| 3' end formation of mRNA precursors is catalyzed by a large multisubunit complex of proteins and is a highly regulated process. We show multiple important roles for the small ubiquitin related modifier (SUMO), in regulating mammalian 3' end formation. SUMO targets multiple key proteins in the cleavage polyadenylation complex, namely, CPSF-73, the endonuclease, symplekin, a factor helping in assembly and poly(A) polymerase (PAP). The sites of modification in all these proteins were found to be conserved across species. The role of sumoylation in the 3' processing reaction was assessed by altering the sumoylation status of nuclear extracts. Downregulating sumoylation by addition of a SUMO protease or RNAi mediated depletion of the E2 enzyme ubc9 resulted in the inhibition of cleavage and polyadenylation activity. Activity of the SUMO protease inhibited the formation of functional complexes and this correlates with the ability of the protease to interact with symplekin and CPSF-73.; SUMO plays a unique role in regulating PAP function. PAP interacts strongly with ubc9 and can be modified both in vivo and in vitro, even though it lacks a consensus sumoylation site. In addition, we discovered that modified forms of PAP accumulate to high levels in certain cell lines and tissues. Most of the sumoylation sites mapped to the C-terminal regulatory region and in particular, two sites overlapped the nuclear localization signal. Strikingly, we found that sumoylation at these sites is necessary for PAP nuclear localization, as mutating these sites or coexpressing a SUMO protease with wt PAP relocalized PAP to the cytoplasm. Downregulating the sumoylation pathway reduced PAP levels. Finally in vitro sumoylation was shown to inhibit enzymatic activity of purified PAP.; Together, our results have established not only that sumoylation plays an important role in pre-mRNA 3' processing, but that it can do so in multiple, distinct ways, with positive as well as negative influences on activity. Sumoylation thus has the potential to be a potent and multi-faceted regulator of pre-mRNA processing. |
