ABSTRACT
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.
Subject(s)
Polyadenylation , RNA, Messenger/metabolism , Antisense Elements (Genetics) , Base Sequence , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Gene Expression , Genetic Vectors , HeLa Cells , Humans , Luciferases/biosynthesis , Luciferases/genetics , Plasmids/genetics , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , Ribonucleases , Transcription, Genetic , TransfectionABSTRACT
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.
Subject(s)
3' Untranslated Regions/metabolism , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Polyadenylation , RNA 3' Polyadenylation Signals , RNA, Messenger/metabolism , DNA-Binding Proteins , Gene Deletion , HeLa Cells , Humans , Models, Biological , Nuclear Matrix-Associated Proteins/metabolism , Octamer Transcription Factors/metabolism , PTB-Associated Splicing Factor , Polypyrimidine Tract-Binding Protein/metabolism , Protein Binding , RNA-Binding Proteins/metabolism , Ribonucleoprotein, U1 Small Nuclear/metabolism , Substrate SpecificityABSTRACT
Human CstF-77 is one of the three subunits of cleavage stimulation factor (CstF) that is essential for mRNA polyadenylation. Its Drosophila homologue, suppressor of forked [su(f)], contains an intronic poly(A) site, which can lead to a short transcript without a stop codon. By both bioinformatic searches and validation with molecular biology experiments, we found that human and mouse CstF-77 genes also contain an intronic poly(A) site, which can be utilized to produce short CstF-77 transcripts lacking sequences encoding domains that are involved in many of the CstF-77 functions. The genomic sequence surrounding the poly(A) site is highly conserved among all vertebrates, but is not present in non-vertebrate species. Using public Serial Analysis of Gene Expression (SAGE) data, we found that the intronic poly(A) site is utilized in a wide range of tissues. This finding indicates that vertebrates may employ a similar alternative polyadenylation mechanism to modulate CstF-77, highlighting the importance of the regulation of CstF-77 in various species.
Subject(s)
Alternative Splicing/genetics , Cleavage Stimulation Factor/genetics , Evolution, Molecular , Introns/genetics , Polyadenylation/genetics , Animals , Humans , Mice , Organ Specificity , Protein Subunits/genetics , RNA, Messenger/genetics , Species SpecificityABSTRACT
Topoisomerase IIalpha plays essential roles in chromosome segregation. However, it is not well understood how topoisomerase IIalpha exerts its function during mitosis. In this report, we find that topoisomerase IIalpha 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 IIalpha. 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 G2/M phase but drastically diminishes in G1/S phase. These results suggest that toposome is a dynamic complex whose assembly or activation is subject to cell cycle regulation.
