Mapping the 5′ and 3′ ends ofTetrahymena thermophilamRNAs using RNA ligase mediated amplification of cDNA ends (RLM-RACE)

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Abstract
A procedure is described for mapping the ends of RNAs. Using T4 RNA ligase, a DNA (3′ end) or RNA (5′ end) oligonucleotide is ligated to RNA ends followed by cDNA synthesis, PCR amplification, cloning and sequencing. This method determines 5′ ends, 3′ polyadenylation sites and the size of poly(A) tails, and should be applicable to non-polyadenylated mRNAs and to non-message RNAs. Analysis of four Tetrahymena thermophila histone mRNAs revealed multiple, closely spaced 5′ ends consistent with those determined by other methods. Except for a ′CCAAT′ box ineither orientation 100–200 nucleotides upstream of the transcription start site, no conserved sequence elements were observed in the untranslated 5′ region or in sequences immediately flanking the transcription start site. Analysis of the 3′ ends of mRNAs encoding four histones, two tubutins and the Tetrahymena TATA binding protein confirmed the observations that Tetrahymena histone messages are polyadenylated and that poly(A) tails in this organism are short (ñ 50 nt). No canonical poly(A) addition signal was identified.The four histone messages analyzed here contained three sequence elements, TGTGT-TAAAAGTATT, not found in non-histone messages. Two non-histone messages contained GCATT(N)15ATACC near the poly(A) addition site.