A point mutation in bioactive RNA results in the failure of mutant heart correction in mexican axolotls

Abstract

Ambystoma mexicanum is an intriguing animal model for studying heart development because it carries a mutation in gene c. Hearts of homozygous recessive (c/c) mutant embryos do not contain organized myofibrils and fail to beat. The defect can be corrected by organ-culturing the mutant heart in the presence of RNA from anterior endoderm or endoderm/mesoderm-conditioned medium. By screening a cDNA library made of total conditioned medium RNA from normal axolotl embryonic endoderm, we isolated a single clone (MIR), the synthetic RNA from which corrects the mutant heart defect by promoting myofibrillogenesis and thus was named MIR (myofibrillogenesis inducing RNA). In the present study, we have examined MIR gene expression in mutant axolotl hearts at early pre-heart-beat developmental stages and found its quantitative expression, as detected by RT-PCR, to be the same as in normal hearts. However, careful analysis of sequence data revealed a G-->U point mutation in the mutant MIR RNA. Further computational analyses, using GENEBEE software to compare normal and mutant MIR RNAs show a significant alteration in RNA secondary structure of the point-mutated MIR RNA. The results from bioassay and confocal microscopy immunofluorescent studies demonstrate that, unlike MIR RNA derived from normal embryos, the mutated MIR RNA does not promote myofibrillogenesis in mutant embryonic hearts and fails to rescue/correct the mutant heart defect.

Role of myofibril-inducing RNA in cardiac TnT expression in developing Mexican axolotl

“…The uniqueness of MIR is that in addition to being involved in cTnT splicing, it also can increase the total cTnT mRNA concentration in the cells. We have already shown that MIR is capable of binding with one or more proteins present in axolotl hearts [17]. Also, we have already reported that in mutant axolotl hearts MIR is expressed.…”

“…All dissections were carried out under a dissecting microscope. Hearts were then placed in 96 U-well shaped plates containing Steinberg's solution for liposome-mediated RNA transfection following our published methods [15][16][17][18]. Mutant hearts were divided into two groups: those treated with sense MIR and those treated with antisense MIR.…”

“…Mutant hearts were divided into two groups: those treated with sense MIR and those treated with antisense MIR. RNA molecules were in vitro synthesized following our published methods (Zhang et al, 2003). Control hearts were treated with Lipofectamine 2000 (Invitrogen, CA) and Steinberg's solution only.…”

“…We have previously reported that treating the mutant hearts with the normal sense strand RNA of Myofibril-Inducing RNA (MIR) rescued the mutant phenotypes by promoting tropomyosin production followed by myofibril formation in mutant hearts in vitro [17,18]. In this study, we have shown for the first time that MIR, a small regulatory RNA, also is capable of promoting cTnT expression at the mRNA level as well as regulating the splicing patterns of cTnT gene in mutant hearts making them more like normal.…”

“…Homozygous mutant (c/c) embryos are first distinguishable from their normal siblings (+/+ or +/c) at stage 34, when the mutant embryos develop hearts that fail to beat and eventually die from lack of circulation. Subsequent studies have demonstrated the defects in myofibrillogenesis and significantly decreased tropomyosin expression in the mutant embryonic hearts [12][13][14][15][16][17]. As a tropomyosin-interacting protein, cardiac troponin T has also been studied in this system in our laboratory.…”