Specific trans-acting proteins interact with auxiliary RNA polyadenylation elements in the COX-2 3′-UTR

Abstract

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.

U1A Regulates 3′ Processing of the Survival Motor Neuron mRNA

“…We find that U1A binds directly to sequences flanking the polyadenylation site in the 3Ј-UTR of the SMN pre-mRNA. In addition to being a component of the U1 snRNP, U1A is also known to bind to several mRNAs and regulates their polyadenylation (35,47,48). We show here that binding of U1A inhibits polyadenylation of the SMN pre-mRNA by specifically blocking 3Ј cleavage of the transcript by the CPSF73 endonuclease.…”

“…Interestingly, we also identified SNRPA (U1A), a bifunctional protein primarily involved in pre-mRNA splicing but also shown to function in regulated polyadenylation. We additionally identified NONO (p54 nrb ), SFPQ (PSF), and PTBP1 (PTB), three factors known to bind snRNP-free U1A and function with it in polyadenylation (48,52,53). In addition to factors involved in polyadenylation, we identified Gemin5, the RNA-binding protein of the SMN complex (54).…”

Novel upstream and downstream sequence elements contribute to polyadenylation efficiency

“…We have previously shown that U1A, PTB, PSF and p54 proteins bind to upstream polyadenylation efficiency elements present in the COX-2 polyadenylation signal 32 and others have demonstrated that a similar complex of proteins binds to similar elements in the prothrombin 3'UTR. 33 These protein factors are especially note-worthy since they are also mRNA splicing factors, suggesting the functional coupling of splicing and 3'end formation.…”

To polyadenylate or to deadenylate

“…Additional cis-acting elements that function to alter the efficiency of polyadenylation have also been identified as auxiliary RNA elements. 117,118 However, once the signal is recognized by the cleavage/ polyadenylation complex in the nucleus, the full-length poly(A) tail is added to the cleaved 3' end. No regulatory mechanisms have been described yet at this first round of polyadenylation, suggesting that addition of a poly(A) tail is a default modification after mRNA 3' cleavage in the nucleus.…”