Comparative analysis of the regulation of the interferon-inducible protein kinase PKR by Epstein-Barr virus RNAs EBER-1 and EBER-2 and adenovirus VAI RNA.

The interferon-inducible protein kinase PKR interacts with a number of small viral RNA species, including adenovirus VAI RNA and the Epstein-Barr virus-encoded RNA EBER-1. These RNAs bind to PKR and protect protein synthesis from inhibition by double-stranded RNA in the reticulocyte lysate system. Using a peptide phosphorylation assay we show here that EBER-1, like VAI, directly inhibits the activation of purified PKR. A second Epstein-Barr virus RNA, EBER-2, also regulates PKR. EBER-1, EBER-2 and VAI RNA exhibit mutually competitive binding to the native or recombinant enzyme, as assessed by U.V. crosslinking experiments and filter binding assays. The affinities of all three RNAs for PKR in vitro are similar (Kd = ca. 0.3 nM). Since this protein kinase has been proposed to exert a tumour suppressor function in vivo, the ability of EBER-1 to inhibit its activation suggests a role for this small RNA in cell transformation by Epstein-Barr virus.

Localization of Epstein-Barr virus-encoded RNAs EBER-1 and EBER-2 in interphase and mitotic Burkitt lymphoma cells.

The subcellular distribution of the small Epstein-Barr virus-encoded RNAs EBER-1 and EBER-2 has been investigated by using a high-resolution in situ hybridization technique. The distribution patterns in Raji cells of fluorescent oligodeoxynucleotides complementary to each RNA were detected by confocal laser scanning microscopy. Both RNAs were found in the cytoplasm as well as in the nuclei of interphase cells. In contrast, use of the same technique indicated an exclusively nuclear location for cellular U2 RNA. In the cytoplasm distribution of the EBERs was similar to that of the double-stranded RNA-dependent protein kinase, to which these RNAs can bind, and was coincident with the rough endoplasmic reticulum. In cells undergoing mitosis the EBERs became localized around the chromosomes, whereas the protein kinase remained uniformly distributed in the cytoplasm. A cytoplasmic location for EBER-1 and EBER-2 in interphase cells is consistent with the evidence for a role for these small RNAs in translational control.

A 60-kDa protein from rabbit reticulocytes specifically recognizes the capped 5' end of beta-globin mRNA.

The binding of proteins from rabbit reticulocyte lysate to in-vitro-generated beta-globin mRNA and its defined segments was investigated using ultraviolet-cross-linking experiments as well as gel-retardation assays. Under stringent conditions, only three proteins (72, 60 and 50 kDa) were found associated with full-length beta-globin mRNA at different positions. The 72-kDa protein is most likely the poly(A)-binding protein and binds, as expected, to the poly(A) tail, whereas the 50-kDa protein exhibits affinity for the trailer region of beta-globin mRNA. The binding region of the 60-kDa protein is located at the 5' end of beta-globin mRNA. The interaction of this protein is dependent on the presence of the 5' cap structure, as indicated by competition experiments using an uncapped beta-globin-mRNA leader segment. Further competition experiments with beta-globin mRNA, deleted in part in the leader region, suggest that, besides the cap structure, certain sequence elements are necessary for the interaction of the 60-kDa protein and the beta-globin mRNA leader.

Binding of Epstein-Barr virus small RNA EBER-1 to the double-stranded RNA-activated protein kinase DAI.

Epstein-Barr virus encodes two small RNAs, EBER-1 and -2, that are abundantly expressed in latently infected cells. Recent evidence suggests a role for EBER-1 in regulation of translation since this RNA is able to prevent the inhibition of protein synthesis by double-stranded RNA in rabbit reticulocyte lysates. We show here that EBER-1 that has been synthesized in vitro forms a complex with the dsRNA-activated inhibitor of protein synthesis DAI, a protein kinase that specifically phosphorylates polypeptide chain initiation factor eIF-2. Gel retardation assays and UV crosslinking experiments indicate that complex formation is specific for EBER-1 and requires the presence of some secondary structure in the molecule. RNA competition studies show that EBER-1-DAI complex formation is not inhibited in the presence of other small RNA species, heparin or the synthetic double-stranded RNA, poly(I).poly(C). SDS gel analysis reveals the existence of two forms of the crosslinked complex, of 64-68kDa and 46-53kDa, both of which are recognized by anti-DAI antibodies in immunoprecipitation experiments. These data suggest that EBER-1 regulates protein synthesis through its ability to interact with DAI.

Binding of globin mRNA, beta-globin mRNA segments and RNA homopolymers by immobilized protein of polysomal globin messenger ribonucleoprotein.

The binding of rabbit globin mRNA, in-vitro-generated beta-globin mRNA segments, and RNA homopolymers by proteins of rabbit reticulocyte polysomal messenger ribonucleoproteins (mRNP) after SDS gel electrophoresis and electroblotting was examined. The polysomal mRNP proteins have a higher affinity for mRNA than for rRNA and tRNA while having a higher affinity for polypurine than polypyrimidine homopolymers. Binding experiments with synthetic poly(A) and with segments of beta-globin mRNA transcribed from a cDNA in vitro revealed a set of polysomal mRNP proteins which preferentially bind the poly(A)-free beta-globin mRNA. A protein of Mr 90,000 binds specifically the 3'-nontranslated trailer of the poly(A)-free beta-globin mRNA and not the poly(A)-containing globin mRNA. Another set of proteins preferentially binds poly(A). The latter group of proteins contains a prominent species of Mr 72,000, which is most likely the rabbit poly(A)-binding protein. Three polysomal mRNP proteins which bound rabbit globin mRNA did not bind preferentially any of the other RNA probes used.

Binding of mRNA by an oligopeptide containing an evolutionarily conserved sequence from RNA binding proteins.

Several proteins with an affinity to RNA contain a conserved sequence of 8 amino acids which is postulated as being important for RNA binding. An oligopeptide of 11 amino acids containing this sequence is shown to bind 32P-labelled globin mRNA in a filter binding assay. High concentrations of heparin compete for this binding. 10 other peptides with different sequences do not exhibit affinity to RNA in this assay. These results support the relevance of the conserved peptide sequence in the binding of proteins to RNA.

Stoichiometric association of cap-binding protein I with translated polysomal globin mRNP.

The protein composition of a 12S polysomal globin messenger ribonucleoprotein (pmRNP) from rabbit reticulocytes was examined. The pmRNP was released from purified polysomes by puromycin treatment under run-off conditions of protein synthesis. The protein pattern of this pmRNP depends on the potassium ion concentration used during the run-off and the subsequent isolation. Several proteins show a salt-dependent association with the pmRNP while a few are constituents of the pmRNP at all salt concentrations tested. By cross-linking the pmRNP-derived proteins to [3H]methyl-labelled oxidized vesicular stomatitis virus (VSV) mRNA and by immunoblotting against anti-cap-binding protein (CBP I) antibodies, it is demonstrated that the association of the CBP I with the pmRNP depends on the ionic strength. At 65 mM KCl, CBP I shows low affinity for the pmRNP; at 140 mM KCl, the affinity of CBP I for the pmRNP is greatly enhanced. At this ionic strength, equimolar amounts of CBP I and mRNA are found in the pmRNP. At 500 mM KCl, the pmRNP is completely devoid of CBP I. In the non-translated free cytoplasmic mRNP (cmRNP) no CBP can be detected by either the cross-link or the immunoblot technique.

Properties of Isoaccepting tRNALys, tRNAGlu and tRNAGln from rabbit Reticulocytes and liver. Multiplicity, Codon Recognition and Inactivation by Iodine.

The three tRNA species from rabbit liver and reticulocytes, each corresponding to the codons XAA and XAG(X equals A, G, C) were investigated. The elution patterns of the isoaccepting tRNA subspecies after separation by reversedphase chromatography followed by determination of amino acid acceptance have common and differing characteristics. Three subspecies of tRNALys, two of tRNAGlu and three of tRNAGln have been found in reticulocytes, wheras two subspecies of tRNALys, three of tRNAGlu and four of tRNAGln have been determined in rabbit liver. Examination of codon recognition by means of a ribosome binding assay showed that each subspecies binds exclusively in the presence of only one of the two possible triplets; i.e. wobbling was not found. It has been assumed that this high specificity, which is not to be expected according to the "wobble hypothesis", is due to a 2-thiouracil base in the first position of the anticodon. This was supported by oxidation experiments with iodine. Treatment with iodine significantly reduces the aminoacylation capacity of all subspecies that show specific binding with triplets ending in adenosine. We describe and compare for the first time the characteristics of eucaryotic tRNA species which occupy homologous positions in the codon table. Thus it can be seen that all the investigated subspecies have rigorously specific codon recognition in common, wherby "wobbling" can probably be excluded by a principle that is valid for all the investigated species.