Protein Synthesis Machinery Of Cells Research Articles

Ataluren and aminoglycosides stimulate read-through of nonsense codons by orthogonal mechanisms

During protein synthesis, nonsense mutations, resulting in premature stop codons (PSCs), produce truncated, inactive protein products. Such defective gene products give rise to many diseases, including cystic fibrosis, Duchenne muscular dystrophy (DMD), and some cancers. Small molecule nonsense suppressors, known as TRIDs (translational read-through-inducing drugs), stimulate stop codon read-through. The best characterized TRIDs are ataluren, which has been approved by the European Medicines Agency for the treatment of DMD, and G418, a structurally dissimilar aminoglycoside. Previously [1], we applied a highly purified in vitro eukaryotic translation system to demonstrate that both aminoglycosides like G418 and more hydrophobic molecules like ataluren stimulate read-through by direct interaction with the cell's protein synthesis machinery. Our results suggested that they might do so by different mechanisms. Here, we pursue this suggestion through a more-detailed investigation of ataluren and G418 effects on read-through. We find that ataluren stimulation of read-through derives exclusively from its ability to inhibit release factor activity. In contrast, G418 increases functional near-cognate tRNA mispairing with a PSC, resulting from binding to its tight site on the ribosome, with little if any effect on release factor activity. The low toxicity of ataluren suggests that development of new TRIDs exclusively directed toward inhibiting termination should be a priority in combatting PSC diseases. Our results also provide rate measurements of some of the elementary steps during the eukaryotic translation elongation cycle, allowing us to determine how these rates are modified when cognate tRNA is replaced by near-cognate tRNA ± TRIDs.

KSHV activates unfolded protein response sensors but suppresses downstream transcriptional responses to support lytic replication.

Herpesviruses usurp host cell protein synthesis machinery to convert viral mRNAs into proteins, and the endoplasmic reticulum (ER) to ensure proper folding, post-translational modification and trafficking of secreted and transmembrane viral proteins. Overloading ER folding capacity activates the unfolded protein response (UPR), whereby sensor proteins ATF6, PERK and IRE1 initiate a stress-mitigating transcription program that accelerates catabolism of misfolded proteins while increasing ER folding capacity. Kaposi's sarcoma-associated herpesvirus (KSHV) can be reactivated from latency by chemical induction of ER stress, which causes accumulation of the XBP1s transcription factor that transactivates the viral RTA lytic switch gene. The presence of XBP1s-responsive elements in the RTA promoter suggests that KSHV evolved a mechanism to respond to ER stress. Here, we report that ATF6, PERK and IRE1 were activated upon reactivation from latency and required for efficient KSHV lytic replication; genetic or pharmacologic inhibition of each UPR sensor diminished virion production. Despite UPR sensor activation during KSHV lytic replication, downstream UPR transcriptional responses were restricted; 1) ATF6 was cleaved to activate the ATF6(N) transcription factor but ATF6(N)-responsive genes were not transcribed; 2) PERK phosphorylated eIF2α but ATF4 did not accumulate; 3) IRE1 caused XBP1 mRNA splicing, but XBP1s protein did not accumulate and XBP1s-responsive genes were not transcribed. Ectopic expression of the KSHV host shutoff protein SOX did not affect UPR gene expression, suggesting that alternative viral mechanisms likely mediate UPR suppression during lytic replication. Complementation of XBP1s deficiency during KSHV lytic replication inhibited virion production in a dose-dependent manner in iSLK.219 cells but not in TREx-BCBL1-RTA cells. However, genetically distinct KSHV virions harvested from these two cell lines were equally susceptible to XBP1s restriction following infection of naïve iSLK cells. This suggests that cell-intrinsic properties of BCBL1 cells may circumvent the antiviral effect of ectopic XBP1s expression. Taken together, these findings indicate that while XBP1s plays an important role in reactivation from latency, it can inhibit virus replication at a later step, which the virus overcomes by preventing its synthesis. These findings suggest that KSHV hijacks UPR sensors to promote efficient viral replication while sustaining ER stress.

Dengue Virus Selectively Annexes Endoplasmic Reticulum-Associated Translation Machinery as a Strategy for Co-opting Host Cell Protein Synthesis.

A primary question in dengue virus (DENV) biology is the molecular strategy for recruitment of host cell protein synthesis machinery. Here, we combined cell fractionation, ribosome profiling, and transcriptome sequencing (RNA-seq) to investigate the subcellular organization of viral genome translation and replication as well as host cell translation and its response to DENV infection. We report that throughout the viral life cycle, DENV plus- and minus-strand RNAs were highly partitioned to the endoplasmic reticulum (ER), identifying the ER as the primary site of DENV translation. DENV infection was accompanied by an ER compartment-specific remodeling of translation, where ER translation capacity was subverted from host transcripts to DENV plus-strand RNA, particularly at late stages of infection. Remarkably, translation levels and patterns in the cytosol compartment were only modestly affected throughout the experimental time course of infection. Comparisons of ribosome footprinting densities of the DENV plus-strand RNA and host mRNAs indicated that DENV plus-strand RNA was only sparsely loaded with ribosomes. Combined, these observations suggest a mechanism where ER-localized translation and translational control mechanisms, likely cis encoded, are used to repurpose the ER for DENV virion production. Consistent with this view, we found ER-linked cellular stress response pathways commonly associated with viral infection, namely, the interferon response and unfolded protein response, to be only modestly activated during DENV infection. These data support a model where DENV reprograms the ER protein synthesis and processing environment to promote viral survival and replication while minimizing the activation of antiviral and proteostatic stress response pathways.IMPORTANCE DENV, a prominent human health threat with no broadly effective or specific treatment, depends on host cell translation machinery for viral replication, immune evasion, and virion biogenesis. The molecular mechanism by which DENV commandeers the host cell protein synthesis machinery and the subcellular organization of DENV replication and viral protein synthesis is poorly understood. Here, we report that DENV has an almost exclusively ER-localized life cycle, with viral replication and translation largely restricted to the ER. Surprisingly, DENV infection largely affects only ER-associated translation, with relatively modest effects on host cell translation in the cytosol. DENV RNA translation is very inefficient, likely representing a strategy to minimize disruption of ER proteostasis. Overall these findings demonstrate that DENV has evolved an ER-compartmentalized life cycle; thus, targeting the molecular signatures and regulation of the DENV-ER interaction landscape may reveal strategies for therapeutic intervention.

“Buzz Off” Zika

Zika virus, or ZIKV, a mosquito‐borne pathogen, is a current public health concern due to its link to microcephaly and Guillain‐Barré syndrome. A flavivirus, ZIKV contains an RNA genome translated using the host cell's protein synthesis machinery. The 5′ end of the viral RNA possesses a “cap” of methylated bases that protect it from being destroyed by the host's immune system. This cap is produced by a virally‐encoded methyltransferase enzyme, the NS5 methyltransferase (MTase). Since the 5′‐RNA cap is required for viral reproduction, inactivating the NS5 MTase with a small molecule inhibitor should be a viable treatment for ZIKV infections. The NS5 MTase transfers CH3 groups from the ubiquitous methyl donor molecule S‐adenosylmethionine (SAM) to specific positions on bases at the 5′ end of the viral RNA. Based on the binding mode of SAM observed in a crystal structure (5KQR), an analog of SAM was developed that could inhibit the enzymatic activity of the MTase. SAM binds in a pocket composed of Ser56, Arg84, Trp87, Thr104, Lys105, His110, Glu111, Asp131, Val132, Phe133, Asp146, and Ile147. The Grafton SMART (Students Modeling A Research Topic) Team has modeled the NS5 MTase with either SAM or the hypothetical inhibitor MO2 bound using 3D printing technology. Models with such compounds bound to the enzyme provide information that might help increase the potency of potential inhibitors and improve their selectivity for the ZIKV MTase.Support or Funding InformationThis work was supported by the National Institutes of of Health Science Education Partnership Award (NIH‐SEPA 1R25OD010505‐01) and the National Institutes of Health Clinical and Translational Science Award (NIH‐CTSA UL1RR031973).

Small nucleolar RNAs functioning and potential roles in cancer.

Non-coding RNAs (ncRNAs) are important regulatory molecules involved in various physiological and pathological cellular processes. Small nucleolar RNAs (snoRNAs), subclass of small ncRNAs, have been considered important but unglamorous elements in the production of protein synthesis machinery of cells. However, recent evidence has indicated that these non-coding RNAs might have a crucial role also in controlling cell behavior, and snoRNAs dysfunction could significantly contribute to carcinogenesis. Here, we summarize the most important aspects of snoRNAs biology, their functioning in cancer cell, and potential usage in diagnosis or as a new class of therapeutic targets in cancer.

Extensive tRNA Gene Changes in Synthetic Brassica napus

Allopolyploidization, where two species come together to form a new species, plays a major role in speciation and genome evolution. Transfer RNAs (abbreviated tRNA) are typically 73-94 nucleotides in length, and are indispensable in protein synthesis, transferring amino acids to the cell protein synthesis machinery (ribosome). To date, the regularity and function of tRNA gene sequence variation during the process of allopolyploidization have not been well understood. In this study, the inter-tRNA gene corresponding to tRNA amplification polymorphism method was used to detect changes in tRNA gene sequences in the progeny of interspecific hybrids between Brassica rapa and B. oleracea, mimicking the original B. napus (canola) species formation event. Cluster analysis showed that tRNA gene variation during allopolyploidization did not appear to have a genotypic basis. Significant variation occurred in the early generations of synthetic B. napus (F1 and F2 generations), but fewer alterations were observed in the later generation (F3). The variation-prone tRNA genes tended to be located in AT-rich regions. BlastN analysis of novel tRNA gene variants against a Brassica genome sequence database showed that the variation of these tRNA-gene-associated sequences in allopolyploidization might result in variation of gene structure and function, e.g., metabolic process and transport.

H5N1 Virus Causes Significant Perturbations in Host Proteome Very Early in Influenza Virus-Infected Primary Human Monocyte-Derived Macrophages

H5N1 influenza viruses, which cause disease in humans, have unusually high pathogenicity. The temporal response of primary human monocyte-derived macrophages infected with highly pathogenic H5N1 and seasonal H1N1 influenza viruses was evaluated using mass spectrometry-based quantitative proteomic profiling. This was done in order to demonstrate significant perturbation of the host proteome upon viral infection, as early as 1 hour after infection. This early host response distinguished H5N1 infection from H1N1 infection, the latter inducing less of a response. The most pronounced effect was observed on the translational machinery, suggesting that H5N1 might gain advantage in replication by using the cell protein synthesis machinery early in the infection.

An interactome map of the nucleocapsid protein from a highly pathogenic North American porcine reproductive and respiratory syndrome virus strain generated using SILAC‐based quantitative proteomics

Positive strand RNA viruses replicate in the cytoplasm of an infected cell and encode nucleocapsid proteins. These proteins function to promote encapsidation of the RNA genome and virus particle assembly as well as playing potential roles in viral RNA synthesis. Nucleocapsid proteins can also associate with cellular proteins and signaling cascades. The arterivirus nucleocapsid (N) protein is no exception and localizes to both the cytoplasm and the nucleolus in virus‐infected cells. This study generated an interactome map of the N protein from a highly virulent North American strain of porcine reproductive and respiratory syndrome virus (PRRSV). This is a major pathogen of swine resulting in significant morbidity and mortality. Crucial to the study was the use of SILAC coupled to affinity purification using GFP‐traps and LC‐MS/MS. This approach has not been applied before to the investigation of host/viral protein interactomes and this study revealed that the PRRSV N protein interacts with the host cell protein synthesis machinery especially at the level of translation initiation as well as with the RNA post‐transcriptional modification machinery. Applications of the dataset can include studies of virus/host interactions and the design of live attenuated recombinant vaccines.

Proteomic profile of the nucellus of castor bean (Ricinus communis L.) seeds during development

In this study, we performed a proteomic analysis of nucellus from two developmental stages of Ricinus communis seeds by a GeLC-MS/MS approach, using of a high resolution orbitrap mass spectrometer, which resulted in the identification of a total of 766 proteins that were grouped into 553 protein groups. The distribution of the identified proteins in stages III and IV into different Gene Ontology categories was similar, with a remarkable abundance of proteins associated with the protein synthesis machinery of cells, as well as several classes of proteins involved in protein degradation, particularly of peptidases associated with programmed cell death. Consistent with the role of the nucellus in mediating nutrient transfer from maternal tissues to the endosperm and embryo, a significant proportion of the identified proteins are related to amino acid metabolism, but none of the identified proteins are known to have a role as storage proteins. Moreover for the first time, ricin isoforms were identified in tissues other than seed endosperm. Results are discussed in the context of the spatial and temporal distribution of the identified proteins within the nucellar cell layers.

Noncytotoxic Inhibition of Viral Infection through eIF4F-Independent Suppression of Translation by 4EGi-1

The eukaryotic initiation factor eIF4F recruits ribosomes to capped mRNAs while eIF2 mediates start codon recognition to initiate protein synthesis. Increasing interest in targeting translation to suppress tumor growth has led to the development of new classes of inhibitors, including 4EGi-1, which disrupts eIF4F complexes. However, the full effects of this inhibitor and its potential uses in the treatment of other disease states remain unclear. Here, we show that overall rates of protein synthesis in primary human cells were affected only modestly by eIF4F disruption using the mTOR inhibitor Torin1, yet were highly sensitive to 4EGi-1. Translational suppression occurred even at concentrations of 4EGi-1 that were below those required to significantly alter eIF4F levels but were instead found to increase the association of ribosomal complexes containing inactive eIF2α. Although highly stable in culture, the effects of 4EGi-1 on both cellular protein synthesis and ribosome association were readily reversible upon inhibitor removal. In addition, despite potently inhibiting translation, prolonged exposure to 4EGi-1 had only modest effects on cell morphology and protein abundance without affecting viability or stress tolerance to any significant degree, although differential effects on heat shock protein (hsp) expression highlighted distinct 4EGi-1-sensitive modes of hsp induction. In contrast, 4EGi-1 potently suppressed poxvirus replication as well as both reactivation and lytic phases of herpesvirus infection. These findings identify a novel way in which 4EGi-1 affects the host cell's protein synthesis machinery and demonstrate its potential as a noncytotoxic inhibitor of diverse forms of viral infection.

Dengue virus-induced regulation of the host cell translational machinery

Dengue virus (DV)-induced changes in the host cell protein synthesis machinery are not well understood. We investigated the transcriptional changes related to initiation of protein synthesis. The human hepatoma cell line, HepG2, was infected with DV serotype 2 for 1 h at a multiplicity of infection of one. RNA was extracted after 6, 24 and 48 h. Microarray results showed that 36.5% of the translation factors related to initiation of protein synthesis had significant differential expression (Z-score >or= +/-2.0). Confirmation was obtained by quantitative real-time reverse transcription-PCR. Of the genes involved in the activation of mRNA for cap-dependent translation (eIF4 factors), eIF4A, eIF4G1 and eIF4B were up-regulated while the negative regulator of translation eIF4E-BP3 was down-regulated. This activation was transient since at 24 h post-infection levels were not significantly different from control cells. However, at 48 h post-infection, eIF4A, eIF4E, eIF4G1, eIF4G3, eIF4B, and eIF4E-BP3 were down-regulated, suggesting that cap-dependent translation could be inhibited during the progression of infection. To test this hypothesis, phosphorylation of p70S6K and 4E-BP1, which induce cap-dependent protein synthesis, was assayed. Both proteins remained phosphorylated when assayed at 6 h after infection, while infection induced dephosphorylation of p70S6K and 4E-BP1 at 24 and 48 h of infection, respectively. Taken together, these results provide biological evidence suggesting that in HepG2 cells DV sustains activation of the cap-dependent machinery at early stages of infection, but progression of infection switches protein synthesis to a cap-independent process.

Regulation of translation initiation by herpesviruses

Viruses are dependent upon the host cell protein synthesis machinery, thus they have developed a range of strategies to manipulate host translation to favour viral protein synthesis. Consequently, the study of viral translation has been a powerful tool for illuminating many aspects of cellular translational control. Although much work to date has focused on translational regulation by RNA viruses, DNA viruses have also evolved complex mechanisms to regulate protein synthesis. Here we summarize work on a large family of DNA viruses, the Herpesviridae, which have evolved mechanisms to sustain efficient cap-dependent translation and to regulate the translation of specific viral mRNAs.

The RNA polymerase I transcription machinery

The rRNAs constitute the catalytic and structural components of the ribosome, the protein synthesis machinery of cells. The level of rRNA synthesis, mediated by Pol I (RNA polymerase I), therefore has a major impact on the life and destiny of a cell. In order to elucidate how cells achieve the stringent control of Pol I transcription, matching the supply of rRNA to demand under different cellular growth conditions, it is essential to understand the components and mechanics of the Pol I transcription machinery. In this review, we discuss: (i) the molecular composition and functions of the Pol I enzyme complex and the two main Pol I transcription factors, SL1 (selectivity factor 1) and UBF (upstream binding factor); (ii) the interplay between these factors during pre-initiation complex formation at the rDNA promoter in mammalian cells; and (iii) the cellular control of the Pol I transcription machinery.

11. Cloning the β-hexosaminidase genes

This chapter presents the author's experience in cloning the β-hexosaminidase genes based on a polysome immunopurification approach. In this procedure, metabolically active cells are cracked open and polysomes—the cell's protein synthesis machinery—are isolated. The polysome contains an attached polypeptide chain in the process of translation that can be used as a tag for isolation of the β-hexosaminidase messenger RNA (mRNA) using specific antibodies. The isolated polysomes can be dissociated and the recovered mRNA reverse transcribed in cDNA for cloning. A drawback in the approach was that the antibodies were made against the human enzyme. The approach was to prepare two hybridization probes— one was a β-hexosaminidase- “enriched” probe made from the highly purified polysomal mRNA preparation, and the other was a β-hexosaminidase-“depleted” probe made from the polysomal mRNA preparation with the β-hexosaminidase mRNA removed. In addition to the identification of mutations, the cloned β-hexosaminidase sequences were fundamental for the elucidation of important structural and functional features of the enzyme. The cloning of the β-hexosaminidase genes has stimulated great progress in understanding the defects underlying Tay-Sachs disease (TSD) and the molecular biology of the β-hexosaminidase system.

Mechanism of genome transcription in segmented dsRNA viruses

This chapter discusses the mechanism of genome transcription in segmented double-stranded RNA (dsRNA) viruses. Genome transcription is a critical stage in the life cycle of a virus, as this is the process by which the viral genetic information is presented to the host cell protein-synthesis machinery for the production of the viral proteins needed for genome replication and progeny virion assembly. Viruses with dsRNA genomes face a particular challenge in that host cells do not produce proteins that can transcribe from a dsRNA template. One of the more striking observations about genome transcription in dsRNA viruses is that this process occurs efficiently only when the transcriptionally competent particle is fully intact. This observation suggests that all of the components of the transcriptionally competent particle, including the viral genome, the transcription enzymes, and the viral capsid, function together to produce and release messenger RNA transcripts and that each component has a specific and critical role to play in promoting the efficiency of this process.

Assignment of the human tryptophanyl-tRNA synthetase gene (WARS) to chromosome 14q32.2 --> q32.32.

Tryptophanyl-tRNA synthetase catalyzes the aminoacylation of tRNAtrp with tryptophan, an essential function in the cell's protein synthesis machinery. It has been shown that tryptophanyl-tRNA synthetase is induced by interferon, and this has led to hypotheses about other possible functions of tryptophanyl-tRNA synthetase. We have mapped a cDNA probe of the tryptophanyl-tRNA synthetase gene (WARS) by a combination of somatic cell hybrid analysis, fluorescence in situ hybridization (FISH), and linkage analysis. Both FISH and linkage analysis independently supported a more distal position of WARS than had been previously reported. FISH mapping indicated a most likely location at 14q32.31. Linkage analysis was based on the 40 reference families from the CEPH collaboration and resulted in a 13-point map, placing WARS, with odds of more than 1,000:1, within an area of approximately 10 cM and, with odds of 198:1, in an approximately 6 cM interval between pCMM101 and D14S27. The study provides additional integration of the physical and genetic maps of the distal part of 14q, as well as genetic tools enabling a more complete understanding of the function of tryptophanyl-tRNA synthetase, especially with regard to the cryptic inducibility of interferon.