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.

Polysome Fractionation for Transcriptome-Wide Studies of mRNA Translation.

Protein synthesis and degradation determine the relationship between mRNA and corresponding protein amounts. This relationship can change in a dynamic and selective fashion when translational efficiencies of transcript subsets are altered downstream of, for example, translation factors and/or RNA binding proteins. Notably, even transcription factors such as estrogen receptor alpha (ERα) can modulate mRNA translation in a transcript-selective manner. Yet, despite ample evidence suggesting a key role for mRNA translation in shaping the proteome in health and disease, it remains largely unexplored. Here, we present a guide for the extraction of mRNA engaged in translation using polysome fractionation with linear and optimized sucrose gradients. The isolated polysome-associated RNA is then quantified, in parallel with total mRNA from the same conditions, using methods such as RNA sequencing; and the resulting data set is analyzed to derive transcriptome-wide insights into how mRNA translation is modulated. The methods we describe are applicable to cultured cells, small numbers of FACS-isolated primary cells, and small tissue samples from biobanks or animal studies. Accordingly, this approach can be applied to study in detail how ERα and other factors control gene expression by selectively modulating mRNA translation both in vitro and in vivo.

The pioneer round of translation ensures proper targeting of ER and mitochondrial proteins.

The pioneer (or first) round of translation of newly synthesized mRNAs is largely mediated by a nuclear cap-binding complex (CBC). In a transcriptome-wide analysis of polysome-associated and CBC-bound transcripts, we identify RN7SL1, a noncoding RNA component of a signal recognition particle (SRP), as an interaction partner of the CBC. The direct CBC-SRP interaction safeguards against abnormal expression of polypeptides from a ribosome-nascent chain complex (RNC)-SRP complex until the latter is properly delivered to the endoplasmic reticulum. Failure of this surveillance causes abnormal expression of misfolded proteins at inappropriate intracellular locations, leading to a cytosolic stress response. This surveillance pathway also blocks protein synthesis through RNC-SRP misassembled on an mRNA encoding a mitochondrial protein. Thus, our results reveal a surveillance pathway in which pioneer translation ensures proper targeting of endoplasmic reticulum and mitochondrial proteins.

Single Molecule RNA Localization and Translation in the Mammalian Oocyte and Embryo.

During oocyte growth the cell accumulates RNAs to contribute to oocyte and embryo development which progresses with ceased transcription. To investigate the subcellular distribution of specific RNAs and their translation we developed a technique revealing several instances of localized translation with distinctive regulatory implications. We analyzed the localization and expression of candidate non-coding and mRNAs in the mouse oocyte and embryo. Furthermore, we established simultaneous visualization of mRNA and in situ translation events validated with polysomal occupancy. We discovered that translationally dormant and abundant mRNAs CyclinB1 and Mos are localized in the cytoplasm of the fully grown GV oocyte forming cloud-like structures with consequent abundant translation at the center of the MII oocyte. Coupling detection of the localization of specific single mRNA molecules with their translation at the subcellular context is a valuable tool to quantitatively study temporal and spatial translation of specific target mRNAs to understand molecular processes in the developing cell.

Cytoplasmic long noncoding RNAs are differentially regulated and translated during human neuronal differentiation.

The expression of long noncoding RNAs is highly enriched in the human nervous system. However, the function of neuronal lncRNAs in the cytoplasm and their potential translation remains poorly understood. Here we performed Poly-Ribo-Seq to understand the interaction of lncRNAs with the translation machinery and the functional consequences during neuronal differentiation of human SH-SY5Y cells. We discovered 237 cytoplasmic lncRNAs up-regulated during early neuronal differentiation, 58%-70% of which are associated with polysome translation complexes. Among these polysome-associated lncRNAs, we find 45 small ORFs to be actively translated, 17 specifically upon differentiation. Fifteen of 45 of the translated lncRNA-smORFs exhibit sequence conservation within Hominidea, suggesting they are under strong selective constraint in this clade. The profiling of publicly available data sets revealed that 8/45 of the translated lncRNAs are dynamically expressed during human brain development, and 22/45 are associated with cancers of the central nervous system. One translated lncRNA we discovered is LINC01116, which is induced upon differentiation and contains an 87 codon smORF exhibiting increased ribosome profiling signal upon differentiation. The resulting LINC01116 peptide localizes to neurites. Knockdown of LINC01116 results in a significant reduction of neurite length in differentiated cells, indicating it contributes to neuronal differentiation. Our findings indicate cytoplasmic lncRNAs interact with translation complexes, are a noncanonical source of novel peptides, and contribute to neuronal function and disease. Specifically, we demonstrate a novel functional role for LINC01116 during human neuronal differentiation.

Molecular mechanisms of stress granule assembly and disassembly.

Stress granules (SGs) are membrane-less ribonucleoprotein (RNP)-based cellular compartments that form in the cytoplasm of a cell upon exposure to various environmental stressors. SGs contain a large set of proteins, as well as mRNAs that have been stalled in translation as a result of stress-induced polysome disassembly. Despite the fact that SGs have been extensively studied for many years, their function is still not clear. They presumably help the cell to cope with the encountered stress, and facilitate the recovery process after stress removal upon which SGs disassemble. Aberrant formation of SGs and impaired SG disassembly majorly contribute to various pathological phenomena in cancer, viral infections, and neurodegeneration. The assembly of SGs is largely driven by liquid-liquid phase separation (LLPS), however, the molecular mechanisms behind that are not fully understood. Recent studies have proposed a novel mechanism for SG formation that involves the interplay of a large interaction network of mRNAs and proteins. Here, we review this novel concept of SG assembly, and discuss the current insights into SG disassembly.

The Use of B-Cell Polysome Profiling to Validate Novel RPL5 (uL18) and RPL26 (uL24) Variants in Diamond-Blackfan Anemia.

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure syndrome usually caused by heterozygous variants in ribosomal proteins (RP) and which leads to severe anemia. Genetic studies in DBA rely primarily on multigene panels that often result in variants of unknown significance. Our objective was to optimize polysome profiling to functionally validate new large subunit RP variants. We determined the optimal experimental conditions for B-cell polysome profiles then performed this analysis on 2 children with DBA and novel missense RPL5 (uL18) and RPL26 (uL24) variants of unknown significance. Both patients had reduced 60S and 80S fractions when compared with an unaffected parent consistent with a large ribosomal subunit defect. Polysome profiling using primary B-cells is an adjunctive tool that can assist in validation of large subunit RP variants of uncertain significance. Further studies are necessary to validate this method in patients with known DBA mutations, small RP subunit variants, and silent carriers.

FUS contributes to mTOR-dependent inhibition of translation.

The amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)-linked RNA-binding protein called FUS (fused in sarcoma) has been implicated in several aspects of RNA regulation, including mRNA translation. The mechanism by which FUS affects the translation of polyribosomes has not been established. Here we show that FUS can associate with stalled polyribosomes and that this association is sensitive to mTOR (mammalian target of rapamycin) kinase activity. Specifically, we show that FUS association with polyribosomes is increased by Torin1 treatment or when cells are cultured in nutrient-deficient media, but not when cells are treated with rapamycin, the allosteric inhibitor of mTORC1. Moreover, we report that FUS is necessary for efficient stalling of translation because deficient cells are refractory to the inhibition of mTOR-dependent signaling by Torin1. We also show that ALS-linked FUS mutants R521G and P525L associate abundantly with polyribosomes and decrease global protein synthesis. Importantly, the inhibitory effect on translation by FUS is impaired by mutations that reduce its RNA-binding affinity. These findings demonstrate that FUS is an important RNA-binding protein that mediates translational repression through mTOR-dependent signaling and that ALS-linked FUS mutants can cause a toxic gain of function in the cytoplasm by repressing the translation of mRNA at polyribosomes.

A Dual Protein-mRNA Localization Screen Reveals Compartmentalized Translation and Widespread Co-translational RNA Targeting.

Local translation allows spatial control of gene expression. Here, we performed a dual protein-mRNA localization screen, using smFISH on 523 human cell lines expressing GFP-tagged genes. 32 mRNAs displayed specific cytoplasmic localizations with local translation at unexpected locations, including cytoplasmic protrusions, cell edges, endosomes, Golgi, the nuclear envelope, and centrosomes, the latter being cell-cycle-dependent. Automated classification of mRNA localization patterns revealed a high degree of intercellular heterogeneity. Surprisingly, mRNA localization frequently required ongoing translation, indicating widespread co-translational RNA targeting. Interestingly, while P-body accumulation was frequent (15 mRNAs), four mRNAs accumulated in foci that were distinct structures. These foci lacked the mature protein, but nascent polypeptide imaging showed that they were specialized translation factories. For ß-catenin, foci formation was regulated by Wnt, relied on APC-dependent polysome aggregation, and led to nascent protein degradation. Thus, translation factories uniquely regulate nascent protein metabolism and create a fine granular compartmentalization of translation.

Hypoxia influences polysome distribution of human ribosomal protein S12 and alternative splicing of ribosomal protein mRNAs.

Ribosomes were once considered static in their composition because of their essential role in protein synthesis and kingdom-wide conservation. The existence of tolerated mutations in select ribosomal proteins (RPs), such as in Diamond-Blackfan anemia, is evidence that not all ribosome components are essential. Heterogeneity in the protein composition of eukaryotic ribosomes is an emerging concept with evidence that different pools of ribosomes exist with transcript-specificity. Here, we show that the polysome association of ribosomal proteins is altered by low oxygen (hypoxia), a feature of the tumor microenvironment, in human cells. We quantified ribosomal protein abundance in actively translating polysomes of normoxic and hypoxic HEK293 cells by tandem mass tags mass spectrometry. Our data suggest that RPS12 (eS12) is enriched in hypoxic monosomes, which increases the heavy polysome association of structured transcripts APAF-1 and XIAP. Furthermore, hypoxia induced five alternative splicing events within a subset of RP mRNAs in cell lines. One of these events in RPS24 (eS24 protein) alters the coding sequence to produce two protein isoforms that can incorporate into ribosomes. This splicing event is greatly induced in spheroids and correlates with tumor hypoxia in human prostate cancer. Our data suggest that hypoxia may influence the composition of the human ribosome through changes in RP incorporation and the production of hypoxia-specific RP isoforms.

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.

Multiple interaction nodes define the postreplication repair response to UV-induced DNA damage that is defective in melanomas and correlated with UV signature mutation load.

Ultraviolet radiation-induced DNA mutations are a primary environmental driver of melanoma. The reason for this very high level of unrepaired DNA lesions leading to these mutations is still poorly understood. The primary DNA repair mechanism for UV-induced lesions, that is, the nucleotide excision repair pathway, appears intact in most melanomas. We have previously reported a postreplication repair mechanism that is commonly defective in melanoma cell lines. Here we have used a genome-wide approach to identify the components of this postreplication repair mechanism. We have used differential transcript polysome loading to identify transcripts that are associated with UV response, and then functionally assessed these to identify novel components of this repair and cell cycle checkpoint network. We have identified multiple interaction nodes, including global genomic nucleotide excision repair and homologous recombination repair, and previously unexpected MASTL pathway, as components of the response. Finally, we have used bioinformatics to assess the contribution of dysregulated expression of these pathways to the UV signature mutation load of a large melanoma cohort. We show that dysregulation of the pathway, especially the DNA damage repair components, are significant contributors to UV mutation load, and that dysregulation of the MASTL pathway appears to be a significant contributor to high UV signature mutation load.

Translational offsetting as a mode of estrogen receptor α-dependent regulation of gene expression.

Estrogen receptor alpha (ERα) activity is associated with increased cancer cell proliferation. Studies aiming to understand the impact of ERα on cancer-associated phenotypes have largely been limited to its transcriptional activity. Herein, we demonstrate that ERα coordinates its transcriptional output with selective modulation of mRNA translation. Importantly, translational perturbations caused by depletion of ERα largely manifest as "translational offsetting" of the transcriptome, whereby amounts of translated mRNAs and corresponding protein levels are maintained constant despite changes in mRNA abundance. Transcripts whose levels, but not polysome association, are reduced following ERα depletion lack features which limit translation efficiency including structured 5'UTRs and miRNA target sites. In contrast, mRNAs induced upon ERα depletion whose polysome association remains unaltered are enriched in codons requiring U34-modified tRNAs for efficient decoding. Consistently, ERα regulates levels of U34-modifying enzymes and thereby controls levels of U34-modified tRNAs. These findings unravel a hitherto unprecedented mechanism of ERα-dependent orchestration of transcriptional and translational programs that may be a pervasive mechanism of proteome maintenance in hormone-dependent cancers.

Tetrapeptide 60-63 of human ribosomal protein uS3 is crucial for translation initiation.

Conserved ribosomal protein uS3 contains a decapeptide fragment in positions 55-64 (human numbering), which has a very specific ability to cross-link to various RNA derivatives bearing aldehyde groups, likely provided by K62. It has been shown that during translation in the cell-free protein-synthesizing system, uS3 becomes accessible for such cross-linking only after eIF3j leaves the mRNA binding channel of the 40S ribosomal subunit. We studied the functional role of K62 and its nearest neighbors in the ribosomal assembly and translation with the use of HEK293T-derived cell cultures capable of producing FLAG-tagged uS3 (uS3FLAG) or its mutant form with amino acid residues at positions 60-63 replaced with alanines. Analysis of polysome profiles from the respective cells and cytosol lysates showed that the mutation significantly affected the uS3 ability to participate in the assembly of 40S subunits, but it was not essential for their maturation and did not prevent the binding of mRNAs to 40S subunits during translation initiation. The most striking effect of the replacement of amino acid residues in the above uS3 positions was that it almost completely deprived the 40S subunits of their ability to form 80S ribosomes, suggesting that the 48S pre-initiation complexes assembled on these subunits were defective in the binding of 60S subunits. Thus, our results revealed the previously unknown crucial role of the uS3 tetrapeptide 60GEKG63 in translation initiation related to maintaining the proper structure of the 48S complex, most likely via the prevention of premature mRNA loading into the ribosomal channel.

Translatome analysis reveals altered serine and glycine metabolism in T-cell acute lymphoblastic leukemia cells.

Somatic ribosomal protein mutations have recently been described in cancer, yet their impact on cellular transcription and translation remains poorly understood. Here, we integrate mRNA sequencing, ribosome footprinting, polysomal RNA sequencing and mass spectrometry datasets from a mouse lymphoid cell model to characterize the T-cell acute lymphoblastic leukemia (T-ALL) associated ribosomal RPL10 R98S mutation. Surprisingly, RPL10 R98S induces changes in protein levels primarily through transcriptional rather than translation efficiency changes. Phosphoserine phosphatase (PSPH), encoding a key serine biosynthesis enzyme, was the only gene with elevated transcription and translation leading to protein overexpression. PSPH upregulation is a general phenomenon in T-ALL patient samples, associated with elevated serine and glycine levels in xenograft mice. Reduction of PSPH expression suppresses proliferation of T-ALL cell lines and their capacity to expand in mice. We identify ribosomal mutation driven induction of serine biosynthesis and provide evidence supporting dependence of T-ALL cells on PSPH.

Transforming activity of an oncoprotein-encoding circular RNA from human papillomavirus.

Single-stranded circular RNAs (circRNAs), generated through 'backsplicing', occur more extensively than initially anticipated. The possible functions of the vast majority of circRNAs remain unknown. Virus-derived circRNAs have recently been described in gamma-herpesviruses. We report that oncogenic human papillomaviruses (HPVs) generate circRNAs, some of which encompass the E7 oncogene (circE7). HPV16 circE7 is detectable by both inverse RT-PCR and northern blotting of HPV16-transformed cells. CircE7 is N6-methyladenosine (m6A) modified, preferentially localized to the cytoplasm, associated with polysomes, and translated to produce E7 oncoprotein. Specific disruption of circE7 in CaSki cervical carcinoma cells reduces E7 protein levels and inhibits cancer cell growth both in vitro and in tumor xenografts. CircE7 is present in TCGA RNA-Seq data from HPV-positive cancers and in cell lines with only episomal HPVs. These results provide evidence that virus-derived, protein-encoding circular RNAs are biologically functional and linked to the transforming properties of some HPV.

Loss of miR-451a enhances SPARC production during myogenesis.

MicroRNAs (miRNAs) are small noncoding RNAs that critically regulate gene expression. Their abundance and function have been linked to a range of physiologic and pathologic processes. In aged monkey muscle, miR-451a and miR-144-3p were far more abundant than in young monkey muscle. This observation led us to hypothesize that miR-451a and miR-144-3p may influence muscle homeostasis. To test if these conserved microRNAs were implicated in myogenesis, we investigated their function in the mouse myoblast line C2C12. The levels of both microRNAs declined with myogenesis; however, only overexpression of miR-451a, but not miR-144-3p, robustly impeded C2C12 differentiation, suggesting an inhibitory role for miR-451a in myogenesis. Further investigation of the regulatory influence of miR-451a identified as one of the major targets Sparc mRNA, which encodes a secreted protein acidic and rich in cysteine (SPARC) that functions in wound healing and cellular differentiation. In mouse myoblasts, miR-451a suppressed Sparc mRNA translation. Together, our findings indicate that miR-451a is downregulated in differentiated myoblasts and suggest that it decreases C2C12 differentiation at least in part by suppressing SPARC biosynthesis.

Concerted expression of a cell cycle regulator and a metabolic enzyme from a bicistronic transcript in plants.

Eukaryotic mRNAs frequently contain upstream open reading frames (uORFs), encoding small peptides that may control translation of the main ORF (mORF). Here, we report the characterization of a distinct bicistronic transcript in Arabidopsis. We analysed loss-of-function phenotypes of the inorganic polyphosphatase TRIPHOSPHATE TUNNEL METALLOENZYME 3 (AtTTM3), and found that catalytically inactive versions of the enzyme could fully complement embryo and growth-related phenotypes. We could rationalize these puzzling findings by characterizing a uORF in the AtTTM3 locus encoding CELL DIVISION CYCLE PROTEIN 26 (CDC26), an orthologue of the cell cycle regulator. We demonstrate that AtCDC26 is part of the plant anaphase promoting complex/cyclosome (APC/C), regulates accumulation of APC/C target proteins and controls cell division, growth and embryo development. AtCDC26 and AtTTM3 are translated from a single transcript conserved across the plant lineage. While there is no apparent biochemical connection between the two gene products, AtTTM3 coordinates AtCDC26 translation by recruiting the transcript into polysomes. Our work highlights that uORFs may encode functional proteins in plant genomes.

Topoisomerase II α Gene alteration in Triple Negative Breast Cancer and Its Predictive Role for Anthracycline-Based Chemotherapy (Egyptian NCI Patients)

Objective: Triple negative breast cancer is an aggressive variant of breast cancer; it forms about 15% of breast cancer cases. It lacks the responsiveness to hormonal and targeted therapies. Anthracyclines remain the treatment option for these patients. Anthracyclines are cardiotoxic, so predicting sensitivity of response by biological predictors may have a role in selecting suitable candidates for these drugs. Material and methods: This study included 50 TNBC cases, from National Cancer Institute, Cairo University(NCI-CU), Egypt, who underwent surgery and received adjuvant chemotherapy. Archived blocks were obtained and immunostaining for Ki-67 LI and Fluorescent In situ Hybridization (FISH) technique to assess TOP2A gene copy number and chromosome 17CEP status were done. Analysis of association between TOP2A alterations and CEP17 polysomy as well as Ki-67 LI with other clinicopathological parameters was done. Associations between the biological markers and event free survival (EFS) and overall survival (OS), were also performed. Results: TOP2A alteration was seen in 9/50 cases (5 amplified and 4 deleted). CEP17 Polysomy was detected in 14% of cases. Most of patients (80%) showed Ki-67 LI ≥20%. There was a significant association between TOP2A gene and CEP17 status. Outcome was better with abnormal TOP2A gene status and CEP17 polysomy, radiotherapy and combined anthracyclines and taxanes in the adjuvant setting, however P-values were not significant. Conclusion: TOP2A gene alterations and CEP17 polysomy may have prognostic and predictive role in TNBC treated with adjuvant Anthracyclines.

Affinity capture of polyribosomes followed by RNAseq (ACAPseq), a discovery platform for protein-protein interactions.

Defining protein-protein interactions (PPIs) is central to the biological sciences. Here, we present a novel platform - Affinity Capture of Polyribosomes followed by RNA sequencing (ACAPseq) - for identifying PPIs. ACAPseq harnesses the power of massively parallel RNA sequencing (RNAseq) to quantify the enrichment of polyribosomes based on the affinity of their associated nascent polypeptides for an immobilized protein 'bait'. This method was developed and tested using neonatal mouse brain polyribosomes and a variety of extracellular domains as baits. Of 92 baits tested, 25 identified one or more binding partners that appear to be biologically relevant; additional candidate partners remain to be validated. ACAPseq can detect binding to targets that are present at less than 1 part in 100,000 in the starting polyribosome preparation. One of the observed PPIs was analyzed in detail, revealing the mode of homophilic binding for Protocadherin-9 (PCDH9), a non-clustered Protocadherin family member.