Gene-specific Translation Research Articles

Phosphorylation of the α subunit of translation initiation factor-2 by PKR mediates protein synthesis inhibition in the mouse brain during status epilepticus

In response to different cellular stresses, a family of protein kinases phosphorylates eIF2alpha (alpha subunit of eukaryotic initiation factor-2), contributing to regulation of both general and genespecific translation proposed to alleviate cellular injury or alternatively induce apoptosis. Recently, we reported eIF2alpha(P) (phosphorylated eIF2alpha) in the brain during SE (status epilepticus) induced by pilocarpine in mice, an animal model of TLE (temporal lobe epilepsy) [Carnevalli, Pereira, Longo, Jaqueta, Avedissian, Mello and Castilho (2004) Neurosci. Lett. 357, 191-194]. We show in the present study that one eIF2alpha kinase family member, PKR (double-stranded-RNA-dependent protein kinase), is activated in the cortex and hippocampus at 30 min of SE, reflecting the levels of eIF2alpha(P) in these areas. In PKR-deficient animals subjected to SE, eIF2alpha phosphorylation was clearly evident coincident with activation of a secondary eIF2alpha kinase, PEK/PERK (pancreatic eIF2alpha kinase/RNA-dependent-protein-kinase-like endoplasmic reticulum kinase), denoting a compensatory mechanism between the two kinases. The extent of eIF2alpha phosphorylation correlated with the inhibition of protein synthesis in the brain, as determined from polysome profiles. We also found that C57BL/6 mice, which enter SE upon pilocarpine administration but are more resistant to seizure-induced neuronal degeneration, showed very low levels of eIF2alpha(P) and no inhibition of protein synthesis during SE. These results taken together suggest that PKR-mediated phosphorylation of eIF2alpha contributes to inhibition of protein synthesis in the brain during SE and that sustained high levels of eIF2alpha phosphorylation may facilitate ensuing cell death in the most affected areas of the brain in TLE.

Antiviral effect of the mammalian translation initiation factor 2α kinase GCN2 against RNA viruses

In mammals, four different protein kinases, heme-regulated inhibitor, double-stranded RNA-dependent protein kinase (PKR), general control non-derepressible-2 (GCN2) and PKR-like endoplasmic reticulum kinase, regulate protein synthesis in response to environmental stresses by phosphorylating the alpha-subunit of the initiation factor 2 (eIF2alpha). We now report that mammalian GCN2 is specifically activated in vitro upon binding of two nonadjacent regions of the Sindbis virus (SV) genomic RNA to its histidyl-tRNA synthetase-related domain. Moreover, endogenous GCN2 is activated in cells upon SV infection. Strikingly, fibroblasts derived from GCN2-/- mice possess an increased permissiveness to SV or vesicular stomatitis virus infection. We further show that mice lacking GCN2 are extremely susceptible to intranasal SV infection, demonstrating high virus titers in the brain compared to similarly infected control animals. The overexpression of wild-type GCN2, but not the catalytically inactive GCN2-K618R variant, in NIH 3T3 cells impaired the replication of a number of RNA viruses. We determined that GCN2 inhibits SV replication by blocking early viral translation of genomic SV RNA. These findings point to a hitherto unrecognized role of GCN2 as an early mediator in the cellular response to RNA viruses.

P2P‐R expression is genetically coregulated with components of the translation machinery and with PUM2, a translational repressor that associates with the P2P‐R mRNA

P2P-R is a nuclear protein with potential functional roles in the control of gene expression and mitosis. The P2P-R protein also interacts with the p53 and Rb1 tumor suppressor proteins. To search for additional functional associations of P2P-R, we employed the WebQTL database that contains the results of cDNA microarray analysis on forebrain, cerebellum, and hematopoietic stem cell (HSC) specimens of multiple BXD recombinant inbred strains of mice. Using WebQTL, gene products were identified that show genetically based coexpression with P2P-R. Initial studies identified general groups of mRNAs that share common functional roles and high covariation in expression with P2P-R. These functional groups involved the regulation of transcription, nucleotide binding, translation control, and ion transport. The findings related to translational mechanisms were further evaluated. In HSCs, expression of P2P-R mRNA demonstrates an impressive expression correlation with a group of gene products associated with translation; high expression of P2P-R specifically was associated with decreased expression of 29 ribosomal protein mRNAs. In all three tissues that were screened using the WebQTL database, a strong positive expression covariance between P2P-R and the Pum2 gene product also was observed. PUM2 is a member of the highly conserved Puf family of RNA binding proteins that often function as gene-specific translation regulators. The ability of Puf proteins to repress translation is mediated by their binding to specific elements located in the 3' untranslated region (UTR) of their target mRNAs. To assess the functional significance of the strong genetic correlation in expression of P2P-R and PUM2, the 3' UTR of the P2P-R mRNA was analyzed and found to contain one perfect consensus and two near-perfect consensus PUM2 binding sequences. PUM2 pull-down methods combined with reverse transcription and RT-PCR confirmed that PUM2 does indeed bind P2P-R mRNA. These results suggest that P2P-R expression may be translationally regulated by PUM2 and that P2P-R may modulate translation by influencing ribosomal protein gene expression. This study represents the first description of a RNA target for mammalian Puf proteins and the first molecular confirmation of information obtained using the WebQTL database.

RNA-binding characteristics of the chloroplast S1-like ribosomal protein CS1.

The chloroplast ribosomal protein CS1, the homolog of the bacterial ribosomal protein S1, is believed to be involved in the process of ribosome binding to mRNA during translation. Since translation control is an important step in chloroplast gene expression, and in order to study initiation complex formation, we studied the RNA-binding properties of CS1 protein. We found that most of the CS1 protein in spinach chloroplast co-purified with the 30S ribosomal subunit. The relative binding affinity of RNA to CS1 was determined using the UV-crosslinking competition assay. CS1 protein binds the ribohomopolymer poly(U) with a relatively high binding affinity. Very low binding affinities were obtained for the other ribohomopolymers, poly(G), poly(A) and poly(C). In addition, no specific binding of CS1, either in the 30S complex or as a recombinant purified protein, was obtained to the 5'-untranslated region of the mRNA in comparison to the other parts. RNA-binding experiments, in which the N- and C-termini of the protein were analyzed, revealed that the RNA-binding site is located in the C-terminus half of the protein. These results suggest that CS1 does not direct the 30S complex to the initiation codon of the translation site by specific binding to the 5'-untranslated region. In bacteria, specific binding is derived by base pairing between 16S rRNA and the Shine-Dalagarno sequences. In the chloroplast, nuclear encoded and gene-specific translation factors may be involved in the determination of specific binding of the 30S subunit to the initiator codon.

ElF-2 kinases: regulators of general and gene-specific translation initiation

Phosphorylation of eukaryotic initiation factor-2 (elF-2) is an important mechanism regulating general translation initiation. Two mammalian elF-2 kinases, the double-stranded-RNA-dependent kinase (PKR) and hemeregulated inhibitor kinase (HRI), have been characterized by sequencing, revealing shared sequence and structural features distinct from other eukaryotic protein kinases. Recent work in yeast has shown that a third related kinase, GCN2, also phosphorylates the regulated site in elF-2. However, unlike the mammalian kinases, this kinase regulates genespecific translation. Current models are presented for the regulation of each elF-2 kinase, and the molecular basis for how this general form of regulation is adapted to control expression of a single species of messenger RNA is discussed.

Two yeast nuclear genes, CBS1 and CBS2, are required for translation of mitochondrial transcripts bearing the 5'-untranslated COB leader.

Mutations in one of the yeast nuclear genes CBS1 or CBS2 both prevent the excision of the maturase-coding introns bI2, bI3 and bI4 from the mitochondrial COB precursor transcript. Mutant strain MK2 (cbs1-1) has recently been reported to be primarily defective in the translation of COB transcripts, as it can be suppressed by a fusion of the COB structural gene with the 5' untranslated leader of the mitochondrial OLI1 gene (G. Rödel, A. Körte and F. Kaudewitz, Curr Genet 9: 641-648). Here I report that the effect of mutation cbs2-1, too, is suppressed by this gene rearrangement. CBS2 is the second nuclear gene identified which is involved in the translation of mitochondrial transcripts bearing the 5' untranslated COB leader. Gene specific translation control appears to be a major mode of regulation of mitochondrial gene expression in Saccharomyces cerevisiae.

Units of functional stability in bacteriophage messenger RNA

The functional stabilities of the messages for seven of the nine genes of bacteriophage S13 were determined by measuring the amount of gene-specific translation at different times after the addition of rifampicin to block further initiation of transcription. Although the decay of mRNA function for each gene product tested was exponential, the functional half-lives of the mRNA were not the same for all phage genes, but fell into at least five groups: 6·2 minutes for genes B and C ; 4·4 minutes for D ;3·8 minutes for F, G and H ; 2·5 minutes for A * and 0·65 minutes for A . The overall mass-decay of bulk phage mRNA as determined by hybridization is much slower, with a half-life of 10·6 minutes. The inactivating event for A messages frequently occurs before the RNA polymerase has reached the 3′-end of the gene. The capsid genes F, G and H are contiguous and exhibit polarity, suggesting that the expression of these three genes is regulated in part by a common mRNA functional-decay rate. For the contiguous genes B and C , the similarity of their inactivation rates suggests that they too might show polarity. Amber mutants in B indeed cause at least an 80% reduction in the amount of C protein synthesis. The two gene clusters, B-C and F-G-H , allow recognition of a new type of mRNA unit, characterized by the similar functional stabilities of the component genes. The existence of units of functional stability suggests possible mechanisms for mRNA degradation and the regulation of protein synthesis.

Identification of the acrylamide gel protein peak for gene C of phages S13 and ∅X174

The functional stabilities of the messages for seven of the nine genes of bacteriophage S13 were determined by measuring the amount of gene-specific translation at different times after the addition of rifampicin to block further initiation of transcription. Although the decay of mRNA function for each gene product tested was exponential, the functional half-lives of the mRNA were not the same for all phage genes, but fell into at least five groups: 6·2 minutes for genes B and C; 4·4 minutes for D;3·8 minutes for F, G and H; 2·5 minutes for A* and 0·65 minutes for A. The overall mass-decay of bulk phage mRNA as determined by hybridization is much slower, with a half-life of 10·6 minutes. The inactivating event for A messages frequently occurs before the RNA polymerase has reached the 3′-end of the gene.The capsid genes F, G and H are contiguous and exhibit polarity, suggesting that the expression of these three genes is regulated in part by a common mRNA functional-decay rate. For the contiguous genes B and C, the similarity of their inactivation rates suggests that they too might show polarity. Amber mutants in B indeed cause at least an 80% reduction in the amount of C protein synthesis. The two gene clusters, B-C and F-G-H, allow recognition of a new type of mRNA unit, characterized by the similar functional stabilities of the component genes. The existence of units of functional stability suggests possible mechanisms for mRNA degradation and the regulation of protein synthesis.