RNA sequence containing hexanucleotide AAUAAA directs efficient mRNA polyadenylation in vitro.

Abstract

To determine whether a specific nucleotide sequence is required to direct polyadenylation of a simian virus 40 early pre-mRNA in a soluble HeLa whole-cell lysate, we constructed a series of rearranged and deleted DNA templates, transcribed them in vitro, and determined whether the resultant RNAs could be polyadenylated when incubated in whole-cell lysate. When a 237-base-pair DNA fragment encoding the 3' end of the simian virus 40 early pre-mRNA was transferred to recombinant plasmids encoding RNAs that were not substrates for polyadenylation, the resultant RNAs could now be polyadenylated efficiently. In one case, the chimeric RNA was polyadenylated even more efficiently than was the original simian virus 40 early transcript. Analysis of the RNAs produced from the deletion mutant templates revealed that only RNAs containing at least one copy of the AAUAAA sequence situated near the 3' end and implicated in 3'-end formation and polyadenylation in vivo could be polyadenylated in vitro. Surprisingly, this sequence directed polyadenylation of pre-mRNAs not only when near the RNA 3' end, i.e., 50 nucleotides or less away, but also when the 3' end was situated over 400 nucleotides downstream. Thus, our results show that a polyadenylic acid polymerase activity in HeLa lysates can recognize a specific nucleotide sequence in pre-mRNA and then, in the absence of the nucleolytic cleavage that presumably occurs in vivo, locate the RNA 3' end and use it as a primer for polyadenylic acid synthesis.

Four factors are required for 3'-end cleavage of pre-mRNAs.

“…One of these factors, SF, is required for both specific cleavage and specific polyadenylation reactions, both of which require the poly(A) signal AAUAAA (Manley 1983;Manley et al 1985;Zarkower et al 1986;Skolnik-David et al 1987). As SF can complement PAP that has only a nonspecific catalytic function, to impart specificity, it is most likely that SF recognizes the polyadenylation signal and interacts in some fashion with PAP.…”

“…In the absence of divalent cation, pre-RNAs are accurately cleaved but not polyadenylated, generating upstream and downstream cleavage products. In the presence of Mg^^, pre-RNAs can be polyadenylated at the 3' ends of either the pre-RNAs, themselves, or upstream cleavage products (Manley 1983;Manley et al 1985;Moore and Sharp 1985;Zarkower et al 1986). Both reactions absolutely require the conserved AAUAAA signal sequence (Proudfoot and Brownlee 1976), and the 'Cuitent address: Department of Biological Sciences, Stanford University, Stanford, California 94305 USA.…”

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

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

“…10,1990 on May 11, 2018 Fig. 1) was loaded onto the column, and the bound material (HS-500) was eluted with buffer A containing 500 mM KCl.…”

Multiple factors are required for poly(A) addition to a mRNA 3' end.

“…First, there is no indication, from either in vivo (Nevins and Darnell 1978;Manley et al 1982), or in vitro studies, for a lag between cleavage and polyadenylation; a cleaved but nonpolyadenylated transcript has never been ob served. Second, one of the sequence elements required for 3'-end cleavage, the AAUAAA, is also required for in vitro polyadenylation (Manley et al 1985;Zarkow^er et al 1986). Indirect RNase protection experiments in vitro suggest the formation of a complex involving the AAUAAA, and competition experiments suggest the possibility that the same complex is involved in both cleavage and polyadenylation (Zarkower and Wickens 1987).…”