The 5' and 3' downstream AUG region elements are required for mosquito-borne flavivirus RNA replication

Flaviviruses require complementarity between the 5' and 3' ends of the genome for RNA replication. For mosquito-borne flaviviruses, the cyclization sequences (CS) and upstream of AUG region (UAR) elements at the genomic termini are necessary for viral RNA replication, and a third motif, the downstream of AUG region (DAR), was recently designated for dengue virus. The 3' DAR sequence is also part of a hairpin (HP-3'SL), and both complementarity between 5' and 3' DAR motifs and formation of the HP-3'SL in the absence of the 5' end are conserved among mosquito-borne flaviviruses. Using West Nile virus as a model, we demonstrate that 5'-3' DAR complementarity and HP-3'SL formation are essential for viral RNA replication.     

Conserved tertiary structural elements in the 5' nontranslated region of cardiovirus, aphthovirus and hepatitis A virus RNAs.

Statistical analyses of RNA folding in 5' nontranslated regions (5'NTR) of encephalomyocarditis virus, Theiler's murine encephalomyelitis virus, foot-and-mouth disease virus, and hepatitis A virus indicate that two highly significant folding regions occur in the 5' and 3' portions of the 5'NTR. The conserved tertiary structural elements are predicted in the unusual folding regions (UFR) for these viral RNAs. The theoretical, common structural elements predicted in the 3' parts of the 5'NTR occur in a cis-acting element that is critical for internal ribosome binding. These structural motifs are expected to be highly significant from extensive Monte Carlo simulations. Nucleotides (nt) in the conserved single-stranded polypyrimidine tract for these RNAs are involved in a distinctively tertiary interaction that is located at about 15 nt prior to the initiator AUG. Intriguingly, the proposed common tertiary structure in this study shares a similar structural feature to that proposed in human enteroviruses and rhinoviruses. Based on these common structural features, plausible base pairing models between these viral RNAs and 18 S rRNA are suggested, which are consistent with a general mechanism for regulation of internal initiation of cap-independent translation.     

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/     

Polymorphisms in the transcribed 3' untranslated region of eukaryotic genes.

In this review we present preliminary evidence for a new class of polymorphism that may be used in a systematic way to map cDNAs efficiently and to expedite the construction of a high-resolution genetic map of the human genome. Ultimately, transcribed 3' untranslated polymorphisms will warrant further study because they should be widely distributed throughout the genome within transcribed sequences, and they can be readily identified as a result of cDNA cloning and sequencing. Furthermore, these markers should be universally available on the basis of the sequence data and highly useful in linkage analyses.     

Functional equivalence of common and unique sequences in the 3' untranslated regions of alfalfa mosaic virus RNAs 1, 2, and 3.

The 3' untranslated regions (UTRs) of alfalfa mosaic virus (AMV) RNAs 1, 2, and 3 consist of a common 3'-terminal sequence of 145 nucleotides (nt) and upstream sequences of 18 to 34 nt that are unique for each RNA. The common sequence can be folded into five stem-loop structures, A to E, despite the occurrence of 22 nt differences between the three RNAs in this region. Exchange of the common sequences or full-length UTRs between the three genomic RNAs did not affect the replication of these RNAs in vivo, indicating that the UTRs are functionally equivalent. Mutations that disturbed base pairing in the stem of hairpin E reduced or abolished RNA replication, whereas compensating mutations restored RNA replication. In vitro, the 3' UTRs of the three RNAs were recognized with similar efficiencies by the AMV RNA-dependent RNA polymerase (RdRp). A deletion analysis of template RNAs indicated that a 3'-terminal sequence of 127 nt in each of the three AMV RNAs was not sufficient for recognition by the RdRp. Previously, it has been shown that this 127-nt sequence is sufficient for coat protein binding. Apparently, sequences required for recognition of AMV RNAs by the RdRp are longer than sequences required for CP binding.     

Aberrant regulation of mRNA 3' untranslated region in cancers and inflammation

Almost 10% of mammalian coding mRNAs contain in their 3' untranslated region a sequence rich in adenine and uridine residues known as AU-rich element (ARE). Many of them encode oncogenes (for instance c-Myc and c-Fos), cell cycle regulators (cyclin D1, A1, B1), cytokines (TNFalpha, IL2) and growth factors (GM-CSF) which are overexpressed in cancer or inflammatory diseases due to increased mRNA stability and/or translation. AREs are recognized by a group of proteins, collectively called AUBPs which display various functions. For instance, HuR/ELAV is mainly known to protect ARE-containing mRNAs from degradation, while AUF1, TTP and KSRP act to destabilize their bound target mRNAs and TIA/TIAR to inhibit their translation. Alterations in ARE sequences or AUBP abundance, cellular localization or activity due to post-translational modifications such as phosphorylation can promote or enhance malignancy or perturb immune homeostasis. Here, c-myc and TNFalpha are chosen as examples to illustrate how altered 3' UTR gene regulation impacts on pathologies.     

The 3' untranslated region of messages in the rumen protozoan Entodinium caudatum

The 3' untranslated regions of a number of cDNAs from the rumen protozoal species Entodinium caudatum were studied with a view to characterising their preference for stop codons, general length, nucleotide composition and polyadenylation signals. Unlike a number of ciliates, Entodinium caudatum uses UAA as a stop codon, rather than as a codon for glutamine. In addition, the 3' untranslated region of the message is generally less than 100 nucleotides in length, extremely A+T rich, and does not appear to utilise any of the conventional polyadenylation signals described in other organisms.     

Structural domains within the 3' untranslated region of Turnip crinkle virus

The genomes of positive-strand RNA viruses undergo conformational shifts that complicate efforts to equate structures with function. We have initiated a detailed analysis of secondary and tertiary elements within the 3′ end of Turnip crinkle virus (TCV) that are required for viral accumulation in vivo. MPGAfold, a massively parallel genetic algorithm, suggested the presence of five hairpins (H4a, H4b, and previously identified hairpins H4, H5, and Pr) and one H-type pseudoknot (Ψ₃) within the 3′-terminal 194 nucleotides (nt). In vivo compensatory mutagenesis analyses confirmed the existence of H4a, H4b, Ψ₃ and a second pseudoknot (Ψ₂) previously identified in a TCV satellite RNA. In-line structure probing of the 194-nt fragment supported the coexistence of H4, H4a, H4b, Ψ₃ and a pseudoknot that connects H5 and the 3′ end (Ψ₁). Stepwise replacements of TCV elements with the comparable elements from Cardamine chlorotic fleck virus indicated that the complete 142-nt 3′ end, and subsets containing Ψ₃, H4a, and H4b or Ψ₃, H4a, H4b, H5, and Ψ₂, form functional domains for virus accumulation in vivo. A new 3-D molecular modeling protocol (RNA2D3D) predicted that H4a, H4b, H5, Ψ₃, and Ψ₂ are capable of simultaneous existence and bears some resemblance to a tRNA. The related Japanese iris necrotic ring virus does not have comparable domains. These results provide a framework for determining how interconnected elements participate in processes that require 3′ untranslated region sequences such as translation and replication.     

RNA-protein interactions within the 3 ' untranslated region of vimentin mRNA.

Several functions have been attributed to protein binding within the 3'untranslated region (3'UTR) of mRNA, including mRNA localization, stability, and translational repression. Vimentin is an intermediate filament protein whose 3'untranslated sequence is highly conserved between species. In order to identify sequences that might play a role in vimentin mRNA function, we synthesized32P-labeled RNA from different regions of vimentin's 3'UTR and assayed for protein binding with HeLa extracts using band shift assays. Sequences required for binding are contained within a region 61-114 nucleotides downstream of the stop codon, a region which is highly conserved from Xenopus to man. As judged by competition assays, binding is specific. Solution probing studies of 32P-labeled RNA with various nucleases and lead support a complex stem and loop structure for this region. Finally, UV cross-linking of the RNA-protein complex identifies an RNA binding protein of 46 kDa. Fractionation of a HeLa extract on a sizing column suggests that in addition to the 46 kDa protein, larger complexes containing additional protein(s) can be identified. Vimentin mRNA has been shown to be localized to the perinuclear region of the cytoplasm, possibly at sites of intermediate filament assembly. To date, all sequences required for localization of various mRNAs have been confined to the 3'UTR. Therefore, we hypothesize that this region and associated protein(s) might be important for vimentin mRNA function such as in localization.     

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.