Site-specific polyadenylation in a cell-free reaction

Abstract

A soluble HeLa cell extract accurately polyadenylates RNA transcribed from DNA templates containing the adenovirus L3 polyadenylation site. Regardless of the length of these DNA templates, the major polyadenylated species had 3' termini corresponding to the in vivo site. Polyadenylated RNA appears after an hour lag and only reaches maximum levels after 4 hr of incubation, a time course similar to that of splicing in this extract. Inhibitor studies suggest that the polyadenylation reaction is not coupled to active transcription. Unlike splicing in this extract where exogenous substrate is processed, addition of purified RNA precursor to the reaction does not yield product polyadenylated at L3 but rather results in addition of poly (A) to termini of the precursor. This suggests that part of the specificity of polyadenylation is established by in situ synthesis of RNA. Surprisingly, synthesis of accurately polyadenylated RNA may involve small nuclear ribonucleoprotein particles (snRNPs). The reaction is inhibited by antisera of Sm and U1 RNP specificities as well as antiserum to the nuclear antigen La, but is not inhibited by control serum and anti-(U2)RNP serum.