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/     

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

The important role the untranslated regions of eukaryotic mRNAs may play in gene regulation and expression is now widely acknowledged. 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 functional patterns already demonstrated by experimental analysis to have some functional role. A collection of such patterns is being collected in UTRsite database (http://bio-www.ba.cnr.it:8000/srs5/) which can also be used with appropriate computational tools to detect known functional patterns contained in mRNA untranslated regions.     

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.     

Lack of secondary structure characterizes the 5' ends of mammalian mitochondrial mRNAs

The mammalian mitochondrial genome encodes 13 proteins, which are synthesized at the direction of nine monocistronic and two dicistronic mRNAs. These mRNAs lack both 5' and 3' untranslated regions. The mechanism by which the specialized mitochondrial translational apparatus locates start codons and initiates translation of these leaderless mRNAs is currently unknown. To better understand this mechanism, the secondary structures near the start codons of all 13 open reading frames have been analyzed using RNA SHAPE chemistry. The extent of structure in these mRNAs as assessed experimentally is distinctly lower than would be predicted by current algorithms based on free energy minimization alone. We find that the 5' ends of all mitochondrial mRNAs are highly unstructured. The first 35 nucleotides for all mitochondrial mRNAs form structures with free energies less favorable than -3 kcal/mol, equal to or less than a single typical base pair. The start codons, which lie at the very 5' ends of these mRNAs, are accessible within single stranded motifs in all cases, making them potentially poised for ribosome binding. These data are consistent with a model in which the specialized mitochondrial ribosome preferentially allows passage of unstructured 5' sequences into the mRNA entrance site to participate in translation initiation.     

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.     

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.     

Functional characterization of the eukaryotic SECIS elements which direct selenocysteine insertion at UGA codons.

We investigated the requirements for selenocysteine insertion at single or multiple UGA codons in eukaryotic selenoproteins. Two functional SECIS elements were identified in the 3' untranslated region of the rat selenoprotein P mRNA, with predicted stem-loops and critical nucleotides similar to those in the SECIS elements in the type I iodothyronine 5' deiodinase (5'DI) and glutathione peroxidase selenoprotein mRNAs. Site-directed mutational analyses of three SECIS elements confirmed that conserved nucleotides in the loop and in unpaired regions of the stem are critical for activity. This indicates that multiple contact sites are required for SECIS function. Stop codon function at any of five out-of-context UGA codons in the 5'DI mRNA was suppressed by SECIS elements from the 5'DI or selenoprotein P genes linked downstream. Thus, the presence of SECIS elements in eukaryotic selenoprotein mRNAs permits complete flexibility in UGA codon position.     

Poly(A)-binding protein facilitates translation of an uncapped/nonpolyadenylated viral RNA by binding to the 3' untranslated region

Viruses employ an alternative translation mechanism to exploit cellular resources at the expense of host mRNAs and to allow preferential translation. Plant RNA viruses often lack both a 5' cap and a 3' poly(A) tail in their genomic RNAs. Instead, cap-independent translation enhancer elements (CITEs) located in the 3' untranslated region (UTR) mediate their translation. Although eukaryotic translation initiation factors (eIFs) or ribosomes have been shown to bind to the 3'CITEs, our knowledge is still limited for the mechanism, especially for cellular factors. Here, we searched for cellular factors that stimulate the 3'CITE-mediated translation of Red clover necrotic mosaic virus (RCNMV) RNA1 using RNA aptamer-based one-step affinity chromatography, followed by mass spectrometry analysis. We identified the poly(A)-binding protein (PABP) as one of the key players in the 3'CITE-mediated translation of RCNMV RNA1. We found that PABP binds to an A-rich sequence (ARS) in the viral 3' UTR. The ARS is conserved among dianthoviruses. Mutagenesis and a tethering assay revealed that the PABP-ARS interaction stimulates 3'CITE-mediated translation of RCNMV RNA1. We also found that both the ARS and 3'CITE are important for the recruitment of the plant eIF4F and eIFiso4F factors to the 3' UTR and of the 40S ribosomal subunit to the viral mRNA. Our results suggest that dianthoviruses have evolved the ARS and 3'CITE as substitutes for the 3' poly(A) tail and the 5' cap of eukaryotic mRNAs for the efficient recruitment of eIFs, PABP, and ribosomes to the uncapped/nonpolyadenylated viral mRNA.     

Tethered function assays using 3' untranslated regions

Proteins that regulate mRNA metabolism are often bipartite: an RNA binding activity confers substrate specificity, and a "functional" domain elicits the biological outcome. In some cases, these two activities reside on different polypeptides that form a complex on the mRNA. Regardless, experimental separation of RNA binding from function facilitates analysis of both properties, liberating each from the constraints of the other. "Tethered function" assays bring a protein to a reporter RNA through a designed RNA-protein interaction. The function of the tethered protein-whether that be stability, translation, localization, or transport, or otherwise-is then assessed. We refer to this approach as a "tethered function" assay, since it can be examined. The approach does not require knowledge of the natural RNA binding sites, or of the composition of the naturally occurring protein complexes. It can be useful in dissecting how proteins that act on RNAs work, and in identifying active components of multiprotein complexes. RNA-binding proteins previously have been linked to foreign RNA-binding specificities, for a wide variety of purposes. We emphasize here the particular value of tethering to the 3' untranslated region of eukaryotic mRNAs, and the opportunities it presents for the analysis of how those mRNAs are regulated. We discuss experimental design, as well as published and potential applications.     

Presence of ATG triplets in 5' untranslated regions of eukaryotic cDNAs correlates with a 'weak' context of the start codon.

MOTIVATION: The context of the start codon (typically, AUG) and the features of the 5' Untranslated Regions (5' UTRs) are important for understanding translation regulation in eukaryotic mRNAs and for accurate prediction of the coding region in genomic and cDNA sequences. The presence of AUG triplets in 5' UTRs (upstream AUGs) might effect the initiation rate and, in the context of gene prediction, could reduce the accuracy of the identification of the authentic start. To reveal potential connections between the presence of upstream AUGs and other features of 5' UTRs, such as their length and the start codon context, we undertook a systematic analysis of the available eukaryotic 5' UTR sequences. RESULTS: We show that a large fraction of 5' UTRs in the available cDNA sequences, 15-53% depending on the organism, contain upstream ATGs. A negative correlation was observed between the information content of the translation start signal and the length of the 5' UTR. Similarly, a negative correlation exists between the 'strength' of the start context and the number of upstream ATGs. Typically, cDNAs containing long 5' UTRs with multiple upstream ATGs have a 'weak' start context, and in contrast, cDNAs containing short 5' UTRs without ATGs have 'strong' starts. These counter-intuitive results may be interpreted in terms of upstream AUGs having an important role in the regulation of translation efficiency by ensuring low basal translation level via double negative control and creating the potential for additional regulatory mechanisms. One of such mechanisms, supported by experimental studies of some mRNAs, includes removal of the AUG-containing portion of the 5' UTR by alternative splicing. AVAILABILITY: An ATG_ EVALUATOR program is available upon request or at www.itba.mi.cnr.it/webgene. CONTACT: rogozin@ncbi.nlm.nih.gov, milanesi@itba.mi.cnr.it.     

Formation of the 3' end of histone mRNA: getting closer to the end

Nearly all eukaryotic mRNAs end with a poly(A) tail that is added to their 3' end by the ubiquitous cleavage/polyadenylation machinery. The only known exceptions to this rule are metazoan replication-dependent histone mRNAs, which end with a highly conserved stem-loop structure. This distinct 3' end is generated by specialized 3' end processing machinery that cleaves histone pre-mRNAs 4-5 nucleotides downstream of the stem-loop and consists of the U7 small nuclear RNP (snRNP) and number of protein factors. Recently, the U7 snRNP has been shown to contain a unique Sm core that differs from that of the spliceosomal snRNPs, and an essential heat labile processing factor has been identified as symplekin. In addition, cross-linking studies have pinpointed CPSF-73 as the endonuclease, which catalyzes the cleavage reaction. Thus, many of the critical components of the 3' end processing machinery are now identified. Strikingly, this machinery is not as unique as initially thought but contains at least two factors involved in cleavage/polyadenylation, suggesting that the two mechanisms have a common evolutionary origin. The greatest challenge that lies ahead is to determine how all these factors interact with each other to form a catalytically competent processing complex capable of cleaving histone pre-mRNAs.     

Deaggregation of eIF4E induced by mRNA 5' cap binding

All eukaryotic mRNAs contain a 5' terminal cap structure, which consists of 7-methylguanosine linked by a 5-5' triphosphate bridge to the first transcribed nucleoside (m7GpppN). Specific recognition of the cap by the eukaryotic initiation factor eIF4E plays a key role in regulation of translation initiation as a rate-limiting step. Using dynamic light scattering (DLS), the apo-form of murine eIF4E (33-217) was shown to aggregate. After addition of m7G7P, progressive deaggregation with the time of incubation in the presence of the cap analogue has been observed.     

Yeast RNA helicases of the DEAD-box family involved in translation initiation

RNA helicases of the DEAD-box and related families have been found to be required for all processes involving RNA molecules. Biochemical and genetic analyses have shown that at least two RNA helicases are required for translation initiation in yeast. Although it is generally believed that these enzymes are necessary to unwind secondary structures in the 5' untranslated region of mRNAs, their exact role has not been convincingly shown. We discuss here our present knowledge of the function of eIF4A and Ded1p, two DEAD-box proteins required for translation in eukaryotic cells.     

5'-to-3' exoribonuclease activity in bacteria: role of RNase J1 in rRNA maturation and 5' stability of mRNA

Although the primary mechanism of eukaryotic messenger RNA decay is exoribonucleolytic degradation in the 5'-to-3' orientation, it has been widely accepted that Bacteria can only degrade RNAs with the opposite polarity, i.e. 3' to 5'. Here we show that maturation of the 5' side of Bacillus subtilis 16S ribosomal RNA occurs via a 5'-to-3' exonucleolytic pathway, catalyzed by the widely distributed essential ribonuclease RNase J1. The presence of a 5'-to-3' exoribonuclease activity in B. subtilis suggested an explanation for the phenomenon whereby mRNAs in this organism are stabilized for great distances downstream of "roadblocks" such as stalled ribosomes or stable secondary structures, whereas upstream sequences are never detected. We show that a 30S ribosomal subunit bound to a Shine Dalgarno-like element (Stab-SD) in the cryIIIA mRNA blocks exonucleolytic progression of RNase J1, accounting for the stabilizing effect of this element in vivo.