Reverse Transcription Slippage over the mRNA Secondary Structure of the LIP1 Gene

The secondary structures in mRNA often cause early termination during the synthesis of cDNA. In an attempt to determine the 5′-untranslated region (UTR) of the gene LIP1 using the RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), we found that reverse transcriptases skipped over the LIP1 RNA secondary structures and continued the DNA synthesis through RNA adapter sequences without early termination. A fragment of only three nucleotides upstream of the LIP1 translation initiation codon was obtained from the initial RACEPCR, which was much shorter than the 57-nucleotide fragment obtained from the cDNA library screening. Analysis of the 5′ end sequence indicates the presence of high G+C content and stem-loop secondary structures. Therefore, optimizations of the reaction with high temperature (70°C) and a thermostable reverse transcriptase were performed to synthesize the first-strand cDNA, which was determined to have 73 nucleotides in the 5′-UTR. These results suggest that, under cDNA synthesis conditions at 42°C and 60°C, the reverse transcriptases skipped over the stem-loop structures of LIP1 mRNA and continued the cDNA synthesis until they reached the RNA adapter sequences. This finding draws attention to adopting optimized conditions for cDNA synthesis on G+C-rich RNA templates.