In this study we provide evidence that CUGBP1 associates to the 5′-UTR of human p27 mRNA and inhibits expression of p27 (). Overexpression of CUGBP1 is associated with lower levels of endogenous p27 and also represses p27 IRES activity. In contrast, knockdown of CUGBP1 using siRNAs leads to enhanced expression of p27 that correlates with increased IRES activity. Recombinant CUGBP1 protein addition specifically represses p27 IRES reporter mRNA translation in vitro in a dose dependent manner. These findings suggest that association of CUGBP1 to the p27 5′-UTR interferes with IRES-dependent initiation of translation. The region of the 5′-UTR that is recognized by CUGBP1 is a U-rich element located ~40 nucleotides upstream of the start codon. This U-rich element is highly similar to the consensus CUGBP1 binding site that was reported by Marquis et al. suggesting a direct interaction between CUGBP1 and the p27 5′-UTR. However, we were unable to detect direct binding of recombinant CUGBP1 to p27 5′-UTR using RNA Electrophoresis Mobility Shift Assay (REMSA) (data not shown). This is probably because that CUGBP1 is indirectly associated to the p27 5′UTR. It may also because that CUGBP1 binding to the mRNA requires other factors. The region of the p27 5′-UTR that includes the U-rich element exists as a large single-stranded loop that functions in ribosome recruitment during initiation of translation.10
CUGBP1 binding may therefore interfere with p27 translation by blocking ribosome recruitment.
In addition to U-rich or UG-rich sequences, CUGBP1 has been shown to interact with specific GC-rich elements. It has been suggested that phosphorylation acts as a switch that determines whether CUGBP1 binds to U-rich elements or GC-rich elements.18,20
Unphosphorylated CUGBP1 may preferentially bind to U-rich elements while phosphorylated CUGBP1 may preferentially bind to GC-rich target sites.17
CUGBP1 is also subject to phosphorylation by multiple protein kinases which further contributes to its binding specificity. Salisbury et al. showed that CUGBP1 is phosphorylated by Akt at serine 28 and that this enhances binding to GC-islands in the 5′ region of the cyclin D1 mRNA in proliferating myocytes. Skeletal muscles from myotonic dystrophy (DM1), which overexpress CUGBP1 and have higher rates of proliferation than normal skeletal muscles, express elevated levels of cyclin D1 but reduced levels of p27.18
The same group showed that CUGBP1 is phosphorylated at serine 302 by cyclin D3/CDK4. Phosphorylation at this site is associated with differentiation and enhances binding of CUGBP1 to the 5′ regions of p21 and C/EBPβ mRNAs.18
It will be of interest to determine if phosphorylation of CUGBP1 also modulates binding to the p27 mRNA 5′-UTR.
Recently, it was shown that CUGBP1 plays a role in mediating decay of short-lived mRNAs by binding to a GU-rich consensus sequence.21
This sequence was found in the 3′-UTR of the transcripts that are destabilized in a CUGBP1-dependent manner. However, unlike destabilized mRNAs, the CUGBP1 binding site in the p27 message is within the 5′-UTR. Thus it appears that the position of the binding site, as well as the specific RNA sequence, is important for CUGBP1 function. CUGBP1 has been shown to play a role in regulating IRES-dependent translation of the cytoplasmic serine hydroxymethyltransferase (cSHMT) mRNA.22
In this case CUGBP1 is thought to bind to an element in the 3′-UTR and interact with H ferritin to promote initiation of translation. This is in contrast to our finding that CUGBP1 binds to the 5′-UTR of the p27 mRNA and inhibits IRES activity. Again this suggests that position of the binding site is an important factor in determining the consequences of CUGBP1 interactions with mRNAs.
CUGBP1 has both nuclear and cytosolic functions.17
In MCF7 cells significant amounts of CUGBP1 were found in the nucleus but only cytosolic CUGBP1 was able to associate with the p27 5′-UTR. The reason for this difference is not yet known. The subcellular localization of CUGBP1 is thought to be regulated by phosphorylation, with the hypophosphorylated form accumulating in the nucleus.23
It is possible that cytosolic and nuclear CUGBP1 are differentially modified and only cytosolic form of CUGBP1 is able to associate with the p27 5′-UTR. It is also possible that CUGBP1 binding to the p27 5′-UTR requires other factors that are also differentially localized in the cytosol and nucleus. The differential binding may also because that all nuclear CUGBP1 was bound to mRNAs and no free CUGBP1 was available.
How CUGBP1 inhibits cap-independent translation and how CUGBP1 cooperates with other p27 5′-UTR-binding proteins in regulating p27 expression are questions that will require additional experiments. Finally, it will be important to explore the possibility that CUGBP1 plays a role in mediating the observed changes in p27 levels during the normal process of cellular differentiation and in diseases such as cancer and DM1.