ISGylation directly modifies hypoxia-inducible factor-2α and enhances its polysome association.

The hypoxia-inducible factors (HIF)-1α and HIF-2α are central regulators of transcriptional programmes in settings such as development and tumour expansion. HIF-2α moonlights as a cap-dependent translation factor. We provide new insights into how the interferon-stimulated gene 15 (ISG15), a ubiquitin-like modifier, and the HIFs regulate one another in hypoxia and interferon-induced cells. We show that upon ISGylation induction and HIF-α stabilization, both HIFs promote protein ISGylates through transcriptional and/or post-transcriptional pathways. We show the first evidence of HIF-2α modification by ISG15. ISGylation induces system-level alterations to the HIF transcriptional programme and increases the cytoplasmic/nuclear fraction and translation activity of HIF-2α. This work identifies ISG15 as a regulator of hypoxic mRNA translation, which has implications for immune processes and disease progression.

Effects of TDP2/VPg Unlinkase Activity on Picornavirus Infections Downstream of Virus Translation.

In this study, we characterized the role of host cell protein tyrosyl-DNA phosphodiesterase 2 (TDP2) activity, also known as VPg unlinkase, in picornavirus infections in a human cell model of infection. TDP2/VPg unlinkase is used by picornaviruses to remove the small polypeptide, VPg (Virus Protein genome-linked, the primer for viral RNA synthesis), from virus genomic RNA. We utilized a CRISPR/Cas-9-generated TDP2 knock out (KO) human retinal pigment epithelial-1 (hRPE-1) cell line, in addition to the wild type (WT) counterpart for our studies. We determined that in the absence of TDP2, virus growth kinetics for two enteroviruses (poliovirus and coxsackievirus B3) were delayed by about 2 h. Virus titers were reduced by ~2 log10 units for poliovirus and 0.5 log10 units for coxsackievirus at 4 hours post-infection (hpi), and by ~1 log10 unit at 6 hpi for poliovirus. However, virus titers were nearly indistinguishable from those of control cells by the end of the infectious cycle. We determined that this was not the result of an alternative source of VPg unlinkase activity being activated in the absence of TPD2 at late times of infection. Viral protein production in TDP2 KO cells was also substantially reduced at 4 hpi for poliovirus infection, consistent with the observed growth kinetics delay, but reached normal levels by 6 hpi. Interestingly, this result differs somewhat from what has been reported previously for the TDP2 KO mouse cell model, suggesting that either cell type or species-specific differences might be playing a role in the observed phenotype. We also determined that catalytically inactive TDP2 does not rescue the growth defect, confirming that TDP2 5' phosphodiesterase activity is required for efficient virus replication. Importantly, we show for the first time that polysomes can assemble efficiently on VPg-linked RNA after the initial round of translation in a cell culture model, but both positive and negative strand RNA production is impaired in the absence of TDP2 at mid-times of infection, indicating that the presence of VPg on the viral RNA affects a step in the replication cycle downstream of translation (e.g., RNA synthesis). In agreement with this conclusion, we found that double-stranded RNA production (a marker of viral RNA synthesis) is delayed in TDP2 KO RPE-1 cells. Moreover, we show that premature encapsidation of nascent, VPg-linked RNA is not responsible for the observed virus growth defect. Our studies provide the first lines of evidence to suggest that either negative- or positive-strand RNA synthesis (or both) is a likely candidate for the step that requires the removal of VPg from the RNA for an enterovirus infection to proceed efficiently.

ILF3 contributes to the establishment of the antiviral type I interferon program.

Upon detection of viral infections, cells activate the expression of type I interferons (IFNs) and pro-inflammatory cytokines to control viral dissemination. As part of their antiviral response, cells also trigger the translational shutoff response which prevents translation of viral mRNAs and cellular mRNAs in a non-selective manner. Intriguingly, mRNAs encoding for antiviral factors bypass this translational shutoff, suggesting the presence of additional regulatory mechanisms enabling expression of the self-defence genes. Here, we identified the dsRNA binding protein ILF3 as an essential host factor required for efficient translation of the central antiviral cytokine, IFNB1, and a subset of interferon-stimulated genes. By combining polysome profiling and next-generation sequencing, ILF3 was also found to be necessary to establish the dsRNA-induced transcriptional and translational programs. We propose a central role for the host factor ILF3 in enhancing expression of the antiviral defence mRNAs in cellular conditions where cap-dependent translation is compromised.

Extraribosomal l13a is a specific innate immune factor for antiviral defense.

UNLABELLED: We report a novel extraribosomal innate immune function of mammalian ribosomal protein L13a, whereby it acts as an antiviral agent. We found that L13a is released from the 60S ribosomal subunit in response to infection by respiratory syncytial virus (RSV), an RNA virus of the Pneumovirus genus and a serious lung pathogen. Unexpectedly, the growth of RSV was highly enhanced in L13a-knocked-down cells of various lineages as well as in L13a knockout macrophages from mice. In all L13a-deficient cells tested, translation of RSV matrix (M) protein was specifically stimulated, as judged by a greater abundance of M protein and greater association of the M mRNA with polyribosomes, while general translation was unaffected. In silico RNA folding analysis and translational reporter assays revealed a putative hairpin in the 3'untranslated region (UTR) of M mRNA with significant structural similarity to the cellular GAIT (gamma-activated inhibitor of translation) RNA hairpin, previously shown to be responsible for assembling a large, L13a-containing ribonucleoprotein complex that promoted translational silencing in gamma interferon (IFN-γ)-activated myeloid cells. However, RNA-protein interaction studies revealed that this complex, which we named VAIT (respiratory syncytial virus-activated inhibitor of translation) is functionally different from the GAIT complex. VAIT is the first report of an extraribosomal L13a-mediated, IFN-γ-independent innate antiviral complex triggered in response to virus infection. We provide a model in which the VAIT complex strongly hinders RSV replication by inhibiting the translation of the rate-limiting viral M protein, which is a new paradigm in antiviral defense. IMPORTANCE: The innate immune mechanisms of host cells are diverse in nature and act as a broad-spectrum cellular defense against viruses. Here, we report a novel innate immune mechanism functioning against respiratory syncytial virus (RSV), in which the cellular ribosomal protein L13a is released from the large ribosomal subunit soon after infection and inhibits the translation of a specific viral mRNA, namely, that of the matrix protein M. Regarding its mechanism, we show that the recognition of a specific secondary structure in the 3' untranslated region of the M mRNA leads to translational arrest of the mRNA. We also show that the level of M protein in the infected cell is rate limiting for viral morphogenesis, providing a rationale for L13a to target the M mRNA for suppression of RSV growth. Translational silencing of a viral mRNA by a deployed ribosomal protein is a new paradigm in innate immunity.

Many needles in a haystack: cell-type specific abiotic stress responses.

Plants react to abiotic stress with a number of physiological, biochemical, and developmental alterations. These responses include changes in signaling components, gene transcription, non-coding RNAs, proteins, and metabolites that occur in a cell-type and tissue-specific manner. Recent advances in cell-type specifically isolating protoplasts and nuclei from plants, extracting mRNA from targeted cells, and whole-genome transcriptional profiling have enabled scientists to gain insight into how cells and tissues respond transcriptionally to abiotic stress. Continued technological advances in profiling the proteomes, metabolomes, and other biological components of specific cells will continue to broaden our understanding of plant stress responses.

Isolation of plant polysomal mRNA by differential centrifugation and ribosome immunopurification methods.

Polyribosomes (polysomes) form as multiple ribosomes engage in translation on a single mRNA. This process is regulated for individual mRNAs by both development and the environment. To evaluate the translation state of an mRNA, ribosomal subunits, ribosomes, and polysomes can be isolated from detergent-treated cell extracts by high-speed differential centrifugation. These ribonucleoprotein complexes can be further purified by centrifugation through sucrose density gradients. By fractionation of the gradient the amount of an individual mRNA in a sub-population of polysomes can be quantitatively determined. Here, we describe methods for the isolation and quantification of polysome complexes from plant tissues. The mRNA obtained can be further analyzed by methods that evaluate polysomal mRNA abundance at the individual transcript or global level. A modification of the conventional polysome isolation procedure is described for transgenic Arabidopsis thaliana that express an epitope-tagged version of ribosomal protein L18 (RPL18) that facilitates capture of ribosomes from crude cell extracts by a one-step immunoprecipitation method.

LPS upregulates MHC class II I-A expression in B lymphocytes at transcriptional and at translational levels.

Major histocompatibility complex (MHC) class II molecules are expressed in a limited number of cell types, including B lymphocytes, dendritic cells and macrophages. Lipopolysaccharide (LPS) increases the surface expression of class II molecules in a murine B-cell line by inducing an increase in I-A protein and I-A mRNA levels. LPS does not modify the rate of mRNA degradation; therefore, the increase in mRNA is due to an increase in transcription. In addition, LPS increases the levels of I-Aalpha protein, which correlates with an increase in ribosome loading for I-Aalpha but not for I-Abeta mRNA after treatment with LPS. Interestingly, in non-induced cells, I-Aalpha messenger RNA shows a significant peak of free mRNA. Therefore, LPS regulates the expression of MHC class II molecules at translational level in B cells, in addition to the transcriptional control. The actual mechanism implies changes of translation initiation rates, as shown by an increase ribosome loading in polysome gradients.

Immunohistochemical evidence for an association of ribosomes with microfilaments in 3T3 fibroblasts.

Ribosome distribution in cultured fibroblasts was investigated immunohistochemically using antibodies which recognize the 60S ribosomal subunit. After treatment of cells with buffer containing 25mM KCl and 0.05% Nonidet-P40 immunostained material was present in punctate patterns and linear arrays consistent with some ribosomes being associated with the cytoskeleton. Treatment of the cells with 130mM KCl caused loss of both the beaded lines of immunostaining and micro-filaments. Double immunostaining showed ribosomes to be closely associated with microfilaments.

[The protector effect of ribosomal preparations against experimental influenza infection in mice]. / Effet protecteur de quelques préparations ribosomales contre l'infection expérimentale à virus de la grippe chez la souris.

A study was conducted on the protective effect of some ribosomal preparations, isolated from chorionic-allantoic membranes of chicken embryos, infected or not with parainfluenza (Sendai) or influenza (AoPR8) virus, in mice experimentally inoculated with influenza virus strain AoPR8 adapted to the mouse. Results showed that the tested preparation, containing ribosomes and polysomes isolated from chorio-allantoic membranes of Sendai virus inoculated chicken embryos, ensure the mice complete protection against AoPR8 virus, if administrated before the control infection.

Purification of mRNA coding for rat-liver fructose-1,6-bisphosphatase by polysome immunoabsorption.

The mRNA coding for rat liver fructose-1,6-bisphosphatase, which represents approx. 0.46% of total hepatic mRNA, has been purified to near homogeneity. Polysomes from rat liver were allowed to react with antibodies to rabbit anti-fructose-1,6-bisphosphatase purified by affinity chromatography. The complex was immobilized on a protein A-Sepharose column. After the removal of unabsorbed polysomes, the specific mRNA was eluted and chromatographed on an oligo(dT)-cellulose column. This method gave a 183-fold enrichment of the fructose-1,6-bisphosphatase mRNA to greater than 80% homogeneity as determined by electrophoreses of immunoprecipitated in vitro translation products on polyacrylamide slab gels in the presence of sodium dodecyl sulphate.

Characterization of the gene and mRNA of the large subunit of ribulose-1,5-bisphosphate carboxylase in pea plants.

mRNA coding for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase was obtained by fractionating chloroplast polysomes on an affinity column, using anti-ribulose-1,5-bisphosphate carboxylase immunoglobulin G. Approximately 20% of the polysomal RNA specifically bound to the affinity column. LS mRNA was also isolated by fractionating chloroplast polysomal RNA on sucrose gradients. The LS mRNA fraction was identified by translation in vitro followed by immunoprecipitation with anti-ribulose-1,5-bisphosphate carboxylase immunoglobulin G. Labeled LS mRNA was hybridized to a genomic digests of pea chloroplast DNA. The LS gene was localized on a 3.55-kilobase pair BamHI fragment in SalI-SmaI DNA fragment 4. The BamHI fragment containing the LS gene was cloned, and a restriction endonuclease map was constructed. The LS gene was localized on a 1.9-kbp KpnI-EcoRI fragment. The LS gene was analyzed by electron microscopy, using the R loop mapping technique. LS mRNA was colinear with the gene, and its size was 1.35 +/- 0.2 kilobase pairs. When the LS mRNA was analyzed on methylmercury agarose gels, it comigrated with the 16S rRNA. The direction of transcription of the LS gene was in the same direction as that of the rRNA genes.

Cloning of developmentally regulated flagellin genes from Caulobacter crescentus via immunoprecipitation of polyribosomes.

Immunoprecipitation of Caulobacter crescentus polyribosomes with antiflagellin antibody provided RNA for the synthesis of cDNA probes that were used to identify three specific EcoRI restriction fragments (6.8, 10, and 22 kilobases) in genomic digests of Caulobacter DNA. The RNA was present only in polyribosomes isolated from a time interval in the Caulobacter cell cycle that was coincident with flagellin polypeptide synthesis. The structural gene for Mr 27,500 flagellin polypeptide was assigned to a region of the 10-kilobase EcoRI restriction fragment by DNA sequence analysis. Analysis of mutants defective in motility further established a correlation between the Mr 27,500 flagellin gene and the flaE gene locus [Johnson, R. C. & Ely, B. (1979) J. Bacteriol. 137, 627-634]. The other EcoRI fragments that hybridize with the immunoprecipitated polyribosome-derived cDNA probe are also temporally regulated and have features that suggest they encode other polypeptides associated with the flagellum. Modifications were required to adapt the procedure of immunoprecipitation of polyribosomes for use with Caulobacter and should be applicable to the production of specific structural gene probes from other prokaryotic systems.

cDNA clones for the heavy chain of HLA-DR antigens obtained after immunopurification of polysomes by monoclonal antibody.

A monoclonal antibody (HC 2.1) directed against the separated heavy chain of HLA-DR has been prepared. By binding HC 2.1 to polysomes from human B lymphoblastoid cells followed by the use of a protein A-Sepharose column as an immunoadsorbent, we have purified the mRNA coding for the HLA-DR heavy chain nearly to homogeneity. The immunopurified mRNA has been used to prepare labeled cDNA with which to probe cDNA libraries. Double-stranded cDNA was also made from the immunopurified mRNA and cloned directly into pBR322. Two clones, one from each of the above procedures, positively selected DR heavy chain message as assayed by cell-free translation and immunoprecipitation. One clone, pDRH-2 [500 base pairs plus 75 base pairs of poly(A)] contains the entire 3' untranslated region as well as coding information for the carboxy-terminal hydrophilic intracellular domain and part of the hydrophobic transmembrane region. Results of carboxypeptidase digestion of the heavy chains from detergent-solubilized (p34) and papain-treated (p33) HLA-DR antigen were consistent with the predicted protein sequence. Specific immunopurification of polysomes by defined monoclonal antibodies followed by direct cloning of cDNA to the highly purified mRNA is a powerful method for obtaining identified cDNA clones.

Isolation and characterization of polysomes from Trypanosoma brucei.

The isolation of polysomes in bulk from bloodstream forms of Trypanosoma brucei is described. The polysomes are active in in vitro protein synthesis in the presence or absence of initiation inhibitors. Nascent variant surface antigen (VSA) has been detected on these polysomes using purified radio-iodinated antibody. EDTA-induced ribosomal sub-units and their large rRNA's are characterized. The 26S rRNA is nicked to produce 2 molecules which are both smaller than the 19S rRNA of the small sub-unit which is larger than that found in the majority of eukaryotic small sub-units.

Tumor-immunotherapeutic efficacy of Serratia marcescens polyribosomes.

The ability of polyribosomes, obtained from several bacterial species, to suppress the development of cutaneous SaD2 fibrosarcomas in DBA/2 mice were evaluated. Suppression of tumor appearance depended upon the tumor load at the time of treatment, dose of polyribosomes, and species source of polyribosomes, with Serratia marcescens being superior to Escherichia coli, Streptococcus pneumoniae, Mycobacterium bovis (Pasteur), Mycobacterium smegmatis, and Propionibacterium acnes (formerly Corynebacterium parvum). A single injection of 5 or 50 microgram of Serratia polyribosomes at the tumor site 72 hr after the intradermal administration of 1.5 X 10(3) SaD2 cells resulted in 66 to 95% survival. All untreated animals expired within 50 days. Tumor suppression occurred at both flank and footpad sites. Presensitization with polyribosomes and incorporation of polyribosomes into adjuvant were not required for the tumor-suppressive effect. Treatment of Serratia polyribosomes with RNase or pronase reduced the number of survivors. Endotoxin was not detectable with the Limulus amebocyte lysate assay.

Immunization of mice with Trypanosoma cruzi polyribosomes.

Studies were carried out with a polyribosomal fraction isolated from Trypanosoma cruzi Y epimastigotes, with the intention to determine both its immunogenic activity and the degree of protection it could induce against experimental T. cruzi infection. This fraction was assayed in four groups of mice by using different schedules of vaccination and varying the dose, intervals, and route of administration. Seven days after the last dose, the animals were sacrificed for immunological studies or subjected to challenge with T. cruzi trypomastigotes. The results obtained in all schedules showed that our polyribosomal fraction only induced a weak antibody response, but was capable of evoking an expressive cellular response. It was also shown that this fraction has the capacity of inducing a high degree of protection against T. cruzi infection, as determined by the decrease of parasitemia and the prolonged survival time of immunized animals.