Evolutionarily conserved elements in the 5' untranslated region of beta globin mRNA mediate site-specific priming of a unique hairpin structure during cDNA synthesis.

AUTOR(ES)

Volloch, V Z

RESUMO

Generation of double-stranded cDNA during reverse transcription of a variety of mRNA molecules is well known to involve the formation of covalently linked antisense and sense strands in a hairpin configuration. In the present study we have examined the sequence of molecular events which occurs during cDNA synthesis from mouse beta globin mRNA, in particular the self-priming event that initiates synthesis of sense-strand DNA. Upon completion of reverse transcription of globin mRNA and the removal of RNA template by RNase H activity associated with reverse transcriptase, the 3' end of cDNA snaps back to form a stable double-stranded structure, which is extended by reverse transcriptase to generate the sense DNA strand. Surprisingly, the fourteen 3' terminal nucleotides of the beta globin antisense DNA strand (cDNA) have strong complementarity with an internal segment of the same molecule corresponding to a portion of the 5'-untranslated region of the mRNA located just upstream of the translation start site. Efficient second strand cDNA synthesis appears to require the occurrence within the cDNA molecule of these two complementary elements, one of which must be 3'-terminal. A second surprising feature is that the strong complementarity between the terminal and the internal portions of the molecule exists in the antisense DNA and not in the sense mRNA strand. This is because A:C mismatches on the sense strand correspond to relatively stable T:G base pairs on the antisense strand. Such an extended region of complementarity within the segment of cDNA corresponding to the short 5' untranslated region of beta globin mRNA is unlikely to occur purely by chance, suggesting some underlying function. In this regard it is of interest that cDNAs of adult beta globin mRNAs from other mammalian species show a very similar arrangement of complementary elements, and that complementarity is heavily conserved, even when there are substitutions in nucleotide sequence.

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