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PubMed1996

A novel collection of accessory factors associated with yeast RNA polymerase II.

A relatively simple subset of general transcription factors is sufficient for transcript initiation by RNA polymerase II. However, a recently identified "holoenzyme" contains additional accessory proteins required for mediating signals from some activators (Y-J. Kim et al., 1994, Cell 77, 599-608; A. Koleske and R. Young, 1994, Nature 368, 466-469). By immobilizing RNA polymerase II and associated proteins (RAPs) from a transcriptionally active yeast extract, we have identified a novel collection of proteins distinct from those found in the holoenzyme. The eluted RAP fraction did not contain the holoenzyme components Srb2,4,5 + 6p, Gal11p, or Sug1p, but did include the known transcription factors TFIIB and TFIIS and the three subunits of yeast TFIIF (Ssu71p/Tfg1p, Tfg2p, and Anc1p/Tfg3p). Also isolated as RAPs are two proteins (Cdc73p and Paf1p) with interesting connections to gene expression. Mutations in CDC73 and PAF1 affect cell growth and the abundance of transcripts from a subset of yeast genes (X. Shi et al., Mol. Cell. Biol., 1996 16, 669-676). The RAP fraction may therefore define one or more functional forms of RNA polymerase II distinct from the activator-mediating holoenzyme.

Amino Acid Sequence↗

PubMed1997

Changes in late-embryogenesis-abundant (LEA) messenger RNAs and dehydrins during maturation and premature drying of Ricinus communis L. seeds.

In Ricinus communis L. (castor bean) endosperms, two classes of Late Embryogenesis Abundant (Lea) transcripts were first detected during mid-development (at 30-35 days after pollination, DAP) and peaked at 50 DAP, just prior to the onset of desiccation. Most of the Class I mRNAs declined substantially during desiccation itself; Class II mRNAs remained abundant in the mature dry (60 DAP) seed. Following imbibition, all Lea mRNAs abundant in the mature dry seed declined rapidly (within 5-24 h). Premature drying of developing 35-DAP seeds resulted in the loss of storage-protein mRNAs (Leg B Mat I); following rehydration, mRNAs encoding post-germinative proteins (Germ D91, D30 and D38) increased in the endosperm. The Lea mRNAs present in the developing fresh seed at 35 DAP were preserved, but did not increase in response to premature desiccation; upon rehydration these Lea mRNAs declined within 5 h. During seed development, substantial changes occurred in the synthesis of a subset of LEA proteins referred to as "dehydrins'; in particular, new dehydrin polypeptides were induced between 40 and 60 DAP. Such proteins were not as evident in prematurely dried endosperms. In contrast to the rapid loss of Lea mRNAs during germination, many of the dehydrin proteins abundant in the dried seed persisted following imbibition or rehydration.

Ricinus communis↗

PubMed1992

Attenuated expression of epithelial cell adhesion molecules in murine polycystic kidney disease.

Polycystic kidney disease is an inherited disorder of parenchymal structure that leads to renal failure. Cysts begin as focal dilations in proximal tubules and collecting ducts, giving rise to cyst walls lined by a phenotypically disturbed epithelium that expresses dysfunctional transport and matrix proteins. We used an mRNA search protocol to probe efficiently for tissue-specific disturbances that might underlie the formation of cysts. This search assessed the relative abundance of transcripts encoding a variety of growth factors (transforming growth factor-beta 1, interleukin-6, tumor necrosis factor, and endothelin-1), structural proteins (collagen IV, nidogen, fibronectin, and laminins A and B1), and cell adhesion molecules (CAMs; E-cadherin, N-CAM, laminin receptor, and fibronectin receptor) in the cystic kidneys of cpk/cpk mice and uncovered a previously unrecognized early reduction in mRNA encoding N-CAM (54%) and E-cadherin (56%) (n = 5; P less than 0.001). Levels of transcripts for growth factors, structural proteins, and for fibronectin and laminin receptors in normal and cystic kidneys were generally similar. The reduction in transcripts for N-CAM and E-cadherin in kidneys from cystic mice was not observed in autologous liver. The immunofluorescent staining of cystic kidneys confirmed that the decrease in N-CAM and E-cadherin was generally confined to regions abundant in developing cystic epithelium. The presence of both N-CAM and E-cadherin appears to guide the sequential differentiation and polarization of normal renal epithelium, and their attenuated expression in the kidney of cpk/cpk mice may be a material factor contributing to the pathogenesis of cyst formation.

Animals↗

PubMed2002

Differential regulation of genes transcribed by nucleus-encoded plastid RNA polymerase, and DNA amplification, within ribosome-deficient plastids in stable phenocopies of cereal albino mutants.

We isolated stable albino plants of barley and maize by inhibiting plastid protein synthesis with streptomycin and propagating bleached seedlings in the absence of antibiotics in vitro. Albino plants are deficient in plastid translation products and plastid ribosomal RNAs, and are stable phenocopies of the barley albostrians and maize iojap mutants, which contain ribosome-free plastids. Once plastid ribosomes are lost they cannot be re-synthesized because about one-third of plastid ribosomal proteins are themselves plastid encoded. The group II/subgroup IIA intron in plastid rpl2 transcripts was not processed in albinos, providing strong evidence for the absence of plastid translation. Photosynthesis-related plastid mRNAs and plastid tRNAs were down-regulated in albino leaves. A differential influence of plastid ribosome deficiency on mRNA levels allowed us to divide genes transcribed by nucleus-encoded plastid RNA polymerase into two groups. Northern analysis revealed increases in the levels of clpP, rpl2, rpl23, rps15 and rpoB mRNAs in total RNA from albino leaves relative to those in green leaves. In contrast, albinism did not increase the band intensities of rps2 and rps4 messages. Plastid ribosome-associated factor(s) or plastid-encoded product(s) play a role in the initiation, termination, processing or stability of transcripts containing trnG(UCC) and rps4. Excision and 100-fold amplification of a 5.2-kb region of plastid DNA encompassing the trnG(UCC) and trnE(UUC) genes was observed in one of four albino barley plants. Gene amplification was correlated with the accumulation of abundant novel transcripts derived from regions flanking the trnG(UCC) gene.

Base Sequence↗

PubMed1991

Retinoic acid treatment alters the distribution of retinoic acid receptor-beta transcripts in the embryonic chick face.

Retinoic acid is a metabolite of vitamin A that can act as a signalling molecule in a number of developmental systems. Retinoic acid is also known to be teratogenic in mammals, causing a range of defects including abnormalities in craniofacial development. Exposure of the developing chick face to retinoic acid released slowly from a bead implanted in the wing bud results in a specific facial defect, in which outgrowth of the frontonasal mass is inhibited. This results in clefting of the primary palate and absence of the upper beak. To investigate the role of nuclear retinoic acid receptors in normal and abnormal chick face morphogenesis, we isolated chick retinoic acid receptor-beta (RAR-beta) cDNA clones and probed northern blots of RNA isolated from chick embryos at stages 22, 24 and 25 and from adults. RAR-beta transcripts of 2.8 and 3.5 kb were present in several regions of the embryo, including the facial primordia, and were also present at much lower levels in adult tissues. In situ hybridisation showed that RAR-beta transcripts were present in all of the facial primordia at embryonic stages 20, 24 and 28, but that their distribution was not uniform. Transcripts were abundant in the lateral nasal processes, at the edges and corners of the frontonasal mass and in the anterior part of the maxillary primordia. Lower levels were present elsewhere. Treatment of stage 20 embryos with retinoic acid altered the distribution of RAR-beta transcripts in the maxillary primordia, such that high levels of transcripts were present throughout, rather than being confined to the anterior part. This change was detectable at stage 24, before any alterations in the morphology of the facial primordia were apparent. By stage 28, when the morphology of the facial primordia was clearly abnormal, there were more widespread changes in the distribution of RAR-beta transcripts. These results show that RAR-beta transcripts are particularly concentrated in regions of the primordia that give rise to the upper beak, the development of which is specifically affected by retinoic acid. In addition, they demonstrate that retinoic acid can induce changes in the pattern of expression of RAR-beta transcripts in vivo.

Amino Acid Sequence↗

PubMed2005

Apical expression of three RhopH1/Clag proteins as components of the Plasmodium falciparum RhopH complex.

The Plasmodium falciparum high molecular mass rhoptry protein ('PfRhopH') complex is important for parasite growth and comprises three distinct gene products: RhopH1, RhopH2 and RhopH3. We have previously shown that P. falciparum RhopH1 is encoded by either PFC0110w (clag3.2) or PFC0120w (clag3.1), members of the previously-named clag (cytoadherence-linked asexual gene) multigene family. In this report, we have further characterized rhoph1/clag members in terms of gene structure, transcription and protein expression. The cDNA sequences for all five rhoph1/clag members were determined, confirming previous in silico predictions of intron-exon boundaries. All member genes were transcribed in HB3 and 3D7 parasite lines, but clag3.2 was not transcribed in Dd2 parasites. The peak abundance of transcripts for all genes was observed during the late schizont stage. Antisera specific to Clag2 and Clag3.1 localized these proteins to the apical end of merozoites in segmented schizonts, and both proteins are found to be components of the PfRhopH complex. PfRhopH complex that was immunoprecipitated with anti-Clag9 antibody contained neither Clag2 nor Clag3.1, thereby suggesting that PfRhopH complexes contain only individual rhoph1/clag gene products. Since the PfRhopH complex binds the erythrocyte surface, and RhopH2 and RhopH3 are encoded by single copy genes, the RhopH1/Clag proteins may serve to confer some degree of specificity to the roles of the individual complexes.

Animals↗

PubMed2005

Identification and expression of Toll-like receptors 1-10 in selected bovine and ovine tissues.

Members of the Toll-like receptor (TLR) family are vital to immune function through the sensing of pathogenic agents and initiation of an appropriate immune response. More specifically, tissue and cell specific TLR expression patterns have been correlated with the ability to respond to various pathogenic challenges. Bovine sequence exists for 4 of the 10 human TLR Reference Sequences and no ovine TLR sequence has been reported. The main goal of this study was to determine if homologues of human TLRs 1-10 exist within the cattle and sheep. Subsequent to this, quantitative real time PCR assays were to be developed to produce transcript expression profiles in cattle skin and sheep gut-associated lymphoid tissue, as these epithelial tissues are the primary sites of host/pathogen interactions for numerous pathogens. Our findings show that homologues of human TLRs 1-10 do indeed exist within both cattle and sheep, with respective bovine and ovine homologues sharing at least 95% nucleotide sequence identity and 83-90% identity to the corresponding human Reference Sequences. Conservation of the amino acid sequence between homologous ruminant and human TLRs ranged between 84 and 97%. Quantitative real time PCR (qPCR) assays confirmed expression of all 10 TLRs within ovine jejunum, Peyer's patch and mesenteric lymph nodes. While in bovine skin all TLRs apart from TLR6 were detected. The most abundant TLR transcripts within the ovine jejunum were TLRs 3, 5 and 6, while TLRs 6, 7 and 10 were abundant in both ovine Peyer's patch and mesenteric lymph node. In bovine skin TLRs 2 and 7 were most abundant. In all tissues tested TLR4 expression was at the lower limit of detection.

Amino Acid Sequence↗

PubMed1996

Effects of progesterone on serum levels of IGF-1 and on femur IGF-1 mRNA in ovariectomized rats.

Local and systemic insulin-like growth factors (IGFs) may be involved in the regulation of bone formation by sex hormones. The present studies describe the in vivo effects of estradiol, progesterone, or both on IGF-1 mRNA abundance in bone, serum IGF-1 levels, and bone formation. Rats were sham-operated (SHAM) or ovariectomized (OVX) at 12 weeks of age and used a week later in three experiments. First, OVX rats were treated with vehicle, estradiol, and/or medroxyprogesterone (MPA) for 3 weeks, and bone formation was assessed in the tibial metaphysis. Second, OVX rats were treated in the same manner and serum IGF-1 levels measured. Third, OVX rats were treated with an injection of vehicle, estradiol, and/or progesterone, and 24 h later, levels of IGF-1 mRNA in the femur were analyzed. The mineralized surface, mineral opposition rate, and bone formation rate (BFR) were higher in OVX than in SHAM rats. The BFR was decreased in estrogen-treated but increased in MPA-treated rats compared with vehicle-treated OVX rats. Circulating levels of IGF-1 were higher in OVX than in SHAM rats but were not affected by sex hormones in a 3-week experiment, whereas these levels were not different among groups in a 24-h experiment. Northern analysis detected 7.5 and 0.8 kb IGF-1 mRNA transcripts. The abundance of IGF-1 mRNA was higher in OVX than in SHAM rats. IGF-1 transcripts 7.5 and 0.8 kb were decreased by 72 and 29%, respectively, in estrogen-treated and increased by 44 and 43%, respectively, in progesterone-treated rats compared with vehicle-treated OVX rats. We conclude that in the short term, estrogen lowers and progesterone raises bone IGF-1 mRNA and these changes are followed by coordinated changes in bone formation rate.

Analysis of Variance↗

PubMed1983

Physical Analysis of Autographa californica Nuclear Polyhedrosis Virus Transcripts for Polyhedrin and 10,000-Molecular-Weight Protein.

In Autographa californica nuclear polyhedrosis virus-infected cells, polyhedrin, the major structural polypeptide of the viral occlusions, and a low-molecular-weight viral protein with a molecular weight of approximately 10,000 (10K) accumulated to high levels late in infection. Two polyadenylated RNAs 1,200 and 630 bases in size were the most abundant viral transcripts present in the cytoplasm at 48 h postinfection. Evidence is presented that these RNAs were the transcripts for polyhedrin and 10K proteins. The 630-base RNA and five other major polyadenylated RNAs present at 48 h postinfection mapped within or near A. californica nuclear polyhedrosis virus EcoRI-P. The DNA sequences that code for polyhedrin mRNA were examined by S1 nuclease analysis. The polyhedrin gene contained no detectable introns and mapped at 3,990 to 5,200 base pairs to the right of the origin of the circular A. californica nuclear polyhedrosis virus physical map. Heterogeneity at the 5' end of polyhedrin mRNA was observed by using S1 nuclease analysis with 5' end-labeled DNA probes.

Journal Article↗

PubMed1998

Characterization of the Mt4 gene from Medicago truncatula.

Previously we identified Mt4, a phosphate starvation inducible cDNA from Medicago truncatula which is down-regulated in roots in response to phosphate fertilization as well as colonization by arbuscular mycorrhizal (AM) fungi (AM). Here we present further studies of Mt4. Expression was highly sensitive to exogenous applications of phosphate fertilizer; transcripts were abundant in roots fertilized with nutrient solution lacking phosphate, reduced when fertilized with 0.02 or 0.1 mM phosphate and undetectable when fertilized with 1 or 5 mM phosphate. A time course experiment, to study the expression of Mt4 following colonization by AM fungi, revealed that Mt4 transcripts increased in uncolonized roots during the first three weeks of growth and then plateaued, while transcript levels in roots colonized with the AM fungus, Glomas versiforme, increased transiently and then decreased. Although the Mt4 gene is expressed exclusively in roots, transcripts were also detected in M. truncatula cell suspension cultures following phosphate starvation. A genomic clone containing the Mt4 gene and 1133 bp of the 5' flanking sequence was identified from a M. truncatula genomic library. The promoter region contains a conserved cis-element found in the promoters of phosphate starvation inducible genes of yeast and tomato. As Mt4 is the first cDNA reported to show independent regulation by both phosphate and mycorrhizal fungi, the genomic clone may provide a starting point from which to analyze the signal transduction pathways involved in these two processes.

Base Sequence↗

PubMed2006

Polyadenylation of ribosomal RNA in human cells.

The addition of poly(A)-tails to RNA is a process common to almost all organisms. In eukaryotes, stable poly(A)-tails, important for mRNA stability and translation initiation, are added to the 3' ends of most nuclear-encoded mRNAs, but not to rRNAs. Contrarily, in prokaryotes and organelles, polyadenylation stimulates RNA degradation. Recently, polyadenylation of nuclear-encoded transcripts in yeast was reported to promote RNA degradation, demonstrating that polyadenylation can play a double-edged role for RNA of nuclear origin. Here we asked whether in human cells ribosomal RNA can undergo polyadenylation. Using both molecular and bioinformatic approaches, we detected non-abundant polyadenylated transcripts of the 18S and 28S rRNAs. Interestingly, many of the post-transcriptionally added tails were composed of heteropolymeric poly(A)-rich sequences containing the other nucleotides in addition to adenosine. These polyadenylated RNA fragments are most likely degradation intermediates, as primer extension (PE) analysis revealed the presence of distal fragmented molecules, some of which matched the polyadenylation sites of the proximal cleavage products revealed by oligo(dT) and circled RT-PCR. These results suggest the presence of a mechanism to degrade ribosomal RNAs in human cells, that possibly initiates with endonucleolytic cleavages and involves the addition of poly(A) or poly(A)-rich tails to truncated transcripts, similar to that which operates in prokaryotes and organelles.

Cell Line, Tumor↗

PubMed1996

T1 alpha protein is developmentally regulated and expressed by alveolar type I cells, choroid plexus, and ciliary epithelia of adult rats.

T1 alpha is the first marker gene known to be expressed in the adult lung solely by the alveolar type I epithelial cell. Previous studies showed that T1 alpha transcripts are abundant in early rat embryos where they are found in the nervous system and in the foregut and certain of its derivatives including the primitive lung. By mid- to late gestation T1 alpha messenger RNA (mRNA) expression is lost from neural tissues but appears to increase in the lung throughout fetal life. To determine whether the T1 alpha transcripts are translated into protein, especially in early embryos which sometimes express transcripts that are translationally silent, we performed immunohistochemistry on embryos and fetal tissues and analyzed certain tissues by western blotting using a monoclonal antibody against T1 alpha protein. T1 alpha protein is present at all sites that have previously been shown to express the mRNA and at similar developmental stages. As estimated from western blots, T1 alpha protein abundance peaks at about fetal day 16 in the brain and decreases thereafter to a relative level in the adult that is lower than that of the neural tube of the day 13 embryo. Relative protein abundance in the lung is very low, although detectable, on embryonic day 13 but increases slowly until fetal day 20 when there is a dramatic increase. At the time of birth, restriction to the type I cell is not complete and therefore must occur during postnatal lung development. Immunostaining reveals additional sites of expression in fetal and adult rats that had not been clearly visualized in previous in situ hybridization studies. T1 alpha is present in mesonephric tubules and apparently in primitive germ cells but is not detectable in specific cells in the adult kidney, ovary, or testis. However, cells of the choroid plexus of the central nervous system and the ciliary epithelium of the eye express T1 alpha in both fetuses and adults. The well-known functions of these epithelia are to elaborate cerebrospinal fluid and aqueous humor respectively by processes of active ion transport and water fluxes, probably through the aquaporin 1 (channel-forming integral membrane protein [CHIP] 28). We speculate therefore that T1 alpha protein may modulate or participate in these types of cellular functions in the lung.

Age Factors↗

PubMed2005

Analysis of gene expression in transgenic plants.

Recent years have seen a huge increase and improvement in techniques for analysis of transgene expression in plants. The analysis of RNA frequently provides a vital link between changes in enzyme levels and/or metabolites and the phenotype. This chapter focuses on RNA-based techniques for the analysis of transgene expression, beginning with the extraction of RNA and its evaluation in terms of purity and integrity by spectrophotometry and gel electrophoresis, respectively. Common methods of transcript analysis by Northern and dot-blot hybridizations using nonradioactive probing methods are described. A protocol for reverse-transcriptase polymerase chain reaction (RT-PCR) as a method of establishing transgene expression qualitatively, as well as a procedure for quantitative RT-PCR for comparing relative abundance of transcript levels of two or more genes are described. Lastly, a protocol for localization of RNA transcripts within tissues by in situ hybridization is included.

Blotting, Northern↗

PubMed1998

Transcripts of the transposon mariner are present in epileptic brain.

Mobile genetic elements termed transposons have been increasingly implicated in human disease. The small transposon mariner is widespread within non-vertebrate genomes and causes mutation by replication, excision, and insertion of itself without an RNA intermediate. We find that human DNA contains about 60 copies of this gene. Mariner transcripts are abundant in RNA prepared from sclerotic epileptic hippocampi. In contrast, typically no mariner-specific RNA is detected in non-sclerotic hippocampi from other epileptic patients or from autopsies. A complete but non-functional copy was obtained using rapid amplification of cDNA ends (RACE). This human mariner transcript is approximately 45% homologous to a functional counterpart active in Drosophila, with a coding region of 1035 bases flanked by 32 base inverted terminal repeats. The differential expression of mariner transcripts within sclerotic hippocampi suggests the probable activity of an autonomous element which by mutating critical genes could establish an epileptogenic substrate in the hippocampus.

Amino Acid Sequence↗

PubMed2004

Transcription of the rod-shaped viruses SIRV1 and SIRV2 of the hyperthermophilic archaeon sulfolobus.

The double-stranded DNA genomes of the crenarchaeal rudiviruses SIRV1 (32 kb) and SIRV2 (35 kb) were previously sequenced. Here we present results of the analysis of gene expression of these viruses at different time points after infection of the host cell, Sulfolobus islandicus, and of the mapping of transcriptional start sites. Transcription of both genomes starts simultaneously at multiple sites spread over the total length of the genome and from both strands. The earliest time point when viral transcripts could be detected in cells was 30 min after infection. At this time point all the viral genes, except one, were transcribed. Many genes were clustered and appeared to be transcribed as polycistronic messengers. Although the coat protein-encoding gene was initially also transcribed as a polycistronic messenger, an abundant monocistronic transcript of this gene was detected 2 to 3 h after infection, just before assembly of viral particles. The expression of a single gene, adjacent to the coat protein gene, was upregulated at the late phase of infection, suggesting that it might be involved in specific processing and activation of the coat protein messenger. Start sites of 13 transcripts from the SIRV1 genome have been mapped by primer extension, and promoter sequences have been identified. Similar to host promoters, these viral promoters all contain potential binding sites for the archaeal transcription factors TATA binding protein and transcription factor B. In addition, most of them contain a virus-specific consensus element, suggesting the involvement of alternative transcription factors.

Base Sequence↗

PubMed2005

Growth arrest of thyrotropic tumors by thyroid hormone is correlated with novel changes in Wnt-10A.

The molecular mechanism underlying thyroid hormone inhibition of thyrotrope cell growth is poorly understood. A comprehensive screen for T3-regulated genes involved in thyrotrope cell regulation was performed by Affymetrix MGU74A Genechip microarray analyses, which compared total RNA from hypothyroid versus 24 h T3-treated TtT-97 tumors. Of the 13,000 genes screened, a number of novel, T3-responsive candidate genes were identified. Within the Wnt family of growth factors, only Wnt-10A transcripts were abundantly expressed in hypothyroid TtT-97 tumors, and were down-regulated with T3 by 6 h of treatment. In addition, nuclear beta-catenin, which is a downstream mediator of canonical Wnt signaling, was decreased at the protein and functional levels. TtT-97 growth suppression was associated with decreased cyclin A transcript levels. We conclude that treatment of thyrotropic TtT-97 tumors with T3 resulted in the decreased expression of Wnt-10A, and that thyroid hormone may inhibit growth via cyclin A regulation.

Animals↗

PubMed1996

Unusual activation of the integrated preS1 promoter of woodchuck hepatitis virus in a liver tumour.

We have analysed abnormal virus RNAs produced from integrated woodchuck hepatitis virus (WHV) sequences in two woodchuck liver tumours. Analysis of cDNA clones revealed that these transcripts consisted of rearranged, virus-specific RNAs encoding the WHV surface antigens. In one tumour, transcription was driven by the major preS2/S promoter and terminated at a cryptic poly(A) signal in the 5' end of the P gene, giving rise to a truncated version of the normal viral S message. In contrast, the integrated preS2/S promoter remained silent in the second tumour. The start sites of two abundant WHV transcripts encoding the large and middle surface proteins were localized about 100 bp upstream and 300 bp downstream of the preS1 translation initiation codon, corresponding to minor start sites of the normal surface protein mRNAs in chronically infected liver. Thus, the preS1 promoter, a weak promoter in episomal replicative forms of the virus, was activated in the integrated state in this tumour. Our results indicate that alternative usage of the preS1 or the preS2/S promoter in the integrated state may yield differential production of the three virus surface proteins in woodchuck liver tumours.

Animals↗

PubMed2006

Reovirus induces and benefits from an integrated cellular stress response.

Following infection with most reovirus strains, viral protein synthesis is robust, even when cellular translation is inhibited. To gain further insight into pathways that regulate translation in reovirus-infected cells, we performed a comparative microarray analysis of cellular gene expression following infection with two strains of reovirus that inhibit host translation (clone 8 and clone 87) and one strain that does not (Dearing). Infection with clone 8 and clone 87 significantly increased the expression of cellular genes characteristic of stress responses, including the integrated stress response. Infection with these same strains decreased transcript and protein levels of P58(IPK), the cellular inhibitor of the eukaryotic initiation factor 2alpha (eIF2alpha) kinases PKR and PERK. Since infection with host shutoff-inducing strains of reovirus impacted cellular pathways that control eIF2alpha phosphorylation and unphosphorylated eIF2alpha is required for translation initiation, we examined reovirus replication in a variety of cell lines with mutations that impact eIF2alpha phosphorylation. Our results revealed that reovirus replication is more efficient in the presence of eIF2alpha kinases and phosphorylatable eIF2alpha. When eIF2alpha is phosphorylated, it promotes the synthesis of ATF4, a transcription factor that controls cellular recovery from stress. We found that the presence of this transcription factor increased reovirus yields 10- to 100-fold. eIF2alpha phosphorylation also led to the formation of stress granules in reovirus-infected cells. Based on these results, we hypothesize that eIF2alpha phosphorylation facilitates reovirus replication in two ways-first, by inducing ATF4 synthesis, and second, by creating an environment that places abundant reovirus transcripts at a competitive advantage for limited translational components.

Activating Transcription Factor 4↗