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 se t of mRNA 3' processing factors in human T24 cells. A key enzyme in RN A 3' processing, poly(A) polymerase (PAP), was found in the cytoplasm and throughout the nucleus in a punctated pattern. A subset of the var ious isoforms of PAP was specifically concentrated at sites of RNA syn thesis in the nucleoplasm. Additionally, the other factors necessary f or RNA 3' processing, such as CstF, CPSF, and PABII, were also found a t these transcription sites. Our data show that the set of 3' processi ng factors that are presumed to be necessary for most RNA 3' cleavage and polyadenylation is indeed found at sites of RNA synthesis in the n ucleoplasm. Furthermore, sites of RNA synthesis that are particularly enriched in both PAP and PABII are found at the periphery of irregular ly shaped domains, called speckles, which are known to contain high co ncentrations of splicing factors and poly(A) RNA. Disruption of RNA 3' processing by the drug 9-beta-D-arabinofuranosyladenine caused the sp eckles to break up into smaller structures. These findings indicate th at there is a spatial and structural relationship between 3' processin g and the nuclear speckles. Our studies reveal a complex and distinct organization of the RNA 3' processing machinery in the mammalian cell nucleus. (C) 1998 Academic Press.