Complementarity between the mRNA 5' untranslated region and 18S ribosomal RNA can inhibit translation

In eubacteria, base pairing between the 3' end of 16S rRNA and the ribosome-binding site of mRNA is required for efficient initiation of translation. An interaction between the 18S rRNA and the mRNA was also proposed for translation initiation in eukaryotes. Here, we used an antisense RNA approach in vivo to identify the regions of 18S rRNA that might interact with the mRNA 5' untranslated region (5' UTR). Various fragments covering the entire mouse 18S rRNA gene were cloned 5' of a cat reporter gene in a eukaryotic vector, and translation products were analyzed after transient expression in human cells. For the largest part of 18S rRNA, we show that the insertion of complementary fragments in the mRNA 5' UTR do not impair translation of the downstream open reading frame (ORF). When translation inhibition is observed, reduction of the size of the complementary sequence to less than 200 nt alleviates the inhibitory effect. A single fragment complementary to the 18S rRNA 3' domain retains its inhibitory potential when reduced to 100 nt. Deletion analyses show that two distinct sequences of approximately 25 nt separated by a spacer sequence of 50 nt are required for the inhibitory effect. Sucrose gradient fractionation of polysomes reveals that mRNAs containing the inhibitory sequences accumulate in the fractions with 40S ribosomal subunits, suggesting that translation is blocked due to stalling of initiation complexes. Our results support an mRNA-rRNA base pairing to explain the translation inhibition observed and suggest that this region of 18S rRNA is properly located for interacting with mRNA.     

Isolation and characterization of Chinese hamster ribosomal DNA clones

We have examined the ribosomal RNA (rRNA) genes of the Chinese hamster ovary (CHO) cell line. A partial EcoRI library of genomic CHO DNA was prepared using lambda Charon-4A. We isolated two recombinants containing the region transcribed as 45S pre-rRNA and 13 kb of external spacer flanking 5' and 3' to the transcribed region. These sequences show restriction site homology with the vast majority of the genomic sequences complementary to rRNA. In addition to this form of rDNA, Southern blot analysis of EcoRI-cut CHO genomic DNA reveals numerous minor fragments ranging from 2 to 19 kb which are complementary to 18S rRNA. We isolated one clone which contains the 18S rRNA gene and sequences 5' which appear to contain length heterogeneity within the non-transcribed spacer region. We have nine additional cloned EcoRI fragments in which the homology with 18S rRNA is limited to a 0.9-kb EcoRI-HindIII fragment. This EcoRI-HindIII fragment is present in each of the cloned EcoRI fragments, and is flanked on both sides by apparently nonribosomal sequences which bear little restriction site homology with each other or the major cloned rDNA repeat.     

The mitochondrial genome of Biomphalaria glabrata (Gastropoda: Basommatophora), intermediate host of Schistosoma mansoni

The complete mitochondrial (Mt) genome of the gastropod Biomphalaria glabrata, a major intermediate host for the human parasite Schistosoma mansoni, was sequenced. The circular genome, the first determined from a basommatophoran snail, is AT rich (74.6%) and the smallest Mt genome (13,670 nucleotides [nt]) characterized from mollusks to date. Sequences from 2 B. glabrata strains, M-line and 1742, differed by only 18 nt. Phylogenetic analysis of 16S and ND1 sequences confirmed the Brazilian ancestry of both B. glabrata strains. Gene predictions indicated 22 transfer RNA, 12S and 16S ribosomal RNA (rRNA), and 13 protein-encoding genes, as is typical for metazoans. Of the mollusk Mt genomes currently known, the gene order was most similar to that of stylommatophoran gastropods, concordant with the monophyly of pulmonate gastropods. Screening of GenBank (expressed sequence tags database [dbEST]) with the Mt sequence identified 108 entries from B. glabrata as Mt-derived sequences, including 12S and 16S rRNA sequences. Moreover, 11 sequences originating from the Mt genome of B. glabrata were identified among EST entries ascribed to intramolluskan stages of S. mansoni. The availability of this Mt sequence will facilitate further molecular investigations into the biology of Biomphalaria sp. and interactions between this intermediate host and intramolluskan stages of S. mansoni.     

Comparison of the nucleotide sequence of soybean 18S rRNA with the sequences of other small-subunit rRNAs

We present the sequence of the nuclear-encoded ribosomal small-subunit RNA from soybean. The soybean 18S rRNA sequence of 1807 nucleotides (nt) is contained in a gene family of approximately 800 closely related members per haploid genome. This sequence is compared with the ribosomal small-subunit RNAs of maize (1805 nt), yeast (1789 nt), Xenopus (1825 nt), rat (1869 nt), and Escherichia coli (1541 nt). Significant sequence homology is observed among the eukaryotic small-subunit rRNAs examined, and some sequence homology is observed between eukaryotic and prokaryotic small-subunit rRNAs. Conserved regions are found to be interspersed among highly diverged sequences. The significance of these comparisons is evaluated using computer simulation of a random sequence model. A tentative model of the secondary structure of soybean 18S rRNA is presented and discussed in the context of the functions of the various conserved regions within the sequence. On the basis of this model, the short base-paired sequences defining the four structural and functional domains of all 18S rRNAs are seen to be well conserved. The potential roles of other conserved soybean 18S rRNA sequences in protein synthesis are discussed.     

The terminal sequences of Bombyx mori 18S ribosomal RNA.

The 5' and 3' terminal T1 oligonucleotides of 32p-labelled B. mori 18S ribosomal RNA were isolated by a two dimensional electrophoretic (diagonal) technique. Nucleotide sequence analysis showed that the 3' terminal fragment, (G)AUCAUUAOH, is identical to that previously obtained from the 18S rRNA of several other eukaryotic species. The sequence of the B. mori 5' terminal fragment is pUCCUCG.     

Comparison of the nucleotide sequence of soybean 18S rRNA with the sequences of other small-subunit rRNAs

Summary We present the sequence of the nuclearencoded ribosomal small-subunit RNA from soybean. The soybean 18S rRNA sequence of 1807 nucleotides (nt) is contained in a gene family of approximately 800 closely related members per haploid genome. This sequence is compared with the ribosomal small-subunit RNAs of maize (1805 nt), yeast (1789 nt),Xenopus (1825 nt), rat (1869 nt), andEscherichia coli (1541 nt). Significant sequence homology is observed among the eukaryotic small-subunit rRNAs examined, and some sequence homology is observed between eukaryotic and prokaryotic small-subunit rRNAs. Conserved regions are found to be interspersed among highly diverged sequences. The significance of these comparisons is evaluated using computer simulation of a random sequence model. A tentative model of the secondary structure of soybean 18S rRNA is presented and discussed in the context of the functions of the various conserved regions within the sequence. On the basis of this model, the short basepaired sequences defining the four structural and functional domains of all 18S rRNAs are seen to be well conserved. The potential roles of other conserved soybean 18S rRNA sequences in protein synthesis are discussed.     

The 26S rRNA binding ribosomal protein equivalent to bacterial protein L11 is encoded by unspliced duplicated genes in Saccharomyces cerevisiae.

Transformant phages expressing L15, a yeast ribosomal protein which binds to 26S rRNA and interacts with the acidic ribosomal proteins, were isolated by screening a yeast cDNA expression library in lambda gt11 with specific monoclonal antibodies. Using yeast DNA HindIII fragments that hybridize with the cDNA insert from the L15-expressing clones, minilibraries were prepared in pUC18, which were afterward screened with the same cDNA probe. In this way, plasmids carrying two different types of genomic DNA inserts were obtained. The inserts were subcloned and sequenced and we found a similar coding sequence in both cases flanked by 5' and 3' regions with very low homology. Sequences homologous to the consensus TUF-binding UAS boxes are present in the 5' flanking regions of both genes. Southern analysis revealed the presence of two copies of the L15 gene in the Saccharomyces cerevisiae genome, which are located in different chromosomes. The encoded amino acid sequence corresponds, as expected, to protein L15 and shows a high similarity to bacterial ribosomal protein L11.     

Isolation from rat liver and sequence of a RNA fragment containing 32 nucleotides from position 5 to 36 from the 3'' end of ribosomal 18S RNA.

Crude tRNA isolated from rat liver by the method of Rogg et al. (Biochem. Biophys. Acta 195, 13-15 1969) contains N6-dimethyladenosine (m6-2A) and was therefore fractionated in order to identify the m6-2A-containing RNAs. A unique species of RNA was purified which contained all the m62A present in the crude tRNA. Sequence analysis by postlabeling with gamma-32p-ATP and polynucleotide kinase revealed that this RNA represents the 32 nucleotides AAGGUUUC(C)U GUAGGUGm62Am62ACCUGCGGAAGGAUC from position 5 to 36 of the 3' terminus of ribosomal 18S RNA. The 36 nucleotide long sequence from the 3' end of rat liver 18S rRNA exhibits extensive homology with the corresponding sequence of E. coli 16S rRNA and with the 21 nucleotide long 3' terminal sequence so far known from Saccharomyces carlsbergensis 17S rRNA. A heterogeneity in this sequence provides the first evidence on the molecular level for the existence of (at least) two sets of redundant ribosomal 18S RNA genes in the rat.     

Isolation of cDNA clones coding for mitochondrial 16S ribosomal RNA from the crustacean Artemia

cDNA clones coding for Artemia mitochondrial 16S ribosomal RNA (rRNA) have been isolated. The clones cover from nucleotide 650 of the RNA molecule to its 3' end. The comparison of Artemia sequence with both vertebrate and invertebrate mitochondrial 16S rRNA sequences has shown the existence of regions of high similarity between them. A model for the secondary structure of the 3' half of Artemia mitochondrial 16S rRNA is proposed. The size of the rRNA molecule has been estimated at 1.35 kb. Despite the similarity of the Artemia gene to insect rRNA in size, sequence and secondary structure, the G + C content of the Artemia gene (42%) is closer to that of mammals than to the insect genes. The number of mitochondria in Artemia has been estimated at 1500 per diploid genome in the cyst and 4000 in the nauplius. In contrast, the amount of mt 16S rRNA is constant at all stages of Artemia development.     

The murine gene encoding the highly conserved Sm B protein contains a nonfunctional alternative 3' splice site.

The cDNA and partial genomic nucleotide (nt) sequences were derived for the mouse Sm B polypeptide and compared to the cDNA and genomic sequences encoding human Sm B. The deduced amino acid (aa) sequences from the mouse and human genes are identical with the exception of a single conserved aa substitution, accounting for the ability of anti-Sm antibodies to recognize the Sm polypeptides from a broad range of species. The genomic sequence of mouse B gene is similar to the human B genomic locus that extends from exon 6 to exon 7. These loci include conservation of both 3' alternative splice sites and putative branch points required to process B and B' mRNAs in human cells. However, the nt sequence downstream from the putative distal 3' splice junction and single nt flanking the 3' splice site consensus sequence, differ between mouse and human B. This results in a murine mRNA with a different predicted secondary structure around the distal 3' splice site when compared to humans. Thus, secondary structural constraints in the mRNA or changes in the exon sequence might prevent recognition of this alternative splice site to form B' mRNA in murine tissues.     

Carp growth hormone: molecular cloning and sequencing of cDNA

cDNA clones of the fish Cyprinus carpio growth hormone (GH) mRNA have been isolated from a cDNA library prepared from carp pituitary gland poly(A)+RNA. The nucleotide sequence of one of the carp GH cDNA clones containing an insert of 1164 nucleotides (nt) was determined. The cDNA sequence was found to encode a polypeptide of 210 amino acids (aa) including a signal peptide of 22 aa and to contain 5' and 3' untranslated regions of the mRNA of 36 and 498 nt, respectively. The carp GH presents a 63% amino acid sequence homology with the salmon GH, has structural features common with other GH polypeptides of mammalian or avian origin and contains domains of conserved sequence near the N- and C-terminal regions. Southern blot hybridization of carp genomic DNA with GH cDNA probes shows the presence of at least two GH-coding sequences in the fish genome.     

Nucleotide sequence of the 5'- and 3'- domains for rabbit 18S ribosomal RNA.

By direct RNA sequence analysis we have determined the primary structures of both the 5' and 3' domains for rabbit 18S ribosomal RNA. Purified 18S rRNA was labeled in vitro at either its 5' or 3' terminus with 32P, base-specifically fragmented enzymatically and chemically, and the resulting fragments electrophoretically fractionated by size in adjacent lanes of 140 cm long polyacrylamide sequencing gels run in 90% formamide. A phylogenetic comparison of both the mammalian 5' proximal 400 residues and the 3' distal 301 nucleotides with the previously determined yeast and Xenopus laevis 18S rRNA sequence shows extensive conservation interspersed with tracts having little homology. Clusters of G + C rich sequences are present within the mammalian 5' domain which are entirely absent in both the Xenopus laevis and yeast 18S rRNAs. Most base differences and insertions within the mammalian 18S rRNA when compared with yeast or Xenopus rRNA result in an increase in the G + C content of these regions. We have found nucleotide sequence analysis of the ribosomal RNA directly permits detection of both cistron heterogeneities and mapping of many of the modified bases.     

Sequence and organization of 5S ribosomal RNA-encoding genes of Arabidopsis thaliana.

We have isolated a genomic clone containing Arabidopsis thaliana 5S ribosomal RNA (rRNA)-encoding genes (rDNA) by screening an A. thaliana library with a 5S rDNA probe from flax. The clone isolated contains seven repeat units of 497 bp, plus 11 kb of flanking genomic sequence at one border. Sequencing of individual subcloned repeat units shows that the sequence of the 5S rRNA coding region is very similar to that reported for other flowering plants. Four A. thaliana ecotypes were found to contain approx. 1000 copies of 5S rDNA per haploid genome. Southern-blot analysis of genomic DNA indicates that 5S rDNA occurs in long tandem arrays, and shows the presence of numerous restriction-site polymorphisms among the six ecotypes studied.