A point mutation in bioactive RNA results in the failure of mutant heart correction in Mexican axolotls.
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.
Zhang
Chi
C
Department of Biomedical Sciences, Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33431, USA.
Dube
Dipak K
DK
Huang
Xupei
X
Zajdel
Robert W
RW
Bhatia
Rajula
R
Foster
Dalton
D
Lemanski
Sharon L
SL
Lemanski
Larry F
LF
D000558:Ambystoma mexicanum / Q000196:embryology*; D000818:Animals; D001483:Base Sequence; D018507:Gene Expression Regulation, Developmental; D015723:Gene Library; D006321:Heart / Q000196:embryology*; D006330:Heart Defects, Congenital / Q000662:veterinary; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D024510:Muscle Development / Q000502:physiology*; D009210:Myofibrils / Q000502:physiology; D009924:Organ Culture Techniques; D017354:Point Mutation; D012313:RNA / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012313:RNA