Degradation of the cleaved leader peptide of thiolase by a peroxisomal proteinase.

A peroxisomal location for insulin-degrading enzyme (IDE) has been defined by confocal immunofluorescence microscopy of stably transfected CHO cells overexpressing IDE and digitonin-permeabilization studies in normal nontransfected fibroblasts. The functional significance of IDE in degrading cleaved leader peptides of peroxisomal proteins targeted by the type II motif was evaluated with a synthetic peptide corresponding to the type II leader peptide of prethiolase. The peptide effectively competed for degradation and cross-linking of the high-affinity substrate 125I-labeled insulin to IDE. Direct proteolysis of the leader peptide of prethiolase was confirmed by HPLC; degradation was inhibited by immunodepletion with an antibody to IDE. Phylogenetic analysis of proteinases related to IDE revealed sequence similarity to mitochondrial processing peptidases.

Trypanosome spliced leader RNA genes contain the first identified RNA polymerase II gene promoter in these organisms

Typical general transcription factors, such as TATA binding protein and TFII B, have not yet been identified in any member of the Trypanosomatidae family of parasitic protozoa. Interestingly, mRNA coding genes do not appear to have discrete transcriptional start sites, although in most cases they require an RNA polymerase that has the biochemical properties of eukaryotic RNA polymerase II. A discrete transcription initiation site may not be necessary for mRNA synthesis since the sequences upstream of each transcribed coding region are trimmed from the nascent transcript when a short m7G-capped RNA is added during mRNA maturation. This short 39 nt m7G-capped RNA, the spliced leader (SL) sequence, is expressed as an ∼100 nt long RNA from a set of reiterated, though independently transcribed, genes in the trypanosome genome. Punctuation of the 5′ end of mRNAs by a m7G cap-containing spliced leader is a developing theme in the lower eukaryotic world; organisms as diverse as Euglena and nematode worms, including Caenorhabditis elegans, utilize SL RNA in their mRNA maturation programs. Towards understanding the coordination of SL RNA and mRNA expression in trypanosomes, we have begun by characterizing SL RNA gene expression in the model trypanosome Leptomonas seymouri. Using a homologous in vitro transcription system, we demonstrate in this study that the SL RNA is transcribed by RNA polymerase II. During SL RNA transcription, accurate initiation is determined by an initiator element with a loose consensus of CYAC/AYR(+1). This element, as well as two additional basal promoter elements, is divergent in sequence from the basal transcription elements seen in other eukaryotic gene promoters. We show here that the in vitro transcription extract contains a binding activity that is specific for the initiator element and thus may participate in recruiting RNA polymerase II to the SL RNA gene promoter.

Processing of the leader mRNA plays a major role in the induction of thrS expression following threonine starvation in Bacillus subtilis.

The threonyl-tRNA synthetase gene, thrS, is a member of a family of Gram-positive genes that are induced following starvation for the corresponding amino acid by a transcriptional antitermination mechanism involving the cognate uncharged tRNA. Here we show that an additional level of complexity exists in the control of the thrS gene with the mapping of an mRNA processing site just upstream of the transcription terminator in the thrS leader region. The processed RNA is significantly more stable than the full-length transcript. Under nonstarvation conditions, or following starvation for an amino acid other than threonine, the full-length thrS mRNA is more abundant than the processed transcript. However, following starvation for threonine, the thrS mRNA exists primarily in its cleaved form. This can partly be attributed to an increased processing efficiency following threonine starvation, and partly to a further, nonspecific increase in the stability of the processed transcript under starvation conditions. The increased stability of the processed RNA contributes significantly to the levels of functional RNA observed under threonine starvation conditions, previously attributed solely to antitermination. Finally, we show that processing is likely to occur upstream of the terminator in the leader regions of at least four other genes of this family, suggesting a widespread conservation of this phenomenon in their control.

In vivo function of mutated spliced leader RNAs in Caenorhabditis elegans

The role of spliced leader RNA (SL RNA) in trans-splicing in Caenorhabditis elegans has been studied through a combination of in vitro mutagenesis and in vivo complementation of rrs-1 mutant nematodes, which lack endogenous SL1 RNA. Three classes of mutant SL1 RNAs have been found—those that rescue the lethal phenotype at low concentration of transforming DNA, those that rescue at high but not low concentration, and those that do not rescue at all. These studies showed that some mutations in the otherwise highly conserved 22-nt spliced leader are tolerated for splicing and post-splicing events. A longer spliced leader also can be tolerated but only when present in high copy number. Changes in the first 16 nucleotides result in the appearance of no SL RNA, consistent with the in vitro studies by others showing that the SL1 RNA promoter partly resides within the spliced leader sequence.

Preferential interaction of the his pause RNA hairpin with RNA polymerase β subunit residues 904–950 correlates with strong transcriptional pausing

RNA secondary structures (hairpins) that form as the nascent RNA emerges from RNA polymerase are important components of many signals that regulate transcription, including some pause sites, all ρ-independent terminators, and some antiterminators. At the his leader pause site, a 5-bp-stem, 8-nt-loop pause RNA hairpin forms 11 nt from the RNA 3′ end and stabilizes a transcription complex conformation slow to react with NTP substrate. This stabilization appears to depend at least in part on an interaction with RNA polymerase. We tested for RNA hairpin interaction with the paused polymerase by crosslinking 5-iodoUMP positioned specifically in the hairpin loop. In the paused conformation, strong and unusual crosslinking of the pause hairpin to β904–950 replaced crosslinking to β′ and to other parts of β that occurred in nonpaused complexes prior to hairpin formation. These changes in nascent RNA interactions may inhibit reactive alignment of the RNA 3′ end in the paused complex and be related to events at ρ-independent terminators.

Plastid-localized acetyl-CoA carboxylase of bread wheat is encoded by a single gene on each of the three ancestral chromosome sets

5′-End fragments of two genes encoding plastid-localized acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) of wheat (Triticum aestivum) were cloned and sequenced. The sequences of the two genes, Acc-1,1 and Acc-1,2, are 89% identical. Their exon sequences are 98% identical. The amino acid sequence of the biotin carboxylase domain encoded by Acc-1,1 and Acc-1,2 is 93% identical with the maize plastid ACCase but only 80–84% identical with the cytosolic ACCases from other plants and from wheat. Four overlapping fragments of cDNA covering the entire coding region were cloned by PCR and sequenced. The wheat plastid ACCase ORF contains 2,311 amino acids with a predicted molecular mass of 255 kDa. A putative transit peptide is present at the N terminus. Comparison of the genomic and cDNA sequences revealed introns at conserved sites found in the genes of other plant multifunctional ACCases, including two introns absent from the wheat cytosolic ACCase genes. Transcription start sites of the plastid ACCase genes were estimated from the longest cDNA clones obtained by 5′-RACE (rapid amplification of cDNA ends). The untranslated leader sequence encoded by the Acc-1 genes is at least 130–170 nucleotides long and is interrupted by an intron. Southern analysis indicates the presence of only one copy of the gene in each ancestral chromosome set. The gene maps near the telomere on the short arm of chromosomes 2A, 2B, and 2D. Identification of three different cDNAs, two corresponding to genes Acc-1,1 and Acc-1,2, indicates that all three genes are transcriptionally active.

Transcript leader regions of two Saccharomyces cerevisiae mRNAs contain internal ribosome entry sites that function in living cells

In higher eukaryotes, translation of some mRNAs occurs by internal initiation. It is not known, however, whether this mechanism is used to initiate the translation of any yeast mRNAs. In this report, we identify naturally occurring nucleotide sequences that function as internal ribosome entry sites (IRESes) within the 5′ leader sequences of Saccharomyces cerevisiae YAP1 and p150 mRNAs. When tested in the 5′ untranslated regions of monocistronic reporter genes, both leader sequences enhanced translation efficiency in vegetatively growing yeast cells. Moreover, when tested in the intercistronic region of dicistronic mRNAs, both sequences were shown to contain IRESes that functioned in living cells. The activity of the p150 leader was much greater than that of the YAP1 leader. The second cistron was not expressed in control dicistronic constructs that lacked these sequences or contained the 5′ leader sequence of the CLN3 mRNA in the intercistronic region. Further analyses of the p150 IRES revealed that it contained several nonoverlapping segments that were able independently to mediate internal initiation. These results suggested a modular composition for the p150 IRES that resembled the composition of IRESes contained within some cellular mRNAs of higher eukaryotes. Both YAP1 and p150 leaders contain several complementary sequence matches to yeast 18S rRNA. The findings are discussed in terms of our understanding of internal initiation in higher eukaryotes.

DsrA RNA regulates translation of RpoS message by an anti-antisense mechanism, independent of its action as an antisilencer of transcription

DsrA RNA regulates both transcription, by overcoming transcriptional silencing by the nucleoid-associated H-NS protein, and translation, by promoting efficient translation of the stress σ factor, RpoS. These two activities of DsrA can be separated by mutation: the first of three stem-loops of the 85 nucleotide RNA is necessary for RpoS translation but not for anti-H-NS action, while the second stem-loop is essential for antisilencing and less critical for RpoS translation. The third stem-loop, which behaves as a transcription terminator, can be substituted by the trp transcription terminator without loss of either DsrA function. The sequence of the first stem-loop of DsrA is complementary with the upstream leader portion of rpoS messenger RNA, suggesting that pairing of DsrA with the rpoS message might be important for translational regulation. Mutations in the Rpos leader and compensating mutations in DsrA confirm that this predicted pairing is necessary for DsrA stimulation of RpoS translation. We propose that DsrA pairing stimulates RpoS translation by acting as an anti-antisense RNA, freeing the translation initiation region from the cis-acting antisense RNA and allowing increased translation.

Dose-response resistance to HIV-1/MuLV pseudotype virus ex vivo in a hairpin ribozyme transgenic mouse model

We have investigated the efficacy of a hairpin ribozyme targeting the 5′ leader sequence of HIV-1 RNA in a transgenic model system. Primary spleen cells derived from transgenic or control mice were infected with HIV-1/MuLV pseudotype virus. A significantly reduced susceptibility to infection in ribozyme-expressing transgenic spleen cells (P = 0.01) was shown. Variation of transgene-expression levels between littermates revealed a dose response between ribozyme expression and viral resistance, with an estimated cut off value below 0.2 copies of hairpin ribozyme per cell. These findings open up possibilities for studies on ribozyme efficacy and anti-HIV-1 gene therapy.

A dynamin GTPase mutation causes a rapid and reversible temperature-inducible locomotion defect in C. elegans

Drosophila shibire and its mammalian homologue dynamin regulate an early step in endocytosis. We identified a Caenorhabditis elegans dynamin gene, dyn-1, based upon hybridization to the Drosophila gene. The dyn-1 RNA transcripts are trans-spliced to the spliced leader 1 and undergo alternative splicing to code for either an 830- or 838-amino acid protein. These dyn-1 proteins are highly similar in amino acid sequence, structure, and size to the Drosophila and mammalian dynamins: they contain an N-terminal GTPase, a pleckstrin homology domain, and a C-terminal proline-rich domain. We isolated a recessive temperature-sensitive dyn-1 mutant containing an alteration within the GTPase domain that becomes uncoordinated when shifted to high temperature and that recovers when returned to lower temperatures, similar to D. shibire mutants. When maintained at higher temperatures, dyn-1 mutants become constipated, egg-laying defective, and produce progeny that die during embryogenesis. Using a dyn-1::lacZ gene fusion, a high level of dynamin expression was observed in motor neurons, intestine, and pharyngeal muscle. Our results suggest that dyn-1 function is required during development and for normal locomotion.

Identification of two short internal ribosome entry sites selected from libraries of random oligonucleotides

Sequences that control translation of mRNA may play critical roles in regulating protein levels. One such element is the internal ribosome entry site (IRES). We previously showed that a 9-nt segment in the 5′ leader sequence of the mRNA encoding Gtx homeodomain protein could function as an IRES. To identify other short sequences with similar properties, we designed a selection procedure that uses a retroviral vector to express dicistronic mRNAs encoding enhanced green and cyan fluorescent proteins as the first and second cistrons, respectively. Expression of the second cistron was dependent upon the intercistronic sequences and was indicative of IRES activity. B104 cells were infected with two retroviral libraries that contained random sequences of 9 or 18 nt in the intercistronic region. Cells expressing both cistrons were sorted, and sequences recovered from selected cells were reassayed for IRES activity in a dual luciferase dicistronic mRNA. Two novel IRESes were identified by this procedure, and both contained segments with complementarity to 18S rRNA. When multiple copies of either segment were linked together, IRES activities were dramatically enhanced. Moreover, these synthetic IRESes were differentially active in various cell types. These properties are similar to those of the previously identified 9-nt IRES module from Gtx mRNA. These results provide further evidence that short nucleotide sequences can function as IRESes and support the idea that some cellular IRESes may be composed of shorter functional modules. The ability to identify IRES modules with specific expression properties may be useful in the design of vectors for biotechnology and gene therapy.

Internal initiation of translation of five dendritically localized neuronal mRNAs

In neurons, translation of dendritically localized mRNAs is thought to play a role in affecting synaptic efficacy. Inasmuch as components of the translation machinery may be limiting in dendrites, we investigated the mechanisms by which translation of five dendritically localized mRNAs is initiated. The 5′ leader sequences of mRNAs encoding the activity-regulated cytoskeletal protein, the α subunit of calcium–calmodulin-dependent kinase II, dendrin, the microtubule-associated protein 2, and neurogranin (RC3) were evaluated for their ability to affect translation in the 5′ untranslated region of a monocistronic reporter mRNA. In both neural and nonneural cell lines, the activity-regulated cytoskeletal protein, microtubule-associated protein 2, and α-CaM Kinase II leader sequences enhanced translation, whereas the dendrin and RC3 5′ untranslated regions slightly inhibited translation as compared with controls. When cap-dependent translation of these constructs was suppressed by overexpression of a protein that binds the cap-binding protein eIF4E, it was revealed that translation of these mRNAs had both cap-dependent and cap-independent components. The cap-independent component was further analyzed by inserting the 5′ leader sequences into the intercistronic region of dicistronic mRNAs. All five leader sequences mediated internal initiation via internal ribosome entry sites (IRESes). The RC3 IRES was most active and was further characterized after transfection in primary neurons. Although translation mediated by this IRES occurred throughout the cell, it was relatively more efficient in dendrites. These data suggest that IRESes may increase translation efficiency at postsynaptic sites after synaptic activation.

An RNA structure involved in feedback regulation of splicing and of translation is critical for biological fitness.

While studies of the regulation of gene expression have generally concerned qualitative changes in the selection or the level of expression of a gene, much of the regulation that occurs within a cell involves the continuous subtle optimization of the levels of proteins used in macromolecular complexes. An example is the biosynthesis of the ribosome, in which equimolar amounts of nearly 80 ribosomal proteins must be supplied by the cytoplasm to the nucleolus. We have found that the transcript of one of the ribosomal protein genes of Saccharomyces cerevisiae, RPL32, participates in such fine tuning. Sequences from exon I of the RPL32 transcript interact with nucleotides from the intron to form a structure that binds L32 to regulate splicing. In the spliced transcript, the same sequences interact with nucleotides from exon II to form a structure that binds L32 to regulate translation, thus providing two levels of autoregulation. We now show, by using a sensitive cocultivation assay, that these RNA structures and their interaction with L32 play a role in the fitness of the cell. The change of a single nucleotide within the 5' leader of the RPL32 transcript, which abolishes the site for L32 binding, leads to detectably slower growth and to eventual loss of the mutant strain from the culture. Experiments designed to assess independently the regulation of splicing and the regulation of translation are presented. These observations demonstrate that, in evolutionary terms, subtle regulatory compensations can be critical. The change in structure of an RNA, due to alteration of just one noncoding nucleotide, can spell the difference between biological success and failure.

Resistance to human immunodeficiency virus type 1 infection conferred by transduction of human peripheral blood lymphocytes with ribozyme, antisense, or polymeric trans-activation response element constructs.

Human peripheral blood lymphocytes (PBLs) were transduced with a number of recombinant retroviruses including RRz2, an LNL6-based virus with a ribozyme targeted to the human immunodeficiency virus (HIV) tat gene transcript inserted within the 3' region of the neomycin-resistance gene; RASH5, and LNHL-based virus containing an antisense sequence to the 5' leader region of HIV-1 downstream of the human cytomegalovirus promoter; and R20TAR, an LXSN-based virus with 20 tandem copies of the HIV-1 trans-activation response element sequence driven by the Moloney murine leukemia virus long terminal repeat. After G418 selection, transduced PBLs were challenged with the HIV-1 laboratory strain IIIB and a primary clinical isolate of HIV-1, 82H. Results showed that PBLs from different donors could be transduced and that this conferred resistance to HIV-1 infection. For each of the constructs, a reduction of approximately 70% in p24 antigen level relative to the corresponding control-vector-transduced PBLs was observed. Molecular analyses showed constitutive expression of all the transduced genes from the retroviral long terminal repeat, but no detectable transcript was seen from the internal human cytomegalovirus transcript was seen from the internal human cytomegalovirus promoter for the antisense construct. Transduction of, and consequent transgene expression in, PBLs did not impact on the surface expression of either CD4+/CD8+ (measured by flow cytometry) or on cell doubling time (examined by [3H]thymidine uptake). These results indicate the potential utility of these anti-HIV-1 gene therapeutic agents and show the preclinical value of this PBL assay system.