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.     

Functional interactions between yeast mitochondrial ribosomes and mRNA 5' untranslated leaders

Translation of mitochondrial mRNAs in Saccharomyces cerevisiae depends on mRNA-specific translational activators that recognize the 5' untranslated leaders (5'-UTLs) of their target mRNAs. We have identified mutations in two new nuclear genes that suppress translation defects due to certain alterations in the 5'-UTLs of both the COX2 and COX3 mRNAs, indicating a general function in translational activation. One gene, MRP21, encodes a protein with a domain related to the bacterial ribosomal protein S21 and to unidentified proteins of several animals. The other gene, MRP51, encodes a novel protein whose only known homolog is encoded by an unidentified gene in S. kluyveri. Deletion of either MRP21 or MRP51 completely blocked mitochondrial gene expression. Submitochondrial fractionation showed that both Mrp21p and Mrp51p cosediment with the mitochondrial ribosomal small subunit. The suppressor mutations are missense substitutions, and those affecting Mrp21p alter the region homologous to E. coli S21, which is known to interact with mRNAs. Interactions of the suppressor mutations with leaky mitochondrial initiation codon mutations strongly suggest that the suppressors do not generally increase translational efficiency, since some alleles that strongly suppress 5'-UTL mutations fail to suppress initiation codon mutations. We propose that mitochondrial ribosomes themselves recognize a common feature of mRNA 5'-UTLs which, in conjunction with mRNA-specific translational activation, is required for organellar translation initiation.     

Detailed analysis of stem I and its 5' and 3' neighbor regions in the trans-acting HDV ribozyme

This prospective community-based study of infants born in inner London was undertaken to examine the association between premorbid airway function and subsequent wheezing in the first year of life and to explore the influence on this association of a family history of asthma and maternal smoking during pregnancy. Healthy Caucasian term infants were recruited shortly after birth, and physician-diagnosed wheezing episodes were identified retrospectively from medical records. Specific airway conductance was determined from plethysmographic measurements of lung volume and airway resistance, before 13 wk and prior to any respiratory illness, in 101 infants, 28 of whom experienced at least one episode of wheezing during the first year. Mean (SD) specific airway conductance was significantly diminished in infants who subsequently wheezed: 2.02 (1.07) s-1. kPa-1 and 2.60 (0.93) s-1. kPa-1, respectively (p < 0.05), and in those with a first-degree relative with asthma: 1.98 (0.83) s-1. kPa-1 and 2.60 (1.0) s-1. kPa-1, respectively (p < 0.05), but not in those whose mothers smoked during pregnancy, in whom airway resistance was, however, significantly elevated (p < 0.05). The odds ratio (95% confidence interval [CI]) for wheezing was 2.1 (1.1 to 3.8) for every unit (s-1. kPa-1) decline in specific airway conductance (p = 0.02). After adjustment for premorbid airway function, the odds of wheezing were significantly increased in those with a family history of asthma (4. 3; 95% CI, 1.3 to 13.8; p = 0.016) and those exposed to maternal smoking during pregnancy (4.9; 95% CI, 1.6 to 15.0; p = 0.005). Our findings confirm previous reports that impaired premorbid airway function precedes and predicts wheezing in the first year. Among those with a genetic predisposition to asthma, alterations in airway geometry or tone may increase susceptibility to wheezing. Maternal smoking has important and potentially preventable adverse effects on somatic growth and respiratory morbidity in early life.     

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.     

The 3' untranslated region of manganese superoxide dismutase RNA contains a translational enhancer element

A redox-sensitive protein that binds to the 3' untranslated region (UTR) of manganese superoxide dismutase (MnSOD) RNA has been described previously [Fazzone, H., Wangner, A., and Clerch, L. B. (1993) J. Clin. Invest. 92, 1278-1281; Chung, D. J., and Clerch, L. B. (1997) Am. J. Physiol. 16, L714-L719]. In the present study, cross-competition gel retardation and RNase H assays were used to identify a 41-base region located 111 bases downstream of the stop codon as the 3' UTR cis element involved in protein binding. The base sequence of this region is approximately 75% conserved among the 3' UTRs of rat, mouse, cow, and human MnSOD mRNAs at approximately the same distance downstream of the stop codon. The role of this protein-binding region in RNA translation was assessed in an in vitro rabbit reticulocyte lysate system. Translation of MnSOD RNA from which the 3' UTR element was deleted decreased 60% compared with translation of MnSOD RNA containing the 3' UTR cis element. In the presence of a specific competitor oligoribonucleotide that inhibits MnSOD RNA protein-binding activity, translation of MnSOD RNA containing the 3' UTR was decreased by 65%. Thus, both the cis element and RNA protein-binding activity were required for more efficient translation of the MnSOD. An analysis of ribosomal profiles suggests the MnSOD RNA-binding protein participates in the formation of the translation initiation complex. When MnSOD RNA-binding activity was inhibited, initiation complex formation was decreased by 50%. From the data obtained in this study, we propose that the 3' UTR cis element of MnSOD through its interaction with MnSOD RNA-binding protein may function as a translational enhancer.     

Analysis of the type 1 pilin gene cluster fim in Salmonella: its distinct evolutionary histories in the 5' and 3' regions

The type 1 pilin encoded by fim is present in both Escherichia coli and Salmonella natural isolates, but several lines of evidence indicate that similarities at the fim locus may be an example of independent acquisition rather than common ancestry. For example, the fim gene cluster is found at different chromosomal locations and with distinct gene orders in these closely related species. In this work we examined the fim gene cluster of Salmonella, the genes of which show high nucleotide sequence divergence from their E. coli counterparts, as well as a different G+C content and codon usage. DNA hybridization analysis revealed that, among the salmonellae, the fim gene cluster is present in all isolates of S. enterica but is absent from S. bongori. Molecular phylogenetic analyses of the fimA and fimI genes yield an estimate of phylogeny that is in satisfactory congruence with housekeeping and other virulence genes examined in this species. In contrast, phylogenetic analyses of the fimZ, fimY, and fimW genes indicate that horizontal transfer of this region has occurred more than once. There is also size variation in the fimZ, fimY, and fimW intergenic regions in the 3' region, and these genes are absent in isolate S2983 of subspecies IIIa. Interestingly, the G+C contents of the fimZ, fimY, and fimW genes are less than 46%, which is considerably lower than those of the other six genes of the fim cluster. This study demonstrates that horizontal transmission of all or part of the same gene cluster can occur repeatedly, with the result that different regions of a single gene cluster may have different evolutionary histories.     

Olfactory Receptor Database: a database of the largest eukaryotic gene family

The Olfactory Receptor Database (ORDB) is a WWW-accessible database that stores data on Olfactory Receptor-like molecules (ORs) and has been open to the public since June 1996. It contains a public and a private area. The public area includes published DNA and protein sequence data for ORs, links to OR models and data on their expression, chromosomal localization and source organism, as well as (i) links to bibliography through PubMed and (ii) interactive WWW-based tools, such as BLAST homology searching. The private area functions as a service to laboratories that are actively cloning receptors. Source laboratories enter the sequences of the receptor clones they have characterized to the private database and can search for identical or near identical OR sequences in both public and private databases. If another laboratory has cloned and deposited an identical or closely matching sequence there are means for communication between the laboratories to help avoid duplication of work. ORDB is available via the WWW at http://crepe.med.yale.edu/ORDB/HTML     

Exonic sequences in the 5' untranslated region of alpha-tubulin mRNA modulate trans splicing in Trypanosoma brucei

Previous studies have identified a conserved AG dinucleotide at the 3' splice site (3'SS) and a polypyrimidine (pPy) tract that are required for trans splicing of polycistronic pre-mRNAs in trypanosomatids. Furthermore, the pPy tract of the Trypanosoma brucei alpha-tubulin 3'SS region is required to specify accurate 3'-end formation of the upstream beta-tubulin gene and trans splicing of the downstream alpha-tubulin gene. Here, we employed an in vivo cis competition assay to determine whether sequences other than those of the AG dinucleotide and the pPy tract were required for 3'SS identification. Our results indicate that a minimal alpha-tubulin 3'SS, from the putative branch site region to the AG dinucleotide, is not sufficient for recognition by the trans-splicing machinery and that polyadenylation is strictly dependent on downstream trans splicing. We show that efficient use of the alpha-tubulin 3'SS is dependent upon the presence of exon sequences. Furthermore, beta-tubulin, but not actin exon sequences or unrelated plasmid sequences, can replace alpha-tubulin exon sequences for accurate trans-splice-site selection. Taken together, these results support a model in which the informational content required for efficient trans splicing of the alpha-tubulin pre-mRNA includes exon sequences which are involved in modulation of trans-splicing efficiency. Sequences that positively regulate trans splicing might be similar to cis-splicing enhancers described in other systems.     

Reduction of syndecan-1 mRNA in cervical-carcinoma cells is involved with the 3' untranslated region

Syndecan-1 is a transmembrane proteoglycan expressed predominantly in epithelial cells. Studies with immunohistochemistry have shown that syndecan-1 expression is reduced in carcinoma derived from human epidermis. Here we show that syndecan-1 mRNA, which is abundant in human primary keratinocyte (HK) and HaCaT spontaneous immortalized keratinocyte, is decreased in cervical-carcinoma cell lines. Further, in relation to a long and well-conserved 3' untranslated region (3' UTR) of syndecan-1 cDNA, we examined whether 3' UTR is involved with syndecan-1-mRNA reduction in cervical-carcinoma cells. A stable transfection experiment showed that addition of the 3' UTR does not affect expression in HaCaT, but that syndecan-1 cDNA containing the 3' UTR is not expressed efficiently selectively in cervical-carcinoma cell lines. The transient assay with CAT reporter plasmids linking the 3' UTR confirmed this, and indicated that the 3' end of the 3' UTR (nt 2285-2410) is required to influence expression in cervical-carcinoma cells. Further excessive expression of syndecan-1 suppressed growth in cervical-carcinoma cells. These results demonstrate that the reduction of syndecan-1 mRNA involved with the 3' untranslated region gives growth advantage to cervical-carcinoma cells.     

Binding of a phosphoprotein to the 3' untranslated region of the mouse protamine 2 mRNA temporally represses its translation

The synthesis of the protamines, the predominant nuclear proteins of mammalian spermatozoa, is regulated during germ cell development by mRNA storage for about 7 days in the cytoplasm of differentiating spermatids. Two highly conserved sequences, the Y and H elements present in the 3' untranslated regions (UTRs) of all known mammalian protamine mRNAs, form RNA-protein complexes and specifically bind a protein of 18 kDa. Here, we show that translation of fusion mRNAs was markedly repressed in reticulocyte lysates supplemented with a mouse testis extract enriched for the 18-kDa protein when the mRNAs contained the 3' UTR of mouse protamine 2 (mP2) or the Y and H elements of mP2. No significant decrease was seen when the fusion mRNAs contained the 3' UTR of human growth hormone. The 18-kDa protein is developmentally regulated in male germ cells, requires phosphorylation for RNA binding, and is found in the ribonucleoprotein particle fractions of a testicular postmitochondrial supernatant. We propose that a phosphorylated 18-kDa protein plays a primary role in repressing translation of mP2 mRNA by interaction with the highly conserved Y and H elements. At a later stage of male gamete differentiation, the 18-kDa protein no longer binds to the mRNA, likely as a result of dephosphorylation, enabling the protamine mRNA to be translated.