Two cyclooxygenase (COX) enzymes, COX-1 and COX-2, are present in human cells. While COX-1 is constitutively expressed, COX-2 is inducible and up-regulated in response to many signals. Since increased transcriptional activity accounts for only part of COX-2 up-regulation, we chose to explore other RNA processing mechanisms in the regulation of this gene. Previously, we showed that COX-2 is regulated by alternative polyadenylation, and that the COX-2 proximal polyadenylation signal contains auxiliary upstream sequence elements (USEs) that are very important in efficient polyadenylation. To explore trans-acting protein factors interacting with these cis-acting RNA elements, we performed pull-down assays with HeLa nuclear extract and biotinylated RNA oligonucleotides representing COX-2 USEs. We identified PSF, p54 nrb , PTB, and U1A as proteins specifically bound to the COX-2 USEs. We further explored their participation in polyadenylation using MS2 phage coat protein-MS2 RNA binding site tethering assays, and found that tethering any of these four proteins to the COX-2 USE mutant RNA can compensate for these cis-acting elements. Finally, we suggest that these proteins (p54 nrb , PTB, PSF, and U1A) may interact as a complex since immunoprecipitations of the transfected MS2 fusion proteins coprecipitate the other proteins.

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Topoisomerase IIα plays essential roles in chromosome segregation. However, it is not well understood how topoisomerase IIα exerts its function during mitosis. In this report, we find that topoisomerase IIα forms a multisubunit complex, named toposome, containing two ATPase/helicase proteins (RNA helicase A and RHII/Gu), one serine/threonine protein kinase (SRPK1), one HMG protein (SSRP1), and two pre-mRNA splicing factors (PRP8 and hnRNP C). Toposome separates entangled circular chromatin DNA about fourfold more efficiently than topoisomerase IIα. Interestingly, this decatenation reaction yields knotted circles, which are not seen in reactions provided with monomeric circular DNA. Our results also show that interaction among toposome-associated proteins is highest in G 2 /M phase but drastically diminishes in G 1 /S phase. These results suggest that toposome is a dynamic complex whose assembly or activation is subject to cell cycle regulation.

Mammalian gene expression can be regulated through various post-transcriptional events, including altered mRNA stability, translational control, and RNA-processing events such as 3'-end formation or polyadenylation (pA). It has become clear in recent years that pA is governed by several core sequence elements and often regulated by additional auxiliary sequence elements. These regulatory events are frequently not reproducible in in vitro assays. Therefore, in vivo methods to measure mRNA pA were developed to meet this need and are described here.

Poly(A) tails are found on most mammalian mRNAs. A “canonical” polyadenylation signal has been defined as an upstream AAUAAA hexamer core element plus a U/UG downstream core element. The reality of polyadenylation signals found in human genes, however, is quite different. Therefore, alternative polyadenylation is an important yet relatively unexplored mechanism of gene regulation. We have focused on alternative polyadenylation of human cyclooxygenase‐2 (COX‐2), which is regulated at many levels. While COX‐1 is constitutively expressed, COX‐2 is inducible and is upregulated in response to many signals. Since increased transcriptional activity accounts for only part of the upregulation of COX‐2, we chose to explore other RNA processing mechanisms in the regulation of this gene. We have previously shown that COX‐2 is regulated by alternative polyadenylation, that the COX‐2 proximal polyadenylation signal contains auxiliary upstream sequence elements (USEs), and that these USEs are very important in efficient polyadenylation (Hall‐Pogar et al., 2005). Our previous results suggest that trans‐acting factors interacting with COX‐2 USEs are very important in COX‐2 polyadenylation. To explore what trans‐acting factors may be involved, we performed a pull‐down assay with HeLa nuclear extract and biotinylated RNA oligonucleotides representing a COX‐2 USE. Specifically bound proteins were identified by mass spectrometry and were verified by Western blotting. We identified PSF, p54nrb, PTB and U1A as proteins which specifically bound to the COX‐2 USEs. We have further explored their participation in polyadenylation using MS2 coat protein tethering assays.