVT, in vitro transcribed Math5 RNA (10 ng); ML, mouse liver RNA (3 µg). C. Similar RT-PCRs performed using internal primers LP6 and LP7. Only the expected 486 bp cDNA was amplified in 3X MA, while spurious products were amplified at lower MA concentrations. The right three panels in B and C represent adjacent lanes in the same gels, displayed separately for clarity. D. Alignment of lacunar cDNAs generated from IVT or E14.5 eye RNA templates. The deletion profile is comparable to the distribution reported by Kanadia and Cepko [18] (cf. Table S1 and Figure 1), using the same primer pairs with no precautions for GC secondary structure. The sequence of breakpoints is given in Table S3, with microhomology at the inferred sites of RT template-switching.
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Related In: Results  -  Collection

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pone-0012315-g004: RT-PCRs of Math5 RNA transcribed in vitro.A. Diagram and agarose gel showing linearized pJN4C and Math5 sense RNA generated by T3 polymerase and treated with DNaseI. B. cDNA products amplified by RT-PCR from IVT-derived RNA with UTR primers LP8 and LP4. Only the full-length 1087 bp Math5 cDNA product was amplified in the presence of 3X Masteramp (MA, indicated above brackets). In the absence of betaine, a variety of weak products were observed, with a heterogeneous deletion profile, reflecting a low level of RT template-switching. This background could be increased by using suboptimal PCR conditions or omitting the mouse liver RNA carrier. IVT, in vitro transcribed Math5 RNA (10 ng); ML, mouse liver RNA (3 µg). C. Similar RT-PCRs performed using internal primers LP6 and LP7. Only the expected 486 bp cDNA was amplified in 3X MA, while spurious products were amplified at lower MA concentrations. The right three panels in B and C represent adjacent lanes in the same gels, displayed separately for clarity. D. Alignment of lacunar cDNAs generated from IVT or E14.5 eye RNA templates. The deletion profile is comparable to the distribution reported by Kanadia and Cepko [18] (cf. Table S1 and Figure 1), using the same primer pairs with no precautions for GC secondary structure. The sequence of breakpoints is given in Table S3, with microhomology at the inferred sites of RT template-switching.
Mentions: Similar experiments were performed with a second pair of primers (LP6 and LP7), which are separated by 486 bp in genomic DNA and flank the GC-rich segment (Figure 3c,d). In the absence of betaine, these primers did not amplify any product. However, when 2–3X Masteramp™ was included in the PCR, only the expected 486 bp amplimer was observed. When we extended the PCR beyond 35 cycles, preincubated the reaction at 25°C (“cold start”) or used crude Taq polymerase preparations in the absence of betaine, a heterogeneous group of deleted (lacunar) products was observed (not shown), with a size and sequence distribution (Figure 4d, Table S3) similar to that reported by Kanadia and Cepko.

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