A subset of poly(A) polymerase is concentrated at sites of RNA synthesis and is associated with domains enriched in splicing factors and poly(A) RNA.

We have performed a detailed study of the spatial distribution of a set of mRNA 3' processing factors in human T24 cells. A key enzyme in RNA 3' processing, poly(A) polymerase (PAP), was found in the cytoplasm and throughout the nucleus in a punctated pattern. A subset of the various isoforms of PAP was specifically concentrated at sites of RNA synthesis in the nucleoplasm. Additionally, the other factors necessary for RNA 3' processing, such as CstF, CPSF, and PABII, were also found at these transcription sites. Our data show that the set of 3' processing factors that are presumed to be necessary for most RNA 3' cleavage and polyadenylation is indeed found at sites of RNA synthesis in the nucleoplasm. Furthermore, sites of RNA synthesis that are particularly enriched in both PAP and PABII are found at the periphery of irregularly shaped domains, called speckles, which are known to contain high concentrations of splicing factors and poly(A) RNA. Disruption of RNA 3' processing by the drug 9-beta-D-arabinofuranosyladenine caused the speckles to break up into smaller structures. These findings indicate that there is a spatial and structural relationship between 3' processing and the nuclear speckles. Our studies reveal a complex and distinct organization of the RNA 3' processing machinery in the mammalian cell nucleus.