Fig. 7

Circular Fmn transcripts are not detected in the brain and kidney of Fmn exon 4- and exon 5-deficient mice. An RT-PCR assay (see Fig. 3) was used to identify circular Fmn transcripts from organs of fmn^4KO and fmn^5KO mice. The locations of the PCR primers used in this RT-PCR assay are shown in the schematic diagrams in parts A and B. The numbers represent the Fmn exon sequences amplified in the PCR reactions. The black bars represent the locations of oligonucleotide probes for Southern blot hybridization of the PCR products shown below the schematic diagram. For this assay, positive controls (Control, A and B), which were PCR amplification of a normal, linear Fmn transcript, show that cDNA was present in each sample. The locations of the PCR primers for these controls are shown in the schematic diagram. The control lanes shown represent RT-PCR products from brain RNA samples. (A) RT-PCR analysis of fmn^4KO samples. First-strand cDNA, primed with a Fmn exon 5 primer, was synthesized from adult brain and kidney from mice with the following genotypes: 1, +/+ littermate of animals 2 and 3; 2, fmn^4KO/+; 3, fmn^4KO/fmn^4KO. RT-PCR products were hybridized with an exon 5 oligonucleotide probe. (B) RT-PCR analysis of fmn^5KO samples. First-strand cDNA, primed with a Fmn exon 4 primer, was synthesized from adult brain and kidney from mice with the following genotypes: 1, +/+ littermate of animals 2 and 3; 2, fmn^5KO/+; 3, fmn^5KO/fmn^5KO. RT-PCR products were hybridized with an exon 4 oligonucleotide probe. (C) A schematic diagram of two circular RNA species (same as in Fig. 3C) represented by the subcloned and sequenced RT-PCR products from the wild-type and heterozygous fmn^4KO and fmn^5KO animals in this assay. Arrows labeled A represent the outward-facing exon 5 PCR primers shown in part A, which were used to analyze the fmn^4KO mice. Arrows labeled B represent the similar primers shown in part B. Numbers represent the complete Fmn exons, which were joined accurately at their consensus splice sites.