Characterization of the 5' and 3' untranslated regions in murine mdm2 mRNAs

The murine double minute 2 (mdm2) gene is essential for embryogenesis in mice that express the p53 tumor suppressor protein. Mdm2 levels must be regulated tightly because overexpression of mdm2 contributes to tumorigenesis. We investigated whether the 5' and 3' untranslated regions (UTRs) of murine mdm2 affect the expression of MDM2 proteins. Induction of mdm2 expression by p53 results in synthesis of an mdm2 mRNA with a short 5' UTR. The long 5' UTR increases internal initiation of translation of a minor MDM2 protein, p76(MDM2), without affecting the efficiency of translation of the full-length p90(MDM2). We discovered two alternative 3' untranslated regions in murine mdm2 mRNA expressed in the testis. The longer 3' UTR contains a consensus instability element, but mdm2 mRNAs containing the long and short 3' UTRs have comparable half-lives. The 3' UTRs do not affect either initiation codon use or translation efficiency. Thus, the murine 5' UTR, but not the 3'UTR, influences the ratio of the two MDM2 proteins but neither UTR affects MDM2 abundance significantly.     

UTRdb: a specialized database of 5' and 3' untranslated regions of eukaryotic mRNAs.

The 5' and 3' untranslated regions of eukaryotic mRNAs may play a crucial role in the regulation of gene expression controlling mRNA localization, stability and translational efficiency. For this reason we developed UTRdb (http://bigarea.area.ba.cnr.it:8000/BioWWW/#U TRdb), a specialized database of 5' and 3' untranslated sequences of eukaryotic mRNAs cleaned from redundancy. UTRdb entries are enriched with specialized information not present in the primary databases including the presence of nucleotide sequence patterns already demonstrated by experimental analysis to have some functional role. All these patterns have been collected in the UTRsite database so that it is possible to search any input sequence for the presence of annotated functional motifs. Furthermore, UTRdb entries have been annotated for the presence of repetitive elements.     

JKTBP1 is involved in stabilization and IRES-dependent translation of NRF mRNAs by binding to 5' and 3' untranslated regions

Heterogeneous nuclear ribonucleoprotein D-like protein (JKTBP) 1 was implicated in cap-independent translation by binding to the internal ribosome entry site in the 5′ untranslated region (UTR) of NF-κB-repressing factor (NRF). Two different NRF mRNAs have been identified so far, both sharing the common 5′ internal ribosome entry site but having different length of 3′ UTRs. Here, we used a series of DNA and RNA luciferase reporter constructs comprising 5′, 3′ or both NRF UTRs to study the effect of JKTBP1 on translation of NRF mRNA variants. The results indicate that JKTBP1 regulates the level of NRF protein expression by binding to both NRF 5′ and 3′ UTRs. Using successive deletion and point mutations as well as RNA binding studies, we define two distinct JKTBP1 binding elements in NRF 5′ and 3′ UTRs. Furthermore, JKTBP1 requires two distinct RNA binding domains to interact with NRF UTRs and a short C-terminal region for its effect on NRF expression. Together, our study shows that JKTBP1 contributes to NRF protein expression via two disparate mechanisms: mRNA stabilization and cap-independent translation. By binding to 5′ UTR, JKTBP1 increases the internal translation initiation in both NRF mRNA variants, whereas its binding to 3′ UTR elevated primarily the stability of the major NRF mRNA. Thus, JKTBP1 is a key regulatory factor linking two pivotal control mechanisms of NRF gene expression: the cap-independent translation initiation and mRNA stabilization.     

Base-pairing between untranslated regions facilitates translation of uncapped, nonpolyadenylated viral RNA

Translationally competent mRNAs form a closed loop via interaction of initiation factors with the 5' cap and poly(A) tail. However, many viral mRNAs lack a cap and/or a poly(A) tail. We show that an uncapped, nonpolyadenylated plant viral mRNA forms a closed loop by direct base-pairing (kissing) of a stem loop in the 3' untranslated region (UTR) with a stem loop in the 5' UTR. This allows a sequence in the 3' UTR to confer translation initiation at the 5'-proximal AUG. This base-pairing is also required for replication. Unlike other cap-independent translation mechanisms, the ribosome enters at the 5' end of the mRNA. This remarkably long-distance base-pairing reveals a novel mechanism of cap-independent translation and means by which mRNA UTRs can communicate.     

The implications of structured 5' untranslated regions on translation and disease

Translational control is a key step in eukaryotic gene expression. The majority of translational control occurs at the level of initiation, thus implicating the 5' untranslated region as a major site of translational regulation. Many growth-related mRNAs have atypical 5' UTRs, which are often long and GC-rich. Such features promote formation of stable secondary structure, and many mRNAs encoding proteins involved in cell growth, proliferation and apoptosis have structured 5' UTRs, which in many cases harbour internal ribosome entry sites (IRESs) and upstream open-reading frames (uORFs). In this review we discuss how secondary structural elements in the 5' UTR can regulate translation and how mutations that perturb these secondary structural elements can have implications for disease and tumourigenesis.     

Ultrabithorax and Antennapedia 5' untranslated regions promote developmentally regulated internal translation initiation.

The 5' untranslated regions (UTRs) of the Drosophila Ubx and Antp genes were tested for their ability to promote cap-independent translation initiation. The Ubx and the Antp 5' UTR were inserted between the CAT and lacZ coding sequences in a dicistronic gene and tested for IRES activity in transgenic Drosophila. Northern analysis of the mRNAs showed the presence of the predicted full-length dicistronic mRNAs. High CAT activity was expressed from the first cistron from all of the dicistronic constructs introduced into the fly genome. The dicistronic transgenic strains bearing the Ubx and Antp IRES elements expressed significant levels of beta-galactosidase (betaGAL) from the second cistron whereas little or no betaGAL was expressed in the controls lacking the IRESs. In situ analysis of betaGAL expression in the transgenic strains indicates that expression of the second cistron is spatially and temporally regulated. Although the developmental patterns of expression directed by the Antp and Ubx IRESs overlap, they exhibit several differences indicating that these IRESs are not functionally equivalent.     

Data mining of imperfect double-stranded RNA in 3' untranslated regions of eukaryotic mRNAs

Recent developments in the study of RNA silencing indicate that double-stranded RNA (dsRNA) can be used in eukaryotes to block expression of a corresponding cellular gene. There is also a large class of small non-coding RNAs having potential to form a distinct, stable stem-loop in numbers of eukaryotic genomes. We had reported that a large imperfect dsRNA structure with hundreds of base-pairs (bp) in the 3' untranslated region (3' UTR) of cytotoxic ribonuclease was correlated with the translation suppression. In this study, we search for such dsRNAs in a 3' UTR database. The occurrence rate of large dsRNA in 3' UTRs ranges from 0.01% in plant to 0.30% in vertebrate mRNAs. However, small imperfect dsRNAs of ~ 30 bp are much more prevalent than large ones. The small dsRNAs are statistically very significant and uniquely well-ordered. Most of them have the conserved structural features of pre-miRNAs. Our data mining of the dsRNAs in the 3' UTR database can be used to explore RNA-based regulation of gene expression.     

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.     

5' untranslated leader sequences of eukaryotic mRNAs encoding heat shock induced proteins.

5' untranslated leaders (5' UTLs) are suggested to play a crucial role in the selective translation of their eukaryotic mRNAs encoding heat shock proteins (HSP) during heat stress conditions. However, the structural features of the HSP mRNAs which cause this effect are mostly unknown. We have compiled the 5' UTLs from about 140 eukaryotic HSP mRNAs including vertebrates, invertebrates, higher and lower plants. A detailed analysis of these sequences according to length, A+T content, context of functional ATGs and presence of upstream non-functional ATGs was made. We observed that all these features were similar to the earlier studies in the literature based on data from HSP as well as non-HSP mRNAs. These observations were reconfirmed by intra-specific comparison of 5' UTLs from HSP and non-HSP genes. Similar to the translation element involved in the selective translation of mRNAs in polioviruses, a search for a short sequence motif complementary to highly conserved 18S rRNA was performed using a HSP mRNA database. The majority of the HSP mRNA sequences (77%) contained one or more small sequence motifs suggesting that they may function as internal ribosome entry sites for selective initiation of translation during heat stress.     

UTRdb and UTRsite: specialized databases of sequences and functional elements of 5' and 3' untranslated regions of eukaryotic mRNAs

The 5' and 3' untranslated regions of eukaryotic mRNAs may play a crucial role in the regulation of gene expression controlling mRNA localization, stability and translational efficiency. For this reason we developed UTRdb, a specialized database of 5' and 3' untranslated sequences of eukaryotic mRNAs cleaned from redundancy. UTRdb entries are enriched with specialized information not present in the primary databases including the presence of nucleotide sequence patterns already demonstrated by experimental analysis to have some functional role. All these patterns have been collected in the UTRsite database so that it is possible to search any input sequence for the presence of annotated functional motifs. Furthermore, UTRdb entries have been annotated for the presence of repetitive elements. All internet resources implemented for retrieval and functional analysis of 5' and 3' untranslated regions of eukaryotic mRNAs are accessible at http://bigarea.area.ba.cnr.it:8000/EmbIT/UTRH ome/     

Small open reading frames in 5' untranslated regions of mRnas

Using the 5'-end sequence data from 'oligo-capped' cDNAs, we generated a representative full-length cDNA dataset for 4870 RefSeq entries, and analyzed the 5' untranslated region (UTR) of these genes. To our surprise, about half of the 4870 genes had an upstream ATG before the ATG that starts the longest open reading frame (ORF), suggesting that about half of them have small ORFs in their 5' UTR of average length of 31 amino acids. They require attention for further analysis to identify their biological role.     

Sequence and translation initiation properties of the xenopus TGFbeta5, PDGF-A, and PDGF-alpha receptor 5' untranslated regions

The properties of the architecturally complex Xenopus laevis TGFbeta5, PDGF-A and PDGF-alpha receptor 5'UTRs were investigated. 5' extended cDNAs were obtained by 5'RACE, resulting in long 5'UTRs (478-710 nt) with multiple upstream AUGs (3-13), andthe potential to fold into stable structures. Injection studies suggested that the cloned PDGF-alphaR 5'UTR contains an intron. Splicing at potential 5' and 3' splice sites would result in a non-complex 5'UTR of 142 nt. The above mentioned 5'UTR characteristics are inhibitory for ribosomal scanning. Indeed, relative to the beta-globin 5'UTR, the complex 5'UTRs strongly repressed initiation of protein synthesis in pre-MBT Xenopus embryos. However, later in embryogenesis, the inhibition was partly relieved. The results show temporal translational control by these 5'UTRs. Transgenic embryos showed that the 5'UTRs allowed translation throughout the embryo; spatial control could not be observed. Interestingly, a fragment in the PDGF-A 5'UTR highly similar to an element in the human PDGF-A 5'UTR is complementary to Xenopus 18S ribosomal RNA. None of these Xenopus 5'UTRs contains an IRES, as determined by injecting bicistronic constructs.     

Pyrimidine tract binding protein and La autoantigen interact differently with the 5' untranslated regions of lentiviruses and oncoretrovirus mRNAs

Retrovirus genomic mRNA exhibits a several hundred nucleotides-long untranslated region (5' UTR) which encloses many control elements required for retrovirus replication. In addition, this 5' UTR contains translation regulatory elements, such as internal ribosome entry sites (IRESes) that have been described in oncoretroviruses, as well as in lentiviruses. UV cross-linking experiments suggested that the pyrimidine tract binding protein (PTB), a cellular protein known to regulate the activity of several picornaviral IRESes, binds to human T-cell leukemia virus (HTLV)-I RNA but not to lentiviral human immunodeficiency virus (HIV)-1, HIV-2 or simian immunodeficiency virus RNAs. To calculate the affinity of such RNA-protein interactions, we developed a new method based on the BIAcore technology. The absence of affinity of PTB for lentiviral RNAs was confirmed, whereas its affinity for HTLV-I RNAs was 1000-fold lower than for picornaviral RNAs. The BIAcore technology also revealed a significant affinity of the La autoantigen, previously described for its involvement in translational control of viral mRNAs, for HIV-1 and HTLV-I RNAs. Addition of recombinant PTB to in vitro translation experiments weakly enhanced translation initiation in the presence of HTLV-I IRES, suggesting that such an IRES requires additional trans-acting factors.