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PubMed1995

Leishmania RNA virus 1-mediated cap-independent translation.

Recently, a group of related Leishmania RNA viruses (Leishmania RNA virus 1 [LRV1]) has been isolated from Leishmania guyanensis and L. brasiliensis. These viruses persist in the cytoplasm and contain double-stranded RNA genomes. Miniexon sequences are absent from the 5' end of the viral RNA, and the 5' end of the viral RNA lacks a cap structure, suggesting that LRV1 has evolved a cap-independent mechanism of translation. Cap-independent translation of picornavirus genomic RNA requires a cis element, within the 5' untranslated region (UTR), referred to as an internal ribosome entry site (IRES). In order to find out if the 5' UTR of LRV1 possessed IRES activity, we modified a Leishmania expression vector, pX63NEO-GUS, so that it would produce a dicistronic transcript in which the neomycin phosphotransferase gene was separated from the downstream beta-glucuronidase (GUS) gene by the LRV1 5' UTR. High levels of GUS activity were detected in L. major stably transformed with this plasmid. Elimination of the first 120 nucleotides of the viral 5' UTR lowered GUS activity 10-fold. Furthermore, when the entire 5' UTR was eliminated, GUS activity was undetectable. These results, together with the absence of trans-spliced GUS transcripts, are consistent with the hypothesis that the 5' UTR of LRV1 functions as an IRES element. The ability to couple expression of genes via an IRES element should prove useful in genetic experiments with Leishmania spp.

Animals↗
PubMed2002

The 3' end formation in small RNAs.

Small RNAs are a major class of RNAs along with transfer RNAs, ribosomal RNAs, and messenger RNAs. They vary in size from less than 100 nucleotides to several thousand nucleotides and have been identified and characterized both in prokaryotes and eukaryotes. Small RNAs participate in a variety of cellular functions including regulating RNA synthesis, RNA processing, guiding modifications in RNA, and in transport of proteins. Small RNAs are generated by a series of posttranscriptional processing steps following transcription. While RNA 5' end structure, 5' cap formation, and RNA processing mechanisms have been fairly well characterized, the 3' end processing is poorly understood. Recent data point to an emerging theme in small RNAs metabolism in which the 3' end processing is mediated by the exosome, a large multienzyme complex. In addition to removal of nucleotides by the exosome, there is simultaneous rebuilding of the 3' end of some small RNA by adenylation and/or uridylation. This review presents a picture of both degradative and rebuilding reactions operative on the 3' end of some small RNA molecules in prokaryotes and eukaryotes.

Animals↗
PubMed1982

Identification of initiation sites for heavy-strand and light-strand transcription in human mitochondrial DNA.

The initiation sites for heavy (H) and light (L) strand transcription in HeLa cell mitochondrial DNA have been investigated by mapping experiments utilizing in vitro "capped" mitochondrial RNA molecules or nascent RNA chains. Mitochondrial poly(A)-containing RNA molecules were labeled at their 5' ends with [alpha-32P]GTP and guanylyltransferase ("capping" enzyme) and mapped on the mitochondrial genome by DNA transfer hybridization and S1 nuclease protection experiments. A mapping site for the capped 5' ends was found on the H strand very near to the 5' terminus of the 12S rRNA gene, and another site was found on the L strand very near to the 5' terminus of the 7S RNA coding sequence. In parallel experiments, the 5' ends of the nascent chains isolated from mitochondrial DNA transcription complexes were similarly mapped very near to the 5' termini of the 12S rRNA gene and of the 7S RNA coding sequence. The in vitro capped RNA molecules and the nascent chains thus presumably identify the same transcriptional initiation sites on the H strand and the L strand. The occurrence of a second possible initiation site for H-strand transcription 90-110 nucleotides upstream of that described above--i.e., 20-40 nucleotides upstream of the tRNAPhe gene--had been previously indicated by a mapping analysis of the nascent RNA chains and has been confirmed in the present work. The presence of two initiation sites for H-strand transcription can be correlated with other types of evidence that point to two different transcription events leading to the synthesis of a polycistronic molecule corresponding to the almost entire H strand and to the synthesis of the rRNA species.

Chromosome Mapping↗
PubMed1983

Association of an RNA 5'-triphosphatase activity with RNA guanylyltransferase partially purified from rat liver nuclei.

An RNA 5'-triphosphatase activity hydrolyzing gamma-phosphate from pppN-RNA was found to be associated with mRNA guanylyltransferase partially purified from rat liver nuclei. The activity specifically removed 32P as inorganic phosphate from [gamma-32P]pppA(pA)n, but not from [beta-32P]pppA(pA)n or from [gamma-32P]ATP. Free SH group(s) were required for its activity, and the reaction was inhibited by N-ethylmaleimide. Divalent cations were not required, but were rather inhibitory for the reaction. The RNA 5'-triphosphatase activity could not be separated from the guanylyltransferase activity through successive chromatographies on Sephadex G-150, CM-Sephadex and blue dextran-Sepharose columns. Both activities remained physically associated during sedimentation in glycerol density gradients after high salt treatment. The heat stability of the RNA 5'-triphosphatase activity was almost identical with that of the guanylyltransferase activity. These results indicate that the 69000 mol. wt. protein purified from rat liver nuclei as guanylyltransferase possesses both mRNA capping and RNA 5'-triphosphatase activities.

Acid Anhydride Hydrolases↗
PubMed1988

Translational control in heat-shocked Drosophila embryos. Evidence for the inactivation of initiation factor(s) involved in the recognition of mRNA cap structure.

Lysates from normally growing (25 degrees C) or heat shocked (37 degrees C, 45 min) Drosophila melanogaster embryos were obtained and the effect of analogues of the mRNA 5'-terminal cap, m7G(5')ppp(5')N structure and of potassium ions on their endogenous protein synthesis activity was studied. At optimal concentration of KCH3COO (75-80 mM), protein synthesis in normal lysates is strongly inhibited by cap analogues (m7GpppG, m7GDP, and m7GMP). At the same ionic conditions, heat shock lysates translate preferentially the heat shock messengers, and this translation is almost unaffected by the cap analogues. In contrast, residual synthesis of normal proteins in heat shock lysates was reduced by these compounds. By lowering the concentration of potassium ions it was possible to gradually reverse the inhibitory effect of the cap analogues in normal lysates and also to increase specifically the translation of normal mRNAs in heat shock lysates. Translation of normal mRNAs is also partial but specifically rescued by supplementing heat shock lysates with polypeptide chain initiation factors partially purified from rabbit reticulocytes. These data are consistent with the notion that the failure of normal mRNAs to be translated under heat shock conditions might be due, at least to some extent, to the inactivation of polypeptide chain initiation factor(s) involved in the recognition of the mRNA 5'-terminal cap structure.

Animals↗
PubMed1983

5'-Conformation of capped alfalfa mosaic virus ribonucleic acid 4 may reflect its independence of the cap structure or of cap-binding protein for efficient translation.

Most eukaryotic mRNAs are characterized by the presence of a 5'-terminal cap structure (m7GpppN), and removal of the cap or translation of capped mRNAs in the presence of cap analogues (m7G) results in most cases in a significant decrease in the translational efficiency of the mRNAs. One way of explaining the importance of the 5'-cap is that cap-binding proteins recognize the cap structure, destabilize the mRNA secondary structure, and thus allow the 40S ribosomal subunit to bind to the mRNA [Sonenberg, N., Guertin, D., Cleveland, D., & Trachsel, H. (1981) Cell (Cambridge, Mass.) 27, 563-572]. Our data and those of others indicate that the translational efficiency of alfalfa mosaic virus RNA 4 (AMV-4 RNA), a naturally capped RNA, is not affected significantly by cap analogues or by removal of the cap. In order to examine the potential relationship between the function of the cap structure and secondary structure at the 5'-mRNA terminus, partial enzymatic digestion of capped AMV-4 RNA with single strand specific and double strand specific nucleases has been performed, and the experimental data have been compared with computer-generated models of AMV-4 secondary structure. In addition, the in vitro translatability of AMV-4 has been examined as a function of increasing potassium concentration, conditions that are likely to increase mRNA secondary structure. The nuclease-digestion results demonstrate that under native ionic conditions, the 5'-terminus of AMV-4 RNA is predominantly single stranded, although computer modeling and double-strand nuclease digestions indicate that the 5'-terminus can form weak base pairs with internal regions of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1985

Isolation and characterization of factors from wheat germ that exhibit eukaryotic initiation factor 4B activity and overcome 7-methylguanosine 5'-triphosphate inhibition of polypeptide synthesis.

Three highly purified preparations (preparations I, II-1, and II-2) have been obtained from wheat germ and shown to support in vitro polypeptide synthesis directed by capped or uncapped mRNAs in a eukaryotic initiation factor 4B (eIF-4B)-deficient system. The three preparations differ, however, in polypeptide composition and in the ability to overcome the inhibitory effect of 7-methylguanosine 5'-triphosphate (m7GTP) on in vitro polypeptide synthesis. Preparation I contains two polypeptides (Mr = 80,000 and 28,000), which are present in a 1:1 molar ratio and are associated in a high molecular weight complex. Preparation II-1 contains two major polypeptides (Mr = 220,000 and 26,000) and preparation II-2 also contains two major polypeptides (Mr = 110,000 and 26,000). Preparations II-1 and II-2 are high molecular weight complexes; neither contains detectable amounts of a Mr 80,000 or a Mr 50,000 component. Preparations II-1 and II-2 both overcome m7GTP inhibition, whereas preparation I does not. These findings raise several questions with regard to the identity of eIF-4B and its relationship to cap recognition factors.

Chromatography, Ion Exchange↗
PubMed1978

HeLa cell RNA (2'-O-methyladenosine-N6-)-methyltransferase specific for the capped 5'-end of messenger RNA.

A novel enzyme involved in the post-transcriptional modification of the 5'-end of mRNA has been partially purified from HeLa cells. Termed an S-adenosyl-L-methionine:RNA(2'-O-methyladenosine-N4)-methyltransferase, the enzyme specifically catalyzes the transfer of a methyl group from S-adenosylmethionine to the N6 position of a 2'-O-methyladenosine residue located within the "capped" 5'-end of mRNA. The dimethylated nucleoside, N6,2'-O-dimethyladenosine, is formed as indicated by the following reaction in which m7G(5')pppAm- represents the capped and methylated 5'-end of mRNA: AdoMet + m7G(5')pppAm- leads to AdoHcy + m7G(5')pppm6A7- Greatest activity is obtained with RNA acceptors ending in m7G(5')pppAm-; less activity is found with RNA ending in m7G(5')pppA-; and barely detectable and no activity is found with RNA ending in G(5')pppA- and ppA-, respectively. Furthermore, no activity is found with oligonucleotides such as m7G(5')pppA, m7G(5')pppAm, and m7G(5')pppAmpN indicating that a longer polymer is required. It can be concluded from the substrate specificity of the enzyme that the formation of N6,2'-O-dimethyladenosine follows the biosynthesis of molecules containing m7G(5')pppAm-N-. The molecular weight of the methyltransferase, as determined by sedimentation in sucrose gradients, is approximately 65,000.

Adenosine↗
PubMed1987

Preferential excision of the 5' proximal intron from mRNA precursors with two introns as mediated by the cap structure.

We have studied the effect of the 5' cap structure on the splicing of precursor mRNAs containing three exons and two introns within a single molecule in a HeLa nuclear extract. When a precursor mRNA was capped, the upstream intron was spliced out more efficiently than the downstream intron. The differential splicing reactions of the two introns are not due to differences in the intrinsic efficiency of splicing of each intron, since the preferential excision of the upstream intron was also observed when the positions of the two introns relative to the cap structure were reversed. When uncapped precursor mRNA was used as substrate, the downstream intron was spliced out appreciably, but splicing of the upstream intron was greatly reduced. Preincubation of the extract with cap analogues inhibited splicing of the upstream intron but not the downstream intron. Thus, the cap structure exerts its effect primarily on the 5' proximal intron.

HeLa Cells↗
PubMed1995

Reaction in alphavirus mRNA capping: formation of a covalent complex of nonstructural protein nsP1 with 7-methyl-GMP.

After the start of transcription, the 5' ends of eukaryotic mRNA molecules are modified by the addition of a guanylyl residue to form a cap structure, G(5')ppp(5')N. The guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) reaction responsible for cap formation usually proceeds via a covalent enzyme-GMP intermediate. We have studied the alphavirus-specific guanylyltransferase by incubating lysates from Semliki Forest and Sindbis virus-infected cells with [alpha-32P]GTP, using vaccinia virus and mock-infected cells as controls. One additional 32P-labeled protein was detected in alphavirus-infected cells but only in the presence of S-adenosylmethionine. This protein was identified as the nonstructural protein nsP1. The properties of the covalent enzyme-guanylate complex were studied with Semliki Forest virus nsP1 expressed in recombinant baculovirus-infected cells. S-Adenosylmethionine and divalent cations were required for the complex formation. The reaction was specific for guanylate nucleotides (GTP, dGTP) and was inhibited by pyrophosphate. nsP1 could be labeled with S-adenosyl[methyl-3H]methionine but only under conditions in which the nsP1-guanylate complex was formed. 7-Methyl-GMP was released from the nsP1-guanylate complex by treatment with acid or acidic hydroxylamine. Similar treatment of vaccinia virus capping enzyme released GMP. These findings suggest that in the capping of alphavirus mRNAs the guanine is methylated before linkage to the mRNA molecule.

Alphavirus↗
PubMed1986

Additional methylation at the N(2)-position of the cap of 26S Semliki Forest virus late mRNA and initiation of translation.

Cap analysis of the late 26S Semliki Forest viral mRNA reveals that almost 30% of the caps possess both a methyl group at the N(7)-position and one at the N(2)-position. We have compared the degree of methylation of the caps of polysomal and non-polysomal 26S mRNA in order to check whether this feature is responsible for its translation late in infection. It was found that extra methyl groups on the caps cause a lower rate of initiation. Polysomal 26S mRNA contained less m2,7G- and m2,2,7G-caps than free 26S. The cap analog m2,2,7Gp was slightly less inhibitory than m7Gp in an in vitro translation system.

Methylation↗
PubMed1989

Effect of the cap structure on pre-mRNA splicing in Xenopus oocyte nuclei.

The effect of the 5' cap structure on the splicing of precursor mRNAs was investigated after the RNAs were injected into Xenopus oocyte nuclei. The precursor mRNAs synthesized in vitro in a prokaryotic transcription system with a dinucleotide, ApppG, as a primer, were extremely stable when injected into the nuclei yet behaved like uncapped pre-mRNAs in the in vitro splicing reaction. The ApppG-primed precursor mRNAs served as a control (uncapped) in the injection experiments, and their splicing reactions were compared with those of their capped (m7GpppG-primed) counterparts. The capped precursors were spliced more efficiently than the uncapped precursors. Examination of splicing of the precursor mRNA that contained three exons and two introns with a single molecule has revealed that the cap structure exerts its effect primarily on the 5'-proximal intron. Thus, the cap structure not only stabilizes precursor mRNAs but also plays a positive role in the splicing of precursor mRNAs in cells.

Animals↗
PubMed2006

Structure-function analysis of Plasmodium RNA triphosphatase and description of a triphosphate tunnel metalloenzyme superfamily that includes Cet1-like RNA triphosphatases and CYTH proteins.

RNA triphosphatase catalyzes the first step in mRNA capping. The RNA triphosphatases of fungi and protozoa are structurally and mechanistically unrelated to the analogous mammalian enzyme, a situation that recommends RNA triphosphatase as an anti-infective target. Fungal and protozoan RNA triphosphatases belong to a family of metal-dependent phosphohydrolases exemplified by yeast Cet1. The Cet1 active site is unusually complex and located within a topologically closed hydrophilic beta-barrel (the triphosphate tunnel). Here we probe the active site of Plasmodium falciparum RNA triphosphatase by targeted mutagenesis and thereby identify eight residues essential for catalysis. The functional data engender an improved structural alignment in which the Plasmodium counterparts of the Cet1 tunnel strands and active-site functional groups are located with confidence. We gain insight into the evolution of the Cet1-like triphosphatase family by noting that the heretofore unique tertiary structure and active site of Cet1 are recapitulated in recently deposited structures of proteins from Pyrococcus (PBD 1YEM) and Vibrio (PDB 2ACA). The latter proteins exemplify a CYTH domain found in CyaB-like adenylate cyclases and mammalian thiamine triphosphatase. We conclude that the tunnel fold first described for Cet1 is the prototype of a larger enzyme superfamily that includes the CYTH branch. This superfamily, which we name "triphosphate tunnel metalloenzyme," is distributed widely among bacterial, archaeal, and eukaryal taxa. It is now clear that Cet1-like RNA triphosphatases did not arise de novo in unicellular eukarya in tandem with the emergence of caps as the defining feature of eukaryotic mRNA. They likely evolved by incremental changes in an ancestral tunnel enzyme that conferred specificity for RNA 5'-end processing.

Acid Anhydride Hydrolases↗