We developed a two-step purification of mammalian
polyadenylation complexes assembled in vitro. Biotinylated
pre-mRNAs containing viral or immunoglobulin poly(A) sites
were incubated with nuclear extracts prepared from mouse
myeloma cells under conditions permissive for in vitro
cleavage and polyadenylation and the mixture was fractionated
by gel filtration; complexes containing biotinylated pre-mRNA
and bound proteins were affinity purified on avidin-agarose
resin. Western analysis of known components of the polyadenylation
complex demonstrated copurification of polyadenylation
factors with poly(A) site-containing RNA but not with control
RNA substrates containing either no polyadenylation signals
or a point mutation of the AAUAAA polyadenylation signal.
Polyadenylation complexes that were assembled on exogenous
RNA eluted from the Sephacryl column in fractions consistent
with their size range extending from 2 to 4 × 106Mr. Complexes endogenous to the extract
were of approximately the same apparent size, but more
heterogeneous in distribution. This method can be used
to study polyadenylation/cleavage complexes that may form
upon a number of different RNA sequences, an important
step towards defining which factors might differentially
associate with specific RNAs.