Accurate and specific polyadenylation of mRNA precursors in a soluble whole-cell lysate

Abstract

Conditions have been developed which allow for the efficient, accurate, and specific polyadenylation of exogenously added mRNA precursors in a whole-cell lysate derived from HeLa cells. Precursors are prepared by in vitro transcription using linear DNA templates in a HeLa whole-cell lysate, purified, and added to another lysate. Under optimal conditions (which are quite precise with respect to several variables), 70% or more of the precursor molecules become polyadenylated, and the length of the poly(A) segment added is controlled much as it is in vivo, giving rise to 150-300-nucleotide long stretches of poly(A). Under suboptimal conditions, both the fraction of precursor RNA that becomes polyadenylated and also the length of the poly(A) segment added are reduced. The in vitro polyadenylation reaction is also remarkably specific: Only in vitro-synthesized pre-mRNAs that contain a 3' end located at or slightly downstream from the corresponding in vivo mRNA 3' end site can be efficiently polyadenylated in vitro. These results suggest that the poly(A) polymerase requires one or more protein (or RNA) factors in order to bring about accurate and specific polyadenylation in vitro, and that the poly(A) polymerase complex must interact with a specific recognition signal at the 3' end of mRNA precursors in order to catalyze subsequent polyadenylation.