The AAUAAA sequence is required both for cleavage and for polyadenylation of simian virus 40 pre-mRNA in vitro.

Abstract

The sequence AAUAAA is found near the polyadenylation site of eucaryotic mRNAs. This sequence is required for accurate and efficient cleavage and polyadenylation of pre-mRNAs in vivo. In this study we show that synthetic simian virus 40 late pre-mRNAs are cleaved and polyadenylated in vitro in a HeLa cell nuclear extract, and that cleavage in vitro is abolished by each of four different single-base changes in AAUAAA. In this same extract, precleaved RNAs (RNAs with 3' termini at the polyadenylation site) are efficiently polyadenylated. This in vitro polyadenylation reaction also requires the AAUAAA sequence.The 3' termini of eucaryotic mRNAs are formed by endonucleolytic cleavage of an mRNA precursor and the addition of approximately 250 adenylate residues [poly(A)] to the newly formed end (8,22,26,34). The structural features of the precursor that are required for cleavage and polyadenylation have not been completely defined, but are known to include two separable elements, the AAUAAA sequence (10,13,23,27,32) and the downstream element (or TG box) (7,12,19,20,28,29,33).The AAUAAA sequence, located 6 to 26 bases before the poly(A) addition site of mammalian mRNAs, is required for cleavage. Deletions (10) or single-base-pair changes (13,23,32) in AAUAAA prevent cleavage in vivo, but do not prevent polyadenylation; thus mRNA precursors containing mutant AAUAAA sequences are cleaved inefficiently, but all precursors that are cleaved also are polyadenylated (23,32). The downstream element is located 10 to 30 bases beyond the polyadenylation site, in the 3' flanking sequence. mRNA precursors from which this sequence has been deleted are cleaved inefficiently (7,12,19,20,29). The AAUAAA sequence is quantitatively more important than the downstream element. For example, in injected oocytes, cleavage is reduced more than 50-fold by point mutations in AAUAAA (32) but only 5-to 10-fold by deletion of the downstream element (7; D. Zarkower, unpublished data). Similarly, the AAUAAA sequence is more highly conserved than the downstream element (4, 20, 32).Recently Moore and Sharp (25) demonstrated cleavage and polyadenylation of a synthetic adenovirus pre-mRNA in an extract prepared from human cells (9, 25). To establish the physiological relevance of these cell-free reactions, they must be compared with the same reactions in vivo; the in vitro reactions must be accurate and efficient and must depend on the same sequences that are required in vivo. Hart et al. (12) cleavage, polyadenylation is dependent on the AAUAAA sequence.MATERIALS AND METHODS RNA substrates. In vitro transcription was performed as described previously (21), except that the GTP concentration was 300 ,uM and 1.2 mM GpppG (P-L Biochemicals) was included to produce capped transcripts (14). After the transcription reaction, DNA was removed with RNase-free DNase (P-L), and the transcripts were purified by repeated ethanol precipitation in the presence of 2 M ammonium

A dissection of the cauliflower mosaic virus polyadenylation signal.

“…Point mutations in this sequence greatly reduce the efficiency of 3'-end formation in vivo (Fitzgerald and Shenk 1981;Montell et al 1983;Wickens and Stephenson 1984) and of both cleavage and polyadenylation in vitro Zarkower et al 1986;Conway and Wickens 1987;Wilusz et al 1989). In addition, a less well-conserved T-rich or TG-rich element, usually situated imiPresent address: Agriculture Canada Research Station, Vancouver, B.C., V6T 1X2 Canada.…”

The enzyme that adds poly(A) to mRNAs is a classical poly(A) polymerase.

“…In vivo, RNA cleavage, not transcription termination, generates the 3' end to which poly(A) is then added (13,14,16). To eliminate the need for cleavage and thus assay polyadenylation in isolation, we prepared RNAs that end at the polyadenylation site by transcription in vitro (23). An RNA containing the last 58 nucleotides of simian virus 40 (SV40) late mRNA up to the polyadenylation site was incubated in a crude HeLa cell nuclear extract (Fig.…”

“…This RNA received approximately 200 nucleotides of poly(A), as judged by its increase in gel mobility (compare lanes 1 and 2) and retention on an oligo(dT)-cellulose column (data not shown). The sequence AAUAAA, located 5 to 30 nucleotides upstream of the polyadenylation site of virtually all mRNAs (15,20), is required for this reaction: AAUCAA-containing RNA is polyadenylated less efficiently (lane 3) (23).…”

“…Genetic analyses (23), competition experiments (24), and biochemical fractionation of the extract (la, 2, 11, 17) further justify the use of this criterion.…”

“…1) (10,23). In vivo, RNA cleavage, not transcription termination, generates the 3' end to which poly(A) is then added (13,14,16).…”