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

Abstract

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.

Antisense globin RNA in mouse erythroid tissues: structure, origin, and possible function.

“…At the 3' end of antisense globin RNA, the terminal 14-nucleotide segment-the same 14-nucleotide segment that is missing in the truncated antisense globin RNA-is strongly complementary to the internal 14-nucleotide segment located within a portion of antisense molecule corresponding to the 5'-untranslated region of globin mRNA. As we reported earlier, strikingly, such complementarity within the antisense strand is highly conserved during evolution of mammalian ,B-globins from marsupials to humans (17) and is also seen in a-globins (unpublished results). Importantly, rather than nucleotide sequence, it is the complementary relationship that is conserved; although nucleotide sequences in this region have diverged during mammalian evolution, the complementary relationship between two elements has been preserved (17).…”

“…As we reported earlier, strikingly, such complementarity within the antisense strand is highly conserved during evolution of mammalian ,B-globins from marsupials to humans (17) and is also seen in a-globins (unpublished results). Importantly, rather than nucleotide sequence, it is the complementary relationship that is conserved; although nucleotide sequences in this region have diverged during mammalian evolution, the complementary relationship between two elements has been preserved (17). (17).…”

“…The relative amount of the antisense globin RNA, which we estimate as 1-3% of the amount of its sense counterpart in spleen cells of anemic mice, is comparable to that of the antisense-strand RNAs of picornaviruses, which typically accumulate to only 1-5% of the level of the sensestrand RNA in infected cells (15,16). The structure of antisense RNA, together with our earlier observations (17), suggests a possible mechanism of the involvement of antisense globin RNA in RNA-directed RNA synthesis. The presence of a poly(U) stretch at the 5' end of antisense globin RNA indicates that, as in the poliovirus system, its synthesis starts within the 3' poly(A) region of mRNA, presumably by a mechanism similar to that employed in viral systems (14).…”

“…Importantly, rather than nucleotide sequence, it is the complementary relationship that is conserved; although nucleotide sequences in this region have diverged during mammalian evolution, the complementary relationship between two elements has been preserved (17). (17).…”

A mechanism for β‐amyloid overproduction in Alzheimer's disease: precursor‐independent generation of β‐amyloid via antisense RNA‐primed mRNA synthesis

“…The strong evolutionary conservation suggested an underlying function, namely the priming of the synthesis of a sense RNA strand by the Y-terminal element of an antisense strand. The strict 3'-terminal location of one of the complementary elements appears to be an absolute requirement, the addition of only one 'noncomplementary' nucleotide to the 3' terminus of the antisense globin strand strongly inhibiting its self-priming capacity [27]. The initial product of such a self-priming process is a chimeric antisense-sense molecule in the form of a hairpin loop (Fig.…”