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 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.     

Similar features in mechanisms of translation initiation of mRNAs in eukaryotic and prokaryotic systems

Using as examples non-canonical features of translation initiation for some bacterial and mammalian mRNAs with unusual 5'- untranslated regions (5'-UTR) or lacking these regions (leaderless mRNAs), the authors of this review discuss similarities in mechanisms of translation initiation on prokaryotic and eukaryotic ribosomes.     

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.     

The untranslated regions of β-globin mRNA evolve at a functional rate in higher primates

We have sequenced the 3′ and 5′ untranslated regions of β-globin mRNAs from cebus monkey, rhesus monkey and chimpanzee. A comparison with the corresponding human sequences reveals that the rate of sequence divergence among the higher primates is the same in the 3′ and 5′ noncoding regions and that this rate is several times lower than the rate for silent substitutions in the coding regions. In addition, the rate of sequence divergence in the 3′ untranslated region of the primate β-globin mRNA is several times lower than the rate calculated for this region from other comparisons. The low rate of sequence divergence in the noncoding 3′ end of the primate β-globin mRNAs may indicate a specialized and significant function for this region in the higher primates.     

Sequence analysis and compositional properties of untranslated regions of human mRNAs

A detailed computer analysis of the untranslated regions, 5′-UTR and 3′- UTR, of human mRNA sequences is reported. The compositional properties of these regions, compared with those of the corresponding coding regions, indicate that 5′-UTR and 3′-UTR are less affected by the isochore compartmentalization than the corresponding third codon positions of mRNAs. The presence of higher functional constraints in 5′-UTR is also reported. Dinucleotide analysis shows a depletion of CpG and TpA in both sequences. A search for significant sequence motifs using the WORDUP algorithm reveals the patterns already known to have a functional role in the mRNA UTR, and several other motifs whose functional roles remain to be demonstrated. This type of analysis may be particularly useful for guiding site-directed mutagenesis experiments. In addition, it can be used for assessing the nature of anonymous sequences now produced in large amounts in megabase sequencing projects.     

Comparative analysis of orthologous eukaryotic mRNAs: potential hidden functional signals

Sequencing of multiple, nearly complete eukaryotic genomes creates opportunities for detecting previously unnoticed, subtle functional signals in non-coding regions. A genome-wide comparative analysis of orthologous sets of mammalian and yeast mRNAs revealed distinct patterns of evolutionary conservation at the boundaries of the untranslated regions (UTRs) and the coding region (CDS). Elevated sequence conservation was detected in ~30 nt regions around the start codon. There seems to be a complementary relationship between sequence conservation in the ~30 nt regions of the 5′-UTR immediately upstream of the start codon and that in the synonymous positions of the 5′-terminal 30 nt of the CDS: in mammalian mRNAs, the 5′-UTR shows a greater conservation than the CDS, whereas the opposite trend holds for yeast mRNAs. Unexpectedly, a ~30 nt region downstream of the stop codon shows a substantially lower level of sequence conservation than the downstream portions of the 3′-UTRs. However, the sequence in this poorly conserved 30 nt portion of the 3′-UTR is non-random in that it has a higher GC content than the rest of the UTR. It is hypothesized that the elevated sequence conservation in the region immediately upstream of the start codon is related to the requirement for initiation factor binding during pre-initiation ribosomal scanning. In contrast, the poorly conserved region downstream of the stop codon could be involved in the post- termination scanning and dissociation of the ribosomes from the mRNA, which requires only the mRNA–ribosome interaction. Additionally, it was found that the choice of the stop codon in mammals, but not in yeasts, and the context in the immediate vicinity of the stop codons in both mammals and yeasts are subject to strong selection. Thus, genome-wide analysis of orthologous gene sets allows detection of previously unrecognized patterns of sequence conservation, which are likely to reflect hidden functional signals, such as ribosomal filters that could regulate translation by modulating the interaction between the mRNA and ribosomes.     

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.     

Short tandem repeats are associated with diverse mRNAs encoding membrane-targeted proteins

Within the genomes of multicellular organisms, short tandem repeating sequences (STRs) are ubiquitous, yet usage patterns remain obscure. The repeats (AC)n and (GU)n appear frequently in the untranslated regions (UTRs) of messenger RNAs (mRNAs). To investigate STR usage patterns, we used three approaches: (1) comparisons of individual mRNA database sequences including annotations and linked references, (2) statistical analysis of complete, UTR databases and (3) study of a large gene family, the aquaporins. Among 500 (AC)n- or (GU)n-containing mRNAs, 58 (12%) had known functions. Of these, 50 (86%) encoded proteins whose activities involved membranes or lipids, including integral membrane proteins, peripheral membrane proteins, ion channels, lipid enzymes, receptors and secreted proteins. A control sequence (AU)n also occurred in mRNAs, but only 5% encoded membrane-related functions. Investigation of all reported 3' UTR sequences, demonstrated that the STR (AC)n was 9 times more common in mRNAs encoding membrane functions than in the total UTR database (P < 0.001). Similarly, (GU)n was 8 times more common in membrane-function mRNAs than in the total database (P < 0.001). These observations suggest that (AC)n and (GU)n may be UTR signals for some mRNAs encoding membrane-targeted proteins.     

Prediction of eukaryotic mRNA translational properties.

MOTIVATION: It is well known that eukaryotic mRNAs are translated at different levels depending on their sequence characteristics. Evaluation of mRNA translatability is of importance in prediction of the gene expression pattern by computer methods and to improve the recognition of mRNAs within cloned nucleotide sequences. It may also be used in biotechnological experiments to optimize the expression of foreign genes in transgenic organisms. RESULTS: The sets of 5' untranslated region characteristics, significantly different between mRNAs encoding abundant and scarce polypeptides, were determined for mammals, dicot plants and monocot plants, and collected in the LEADER_RNA database. Computer tools for the prediction of mRNA translatability are presented. AVAILABILITY: Programs for mRNA translatability prediction are available at http://wwwmgs.bionet.nsc. ru/programs/acts2/mo_mRNA.htm (for monocots), http://wwwmgs.bionet. nsc.ru/programs/acts2/di_mRNA.htm (for dicots) and http://wwwmgs. bionet.nsc.ru/programs/acts2/ma_mRNA.htm (for mammals). The LEADER_RNA database may be accessed at: http://wwwmgs.bionet.nsc. ru/systems/LeaderRNA/.     

Translational control of growth factor and proto-oncogene expression

Control of translation is now understood to be one of the major regulatory events in eukaryotic gene expression. Moreover there is evidence which suggests that aberrant expression of growth-related genes by translational mechanisms makes a significant contribution to cell transformation. However, the mechanisms which regulate translation of specific growth-related mRNAs have yet to be fully elucidated. The majority of these mRNAs have long 5' untranslated regions (UTRs) and three features which are important in translational control have been identified, namely (i) structured regions which inhibit the scanning mechanisms of translation, (ii) regulatory upstream open reading frames and (iii) internal ribosome entry segments which are capable of initiating cap-independent translation. In this review the translational regulation of specific mRNAs encoding growth factors and proto-oncogenes by these three mechanisms will be discussed, together with examples of altered translational regulation in neoplasia.     

Eukaryotic mRNAs encoding abundant and scarce proteins are statistically dissimilar in many structural features

It is well known that non-coding mRNA sequences are dissimilar in many structural features. For individual mRNAs correlations were found for some of these features and their translational efficiency. However, no systematic statistical analysis was undertaken to relate protein abundance and structural characteristics of mRNA encoding the given protein. We have demonstrated that structural and contextual features of eukaryotic mRNAs encoding high- and low-abundant proteins differ in the 5′ untranslated regions (UTR). Statistically, 5′ UTRs of low-expression mRNAs are longer, their guanine plus cytosine content is higher, they have a less optimal context of the translation initiation codons of the main open reading frames and contain more frequently upstream AUG than 5′ UTRs of high-expression mRNAs. Apart from the differences in 5′ UTRs, high-expression mRNAs contain stronger termination signals. Structural features of low- and high-expression mRNAs are likely to contribute to the yield of their protein products.     

The origins of eukaryotic gene structure

Most of the phenotypic diversity that we perceive in the natural world is directly attributable to the peculiar structure of the eukaryotic gene, which harbors numerous embellishments relative to the situation in prokaryotes. The most profound changes include introns that must be spliced out of precursor mRNAs, transcribed but untranslated leader and trailer sequences (untranslated regions), modular regulatory elements that drive patterns of gene expression, and expansive intergenic regions that harbor additional diffuse control mechanisms. Explaining the origins of these features is difficult because they each impose an intrinsic disadvantage by increasing the genic mutation rate to defective alleles. To address these issues, a general hypothesis for the emergence of eukaryotic gene structure is provided here. Extensive information on absolute population sizes, recombination rates, and mutation rates strongly supports the view that eukaryotes have reduced genetic effective population sizes relative to prokaryotes, with especially extreme reductions being the rule in multicellular lineages. The resultant increase in the power of random genetic drift appears to be sufficient to overwhelm the weak mutational disadvantages associated with most novel aspects of the eukaryotic gene, supporting the idea that most such changes are simple outcomes of semi-neutral processes rather than direct products of natural selection. However, by establishing an essentially permanent change in the population-genetic environment permissive to the genome-wide repatterning of gene structure, the eukaryotic condition also promoted a reliable resource from which natural selection could secondarily build novel forms of organismal complexity. Under this hypothesis, arguments based on molecular, cellular, and/or physiological constraints are insufficient to explain the disparities in gene, genomic, and phenotypic complexity between prokaryotes and eukaryotes.     

Expression of the genes coding for the skeletal muscle and cardiac actions in the heart

Several types of evidence indicate that the gene coding for the skeletal muscle actin is expressed in the rat heart: 1) A recombinant plasmid containing an insert with a nucleotide sequence identical to that of the homologous region of skeletal muscle actin gene was isolated from a cDNA library prepared on rat cardiac mRNA template. 2) Using specific probes it was found that the hearts of newborn rats contain a significant amount of skeletal muscle actin mRNA. The quantity of this mRNA in the heart decreases during development. 3) The skeletal muscle actin gene is DNAase I sensitive in nuclei from rat heart tissue. A plasmid containing a cDNA insert homologous to a part of the cardiac actin mRNA was isolated and sequenced. It was found that in spite of the great similarity between the amino acid sequence of the skeletal muscle and cardiac actins, the nucleotide sequences of the two mRNAs are considerably divergent. There is only limited sequence homology between the 3' untranslated regions of the two mRNAs. However, there is an extensive sequence homology between the 3' untranslated regions of the rat and human cardiac mRNAs, suggesting a functional role for this region of the gene or mRNA.