Antisense RNA injections in fertilized frog eggs reveal an RNA duplex unwinding activity

Previous experiments have shown that mRNA translation in frog oocytes can be inhibited by the injection of a complementary antisense RNA. Here we explore the use of antisense RNAs to study the functions of localized maternal mRNAs during postfertilization development. While developmental abnormalities were observed in injected fertilized eggs, these abnormalities could not be attributed to the antisense RNA since they were induced at a similar frequency in control embryos. Biochemical tests show that the injected antisense RNA does not form stable hybrids in vivo with its complementary endogenous mRNA. In addition, a novel activity that unwinds RNA:RNA duplexes was found. This activity exists at high levels in eggs and early embryos and is absent or very much diminished in oocytes and late blastula embryos. These results suggest that antisense RNAs may be of limited use in studying the functions of maternal RNAs in Xenopus.     

Detection of a novel sense-antisense RNA-hybrid structure by RACE experiments on endogenous troponin I antisense RNA

Conformational changes in the troponin/tropomyosin complex significantly alter the mechanical properties of cardiac muscle. Phosphorylation of cardiac troponin I, part of the troponin/tropomyosin complex, reduces calcium affinity, which leads to increased relaxation of cardiac muscle. Because cardiac troponin I plays a central role in tuning the heart to different work demands, detailed knowledge of troponin I protein regulation is required. Our group previously detected naturally occurring antisense RNA for troponin I in human and rat hearts, and here, attempt to unravel the structure of rat cardiac troponin I antisense RNA. We performed rapid amplification of cDNA ends (RACE) experiments and discovered antisense sequences identical to a copy of the sense mRNA, which led us to conclude that the antisense RNA must be transcribed from troponin I mRNA in the cytoplasm. Moreover, we isolated RNA structures comprising sense and antisense sequences in one continuous molecule. As we found no homolog structures described in the literature, we called this "hybrid RNA." Because a duplex formation was demonstrated previously we concluded that hybrid RNA is a consequence of a tight interaction between sense and antisense troponin I RNA in vivo, which we discuss in the article.