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Biomedical subjects

M J Clemens

Publications and source records attributed to M J Clemens.

At least 37 records · Page 2Linked to original sources
PubMed1998

Activation of 2'-5' oligoadenylate synthetase by single-stranded and double-stranded RNA aptamers.

A number of small RNA molecules that are high affinity ligands for the 46-kDa form of human 2'-5' oligoadenylate synthetase have been identified by the SELEX method. Surface plasmon resonance analysis indicates that these RNAs bind to the enzyme with dissociation constants in the nanomolar range. Competition experiments indicate that the binding site for the small RNAs on the 2'-5' oligoadenylate synthetase molecule at least partially overlaps that for the synthetic double-stranded RNA, poly(I).poly(C). Several of the RNAs function as potent activators of 2'-5' oligoadenylate synthetase in vitro, although there is no correlation between binding affinity and ability to activate. The RNA aptamers having the strongest activation potential appear to have few base-paired regions. This suggests that 2'-5' oligoadenylate synthetase, which has previously been believed to be activated only by double-stranded RNA, can also be activated by RNA ligands with little secondary structure. Since 2'-5' oligoadenylate synthetase possesses no homology to other known RNA-binding proteins, the development of small specific ligands by SELEX should facilitate studies of RNA-protein interactions and may reveal novel features of the structure-function relationships involving this enzyme.

2',5'-Oligoadenylate Synthetase↗
PubMed1997

PKR--a protein kinase regulated by double-stranded RNA.

The RNA-regulated protein kinase, (PKR) is an interferon-inducible enzyme of widespread occurrence in eukaryotic organisms. This serine/threonine-specific protein kinase is activated by double-stranded RNA by a mechanism involving autophosphorylation. Once activated, the enzyme phosphorylates the alpha subunit of protein synthesis initiation factor eIF2, thereby inhibiting translation. Recent evidence suggests that there may be additional substrates, and that signal transduction and gene transcription pathways also may be regulated by the protein kinase. As well as being important in mediating the antiviral effects of interferons, PKR is implicated in regulating cell proliferation in uninfected cells and may have a tumour suppressor function under normal conditions. Studies using cell lines expressing inactive mutants of PKR and mice with homozygous disruptions of the PKR gene are leading to greater insights into the biological significance of this enzyme.

Amino Acid Sequence↗
PubMed1997

The double-stranded RNA-dependent protein kinase PKR: structure and function.

This review describes the structure and function of the interferon (IFN)-inducible, double-stranded RNA-activated protein kinase PKR. This protein kinase has been studied extensively in recent years, and a large body of evidence has accumulated concerning its expression, interaction with regulatory RNA and protein molecules, and modes of activation and inhibition. PKR has been shown to play a variety of important roles in the regulation of translation, transcription, and signal transduction pathways through its ability to phosphorylate protein synthesis initiation factor eIF2, I-kappaB (the inhibitor of NF-kappaB), and other substrates. Expression studies involving both the wild-type protein and dominant negative mutants of PKR have established roles for the enzyme in the antiviral effects of IFNs, in the responses of uninfected cells to physiologic stresses, and in cell growth regulation. The possibility that PKR may function as a tumor suppressor and inducer of apoptosis suggests that this IFN-regulated protein kinase may be of central importance to the control of cell proliferation and transformation.

Amino Acid Sequence↗
PubMed1996

Regulation of the double-stranded RNA-dependent protein kinase PKR by RNAs encoded by a repeated sequence in the Epstein-Barr virus genome.

During the initial infection of B lymphocytes by Epstein-Barr virus (EBV) only a few viral genes are expressed, six of which encode the EBV nuclear antigens, EBNAs 1-6. The majority of EBNA mRNAs share common 5'-ends containing a variable number of two alternating and repeated exons transcribed from the BamHI W major internal repeats of the viral DNA. These sequences can also exist as independent small RNA species in some EBV-infected cell types. We present evidence that transcripts from these W repeat regions can exert a trans-acting effect on protein synthesis, through their ability to activate the dsRNA-dependent protein kinase PKR. UV cross-linking and filter binding assays have demonstrated that the W transcripts bind specifically to PKR and can compete with another EBV-encoded small RNA, EBER-1, which was shown previously to bind this kinase. In the reticulocyte lysate system the W RNAs shut off protein synthesis through an ability to activate PKR. In contrast to EBER-1, the W RNAs are unable to block the dsRNA-dependent activation of PKR. Using a purified preparation of the protein kinase we have shown that the W transcripts directly activate PKR in vitro. The results suggest that EBV has the ability both to activate and to inhibit PKR through the actions of different products of viral transcription.

Base Sequence↗
PubMed1996

Detection of double-stranded RNA-protein interactions by methylene blue-mediated photo-crosslinking.

Double-stranded(ds) RNA-binding proteins have diverse functions in the cell. An obstacle to investigating the interactions between these proteins and dsRNA is the relative inefficiency of traditional UV-crosslinking methods for extended regions of dsRNA. We have therefore developed an alternative procedure for RNA-protein photo-crosslinking that efficiently induces RNA-protein crosslinks in double-stranded regions of RNA. We show that dsRNA-protein crosslinks can be induced by visible light in the presence of the dye methylene blue, which most likely mediates crosslinking by intercalating in the dsRNA helix. A recombinant dsRNA binding domain from the Drosophila staufen protein and human protein kinase R were crosslinked by UV or methylene blue to a series of dsRNAs. In each case, the degree of crosslinking was greater with methylene blue, particularly with RNAs with few single-stranded loops. Methylene blue-mediated crosslinking therefore complements and extends the existing repertoire of crosslinking methods for detecting RNA-protein interactions.

Animals↗
PubMed1995

Regulation of the interferon-inducible protein kinase PKR and (2'-5')oligo(adenylate) synthetase by a catalytically inactive PKR mutant through competition for double-stranded RNA binding.

The interferon-inducible double-stranded RNA-dependent protein kinase PKR has been suggested to function as a tumour suppressor gene product. Catalytically inactive mutants of PKR give rise to a tumorigenic phenotype when overexpressed in NIH-3T3 fibroblasts and this has been attributed to a dominant negative effect on the activity of the wild-type enzyme. Here we show that the mutant with Lys296 replaced by Arg, [K296R]PKR, not only inhibits the protein kinase activity of wild-type PKR but is also inhibitory towards another double-stranded RNA-dependent enzyme, the 40-kDa form of (2'-5')oligo(adenylate) synthetase. Inhibition of both wild-type PKR and (2'-5')oligo(adenylate) synthetase is reversed by adding higher concentrations of double-stranded RNA. These results suggest competition between [K296R]PKR and wild-type PKR or (2'-5')oligo(adenylate) synthetase for limiting amounts of double-stranded RNA. Moreover, the data imply that the tumorigenic effect of this PKR mutant could be due to inhibition of additional pathways requiring low levels of double-stranded RNA for activation and cannot be unambiguously attributed to inhibition of endogenous PKR itself.

2',5'-Oligoadenylate Synthetase↗
PubMed1995

Nuclear localization of the interferon-inducible protein kinase PKR in human cells and transfected mouse cells.

The levels and subcellular distribution of the interferon-inducible double-stranded RNA-dependent protein kinase PKR have been measured in human Daudi cells and stably transfected mouse NIH 3T3 cells expressing the human protein kinase. Immunofluorescence of intact cells and quantitative immunoblotting of cell extracts indicate that PKR occurs in both the cytoplasm and the cell nucleus, with staining specifically in the nucleolus. The ratio of cytoplasmic to nuclear PKR is approximately 5:1 in control cells; in response to interferon treatment the protein kinase is induced severalfold in the cytoplasm whereas the level in the nucleus does not increase significantly. Analysis of individual transfected cells by confocal microscopy reveals a pattern of distribution of PKR similar to that in Daudi cells, with immunostaining of cytoplasm and nucleoli. Similar results are observed whether cells expressing wild-type PKR or a catalytically inactive mutant form of the kinase are analyzed, but untransfected 3T3 cells are not stained by the antibody used. Two-dimensional isoelectric focusing analysis of PKR in whole cell extracts reveals the presence of multiple forms with different pI values whereas similar analysis of the nuclear fraction indicates only one predominant species with a relatively basic pI. These results suggest that PKR may have a role in the cell nucleus as well as the cytoplasm and that the subcellular distribution of the protein kinase may be related to post-translational modifications.

3T3 Cells↗
PubMed1994

The La antigen inhibits the activation of the interferon-inducible protein kinase PKR by sequestering and unwinding double-stranded RNA.

The La (SS-B) autoimmune antigen is an RNA-binding protein that is present in both nucleus and cytoplasm of eukaryotic cells. The spectrum of RNAs that interact with the La antigen includes species which also bind to the interferon-inducible protein kinase PKR. We have investigated whether the La antigen can regulate the activity of PKR and have observed that both the autophosphorylation of the protein kinase that accompanies its activation by dsRNA and the dsRNA-dependent phosphorylation of the alpha subunit of polypeptide chain initiation factor eIF-2 by PKR are inhibited in the presence of recombinant La antigen. This inhibition is partially relieved at higher concentrations of dsRNA. Once activated by dsRNA the protein kinase activity of PKR is insensitive to the La antigen. We have demonstrated by a filter binding assay that La is a dsRNA binding protein. Furthermore, when recombinant La is incubated with a 900 bp synthetic dsRNA or with naturally occurring reovirus dsRNA it converts these substrates to single-stranded forms. We conclude that the La antigen inhibits the dsRNA-dependent activation of PKR by binding and unwinding dsRNA and that it may therefore play a role in the regulation of this protein kinase in interferon-treated or virus-infected cells.

Amino Acid Sequence↗
PubMed1994

Regulation of the interferon-inducible eIF-2 alpha protein kinase by small RNAs.

This review describes the structure and function of the double-stranded RNA-dependent protein kinase (PKR) and its interaction with RNA activators and inhibitors. The abilities of small virally-encoded RNAs such as VAI RNA of adenovirus, the Epstein-Barr virus encoded (EBER) RNAs and the Tat-responsive region RNA of HIV-1 to bind to and regulate PKR are reviewed, and the physiological implications of such regulation for the control of viral replication and cell growth are discussed. The potential effects on the activity of PKR of other proteins that bind double-stranded RNA and/or small viral and cellular RNAs are also considered.

Adenoviridae↗
PubMed1993

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.

Adenoviridae↗
PubMed1993

Characterization of the superinduction of the c-myc proto-oncogene in fibroblasts by benzamide derivatives.

In mouse fibroblasts stimulated from quiescence into proliferation by serum the induction of expression of the c-myc proto-oncogene is strongly stimulated by 3-methoxybenzamide. Similar superinduction effects are seen with related compounds such as 3-aminobenzamide and the acid analogues, 3-anisic acid and 3-aminobenzoic acid. Whereas the benzamide derivatives are inhibitors of poly(ADP-ribose) polymerase the acid analogues are not, suggesting that inhibition of this enzyme is not the basis for superinduction of the c-myc gene. Analysis of the kinetics of induction of c-myc mRNA indicates that the RNA accumulates more rapidly as well as to a higher level in the presence of serum plus 3-methoxybenzamide than with serum alone. However the stimulation is transient in both cases. Addition of actinomycin D at 30 min or 1 h after serum stimulation shows the c-myc mRNA to be stable at these times, in the presence or absence of 3-methoxybenzamide. Thus the effect of the latter on c-myc mRNA accumulation is likely to be exerted at the level of transcription or RNA processing rather than turnover of the mRNA.

3T3 Cells↗
PubMed1993

Reversal of the double-stranded-RNA-induced inhibition of protein synthesis by a catalytically inactive mutant of the protein kinase PKR.

The interferon-inducible double-stranded-RNA(dsRNA)-dependent protein kinase PKR has been implicated in both the antiviral and cell growth-regulatory effects of the interferons. Over-expression of the wild-type form of this protein inhibits cell proliferation, whereas over-expression of inactive mutant forms transforms cells to a tumourigenic phenotype. It has been suggested that mutant PKR exerts a dominant negative effect on the activity of the wild-type protein kinase. We have investigated this possibility using the rabbit reticulocyte cell-free translation system in which protein synthesis is inhibited by dsRNA due to activation of PKR and phosphorylation of initiation factor eIF-2. Addition of a highly purified inactive PKR mutant, synthesised in a baculovirus-infected insect cell system, rescues protein synthesis from inhibition by low concentrations of dsRNA in a dose-dependent manner. The PKR mutant has no effect on protein synthesis in the absence of dsRNA or in the presence of another inhibitory protein kinase, the haem-controlled repressor. Inhibition of translation can be re-established in the presence of the mutant PKR by adding a higher concentration of dsRNA. These results suggest that inactive mutant PKR does exert a dominant negative effect on wild-type PKR and that this may be due to competition for dsRNA binding.

Animals↗