Epigenetic regulation of the binding activity of translation inhibitory proteins that bind the 3' untranslated region of beta-F1-ATPase mRNA by adenine nucleotides and the redox state

Here, we describe the binding affinities and the regulation of the binding activities of the fetal liver proteins that interact with the 3' untranslated region of beta-F1-ATPase mRNA (beta-mRNA). These proteins (3'beta FBPs), which are involved in the repression of beta-mRNA translation during fetal development, have poly(A)-binding activity. Reducing agents do not affect the RNA-binding activity of 3'beta FBPs. In contrast, oxidizing and alkylating reagents abolished the binding activity of 3'beta FBPs to its target RNA element, an effect that is partially prevented by the presence of reducing agents. Interestingly, the availability of adenine nucleotides regulates in a concentration-dependent manner the binding activities of 3'beta FBPs. The results suggest that epigenetic changes that occur at the time of birth affecting both the redox and energy state of the liver play a relevant role in the regulation of the binding activities of 3'beta FBPs and therefore in the translation of beta-mRNA.     

Translational control of maskin mRNA by its 3' untranslated region

BACKGROUND INFORMATION: Maskin is a member of the TACC (transforming acidic coiled-coil) domain proteins found in Xenopus laevis oocytes and embryos. It has been implicated in the co-ordination of the spindle and has been reported to mediate translational repression of cyclin B1 mRNA. RESULTS: In the present study, we report that maskin mRNA is translationally repressed at the level of initiation in stage 4 oocytes and becomes activated in stage 6 oocytes. The translational repression of maskin mRNA correlates with the presence of a short poly(A) tail on this mRNA in stage 4 oocytes. The 3'-UTR (untranslated region) of maskin can confer the translational regulation to a reporter mRNA, and so can the 3'-UTR of human TACC3. A conserved GUCU repeat element was found to repress translation in both stage 4 and stage 6 oocytes, but deletion of this element did not abrogate repression in stage 4 oocytes. UV cross-linking experiments indicated that overlapping sets of proteins bind efficiently to both the maskin and the cyclin B1 3'-UTRs. As reported previously, CPEB [CPE (cytoplasmic polyadenylation element)-binding protein] binds to the cyclin B1 3'-UTR, but its binding to the maskin 3'-UTR is minimal. By RNA affinity chromatography and MS, we identified the EDEN-BP [EDEN (embryonic deadenylation element)-binding protein] as one of the proteins binding to both the maskin and the cyclin B1 3'-UTRs. CONCLUSIONS: Maskin mRNA is translationally regulated by at least two repressor elements and an activation element. One of the repessor elements is the evolutionarily conserved GUCU repeat. EDEN-BP binds to both the maskin and cyclin B1 3'-UTRs, indicating it may be involved in the deadenylation of these mRNAs.     

Characterization and evolution of 5' and 3' untranslated regions in eukaryotes

Untranslated regions (UTRs) in eukaryotes play a significant role in the regulation of translation and mRNA half-life, as well as interacting with specific RNA-binding proteins. However, UTRs receive less attention than more crucial elements such as genes, and the basic structural and evolutionary characteristics of UTRs of different species, and the relationship between these UTRs and the genome size and species gene number is not well understood. To address these questions, we performed a comparative analysis of 5' and 3' untranslated regions of different species by analyzing the basic characteristics of 244,976 UTRs from three eukaryote kingdoms (Plantae, Fungi, and Protista). The results showed that the UTR lengths and SSR frequencies in UTRs increased significantly with increasing species gene number while the length and G+C content in 5' UTRs and different types of repetitive sequences in 3' UTRs increased with the increase of genome size. We also found that the sequence length of 5' UTRs was significantly positively correlated with the presence of transposons and SSRs while the sequence length of 3' UTRs was significantly positively correlated with the presence of tandem repeat sequences. These results suggested that evolution of species complexity from lower organisms to higher organisms is accompanied by an increase in the regulatory complexity of UTRs, mediated by increasing UTR length, increasing G+C content of 5' UTRs, and insertion and expansion of repetitive sequences.     

La, PTB, and PAB proteins bind to the 3(') untranslated region of Norwalk virus genomic RNA

Noroviruses are human enteric caliciviruses for which no cell culture is available. Consequently, the mechanisms and factors involved in their replication have been difficult to study. In an attempt to analyze the cis- and trans-acting factors that could have a role in NV replication, the 3(')-untranslated region of the genome was studied. Use of Zuker's mfold-2 software predicted that NV 3(')UTR contains a stem-loop structure of 47 nts. Proteins from HeLa cell extracts, such as La and PTB, form stable complexes with this region. The addition of a poly(A) tail (24 nts) to the 3(')UTR permits the specific binding of the poly(A) binding protein (PABP) present in HeLa cell extracts, as well as the recombinant PABP. Since La, PTB, and PABP are important trans-acting factors required for viral translation and replication, these RNA-protein interactions may play a role in NV replication or translation.     

AP endonuclease 1 has no biologically significant 3(')-->5(')-exonuclease activity

The 3(')-->5(')-exonucleolytic activity of human apurinic/apyrimidinic endonuclease 1 (APE1) on mispaired DNA at the 3(')-termini of recessed, nicked or gapped DNA molecules was analyzed and compared with the primary endonucleolytic activity. We found that under reaction conditions optimal for AP endonuclease activity the 3(')-->5(')-exonuclease activity of APE1 manifests only at enzyme concentration elevated by 6-7 orders of magnitude. This activity does not show a preference to mismatched compared to matched DNA structures as well as to nicked or gapped DNA substrates in comparison to recessed ones. Therefore, the 3(')-->5(')-exonuclease activity associated with APE1 can hardly be considered as key mechanism that improves fidelity of DNA repair.     

Interaction between the SH3 domains and C-terminal proline-rich region in NADPH oxidase organizer 1 (Noxo1)

NADPH oxidase organizer 1 (Noxo1), harboring a PX domain, two SH3 domains, and a proline-rich region (PRR), participates in activation of superoxide-producing Nox-family NADPH oxidases. Here, we show that Noxo1 supports superoxide production in a cell-free system for gp91(phox)/Nox2 activation by arachidonic acid. This lipid enhances an SH3-mediated binding of Noxo1 to p22(phox), a protein complexed with Nox oxidases; the binding is known to be required for Nox activation. We also demonstrate that the bis-SH3 domain directly interacts with the Noxo1 PRR. The interaction appears to prevent the bis-SH3 domain and PRR from binding to their target proteins; disruption of the intramolecular interaction facilitates Noxo1 binding to p22(phox) and also allows the PRR to associate with the Nox activator Noxa1, which association is crucial for Nox activation as well. These findings suggest that Nox activation involves a conformational change leading to disruption of the bis-SH3-PRR interaction in Noxo1.     

Far upstream element binding protein 1 activates translation of p27Kip1 mRNA through its internal ribosomal entry site

The cyclin dependent kinase inhibitor p27 plays an important role in controlling the eukaryotic cell cycle by regulating progression through G1 and entry into S phase. It is often elevated during differentiation and under conditions of cellular stress. In contrast, it is commonly downregulated in cancer cells and its levels are generally inversely correlated with favorable prognosis. The cellular levels of p27 are regulated, in part, by translational control mechanisms. The 5′-untranslated region (5′-UTR) of the p27 mRNA harbors an internal ribosome entry site (IRES) which may facilitate synthesis of p27 in certain conditions. In this study, Far Upstream Element (FUSE) Binding Protein 1 (FBP1) was shown to directly bind to the human p27 5′-UTR and to promote IRES activity. An eight-nucleotide element downstream of a U-rich region within the 5′-UTR was important for FBP1 binding and p27 IRES activity. Overexpression of FBP1 enhanced endogenous p27 levels and stimulated translation initiation. In contrast, repression of FBP1 by siRNA transfection downregulated endogenous p27 protein levels. Using rabbit reticulocyte lysates, FBP1 stimulated p27 mRNA translation in vitro. The central domain of FBP1, containing four K homology motifs, was required for p27 5′-UTR RNA binding and the N terminal domain was important for translational activation. These findings indicate that FBP1 is a novel activator of p27 translation upon binding to the 5′-UTR.     

Multi-level functional genomics reveals molecular and cellular oncogenicity of patient-based 3′ untranslated region mutations

1. Download : Download high-res image (200KB)
2. Download : Download full-size image