Structural organization of the human glutathione reductase gene: determination of correct cDNA sequence and identification of a mitochondrial leader sequence

The primary structure of human glutathione reductase gene (GSR) was determined by genomic cloning. The gene structure of human GSR spans 50 kb, consists of 13 exons, and was found to be highly similar to the mouse GSR gene. The coding sequence of human GSR resides on all 13 exons. An N-terminal arginine-rich mitochondrial leader sequence was present, with high homology to the murine leader sequence, between two in-frame start codons in the first exon. The 5' and 3' intron/exon splice junctions, with one exception, followed the general consensus sequences for intron spliced donor and acceptance sites.     

Effects of alterations in the leader sequence of Rous sarcoma virus RNA on initiation of translation.

The 372-nucleotide leader sequence of Rous sarcoma virus RNA contains three conserved short open reading frames and other sequences responsible for a variety of life cycle functions. We have investigated several aspects of the leader RNA which may influence the translation of the major coding regions to which the leader is juxtaposed. We found that small perturbations of the leader length do not affect the binding and scanning of ribosomal subunits by more than about 10%, that the length and/or structure of the RSV RNA leader is near optimal for translation of the major coding regions of the viral RNA, that inclusion or deletion of open reading frames influences downstream initiation in a manner that is not strictly additive, and that reinitiation of translation at the gag gene is very efficient.     

The HIV-1 leader RNA conformational switch regulates RNA dimerization but does not regulate mRNA translation

The untranslated leader RNA is the most conserved part of the human immunodeficiency virus type I (HIV-1) genome. It contains many regulatory motifs that mediate a variety of steps in the viral life cycle. Previous work showed that the full-length leader RNA can adopt two alternative structures: a long distance interaction (LDI) and a branched multiple-hairpin (BMH) structure. The BMH structure exposes the dimer initiation site (DIS) hairpin, whereas this motif is occluded in the LDI structure. Consequently, these structures differ in their capacity to form RNA dimers in vitro. The BMH structure is dimerization-competent, due to DIS hairpin formation, but also presents the splice donor (SD) and RNA packaging (Psi) hairpins. In the LDI structure, an extended RNA packaging (Psi(E)) hairpin is folded, which includes the splice donor site and gag coding sequences. The gag initiation codon is engaged in a long distance base pairing interaction with sequences in the upstream U5 region in the BMH structure, thus forming the evolutionarily conserved U5-AUG duplex. Therefore, the LDI-BMH equilibrium may affect not only the process of RNA dimer formation but also translation initiation. In this study, we designed mutations in the 3'-terminal region of the leader RNA that alter the equilibrium of the LDI-BMH structures. The mutant leader RNAs are affected in RNA dimer formation, but not in their translation efficiency. These results indicate that the LDI-BMH status does not regulate HIV-1 RNA translation, despite the differential presentation of the gag initiation codon in both leader RNA structures.     

A retroviral RNA secondary structure required for efficient initiation of reverse transcription

Genetic evidence is presented which suggests the existence of an important structural element in the 5' noncoding region of avian retrovirus RNA. The proposed structure, which we term the U5-leader stem, is composed of sequences in the middle of U5 and in the leader, flanking the primer-binding site. U5 and leader mutations which would disrupt this structure caused a partial replication defect. However, nucleotide substitutions in the leader, which would structurally compensate for a U5 deletion mutation, restored normal replication. Analysis of replication intermediates of viruses with the above mutations suggests that the U5-leader stem is required for efficient DNA synthesis in vivo and for initiation of DNA synthesis from the tRNA(Trp) primer in melittin-activated virions. However, this structure does not appear to be required for binding of the tRNA(Trp) primer to viral RNA. These results support a role for the U5-leader stem structure, independent of its primary sequence, in the initiation of retroviral replication.     

The role of intron structures in trans-splicing and cap 4 formation for the Leishmania spliced leader RNA.

A 39-nucleotide leader is trans-spliced onto all trypanosome nuclear mRNAs. The precursor spliced leader RNA was tested for trans-splicing function in vivo by mutating the intron. We report that in Leishmania tarentolae spliced leader RNA 5' modification is influenced by the primary sequence of stem-loop II, the Sm-binding site, and the secondary structure of stem-loop III. The sequence of stem-loop II was found to be important for cap 4 formation and splicing. As in Ascaris, mutagenesis of the bulge nucleotide in stem-loop II was detrimental to trans-splicing. Because restoration of the L. tarentolae stem-loop II structure was not sufficient to restore splicing, this result contrasts the findings in the kinetoplastid Leptomonas, where mutations that restored stem-loop II structure supported splicing. Methylation of the cap 4 structure and splicing was also dependent on both the Sm-binding site and the structure of stem-loop III and was inhibited by incomplete 3' end processing. The critical nature of the L. tarentolae Sm-binding site is consistent with its essential role in the Ascaris spliced leader RNA, whereas in Leptomonas mutation of the Sm-binding site and deletion of stem-loop III did not affect trans-splicing. A pathway for Leishmania spliced leader RNA processing and maturation is proposed.     

The ribosomal RNA genes from chloroplasts of mustard (Sinapis alba L.): mapping and sequencing of the leader region

Summary The genes coding for rRNAs from mustard chloroplasts were mapped within the inverted repeat regions of intact ctDNA and on ctDNA fragments cloned in pBR322. R-loop analysis and restriction endonuclease mapping show that the genes for 16S rRNA map at distances of 17 kb from the junctions of the repeat regions with the large unique region. The genes for 23S rRNA are located at distances of 2.8 kb from the junctions with the small unique region. Genes for 4.5S and 5S rRNA are located in close proximity to the 23S rRNA genes towards the small unique region. DNA sequencing of portions of the 5 terminal third from the mustard 16S rRNA gene shows 96–99% homology with the corresponding regions of the maize, tobacco and spinach chloroplast genes. Sequencing of the region proximal to the 16S rRNA gene reveals the presence of a tRNA^Val gene in nearly the same position and with identical sequence as in maize, tobacco and spinach. Somewhat less but still strong homology is also observed for the tDNA ^Val/16S rDNA intercistronic regions and for the regions upstream of the tRNA^Val gene. However, due to many small and also a few larger deletions and insertions in the leader region, common reading frames coding for homologous peptides larger than 44 amino acids can not be detected; it is therefore unlikely that this region contains a protein coding gene.     

Two sequence classes of kinetoplastid 5S ribosomal RNA gene revealed among bodonid spliced leader RNA gene arrays

The spliced leader RNA genes of Bodo saltans, Cryptobia helicis and Dimastigella trypaniformis were analyzed as molecular markers for additional taxa within the suborder Bodonina. The non-transcribed spacer regions were distinctive for each organism, and 5S rRNA genes were present in Bodo and Dimastigella but not in C. helicis. Two sequence classes of 5S rRNA were evident from analysis of the bodonid genes. The two classes of 5S rRNA genes were found in other Kinetoplastids independent of co-localization with the spliced leader RNA gene.     

Structure of the murine complement factor H gene

Factor H is a regulatory protein of the alternative pathway of complement activation comprised of 20 tandem repeating units of 60 amino acids each. A factor H cDNA clone was used to identify 17 genomic clones from a cosmid library. Four clones were selected for analysis of intron/exon junctions and 5' and 3' regions of the gene and for mapping of the exons. The factor H gene was found to be comprised of 22 exons. Each repeating unit is encoded by one exon, except the second repeat, which is coded by two exons; the leader sequence is encoded by a separate exon. The exons range in size from 77 to 210 base pairs (bp) and average 178 bp. They span a region of approximately 100 kilobases (kb) on chromosome 1. The leader sequence exon is 26 kb upstream of the first repeat exon, representing the largest intron. The other introns range in size from 86 bp to 12.9 kb, and the average intron size is 4.7 kb. Analysis of the genomic organization of the factor H gene has provided insight into the protein structure and will enable the construction of deletion mutants for functional studies.     

The U2 RNA analogue of Trypanosoma brucei gambiense: implications for a splicing mechanism in trypanosomes.

We have isolated the gene coding for the U2 analogue in trypanosomes. The 148 nucleotide long U2 RNA is capped and transcribed from a single copy gene. The 5' half of the molecule is highly homologous to mammalian U2 RNA, while the 3' half does not show any significant sequence homology with the mammalian counterpart. Nevertheless, the trypanosome U2 RNA can be folded into a secondary structure resembling the one proposed for U2 RNA. The presence of a U2 analogue and most likely other U RNAs in trypanosomes suggests that splicing is involved at some point in the maturation of mRNA. Possible interactions of the U2 RNA with the spliced leader RNA are considered.