AKT-dependent nuclear localization of EPRS1 activates PARP1 in breast cancer cells.

Glutamyl-prolyl-tRNA synthetase (EPRS1) is a bifunctional aminoacyl-tRNA-synthetase (aaRS) essential for decoding the genetic code. EPRS1 resides, with seven other aaRSs and three noncatalytic proteins, in the cytoplasmic multi-tRNA synthetase complex (MSC). Multiple MSC-resident aaRSs, including EPRS1, exhibit stimulus-dependent release from the MSC to perform noncanonical activities distinct from their primary function in protein synthesis. Here, we show EPRS1 is present in both cytoplasm and nucleus of breast cancer cells with constitutively low phosphatase and tensin homolog (PTEN) expression. EPRS1 is primarily cytosolic in PTEN-expressing cells, but chemical or genetic inhibition of PTEN, or chemical or stress-mediated activation of its target, AKT, induces EPRS1 nuclear localization. Likewise, preferential nuclear localization of EPRS1 was observed in invasive ductal carcinoma that were also P-Ser473-AKT+. EPRS1 nuclear transport requires a nuclear localization signal (NLS) within the linker region that joins the catalytic glutamyl-tRNA synthetase and prolyl-tRNA synthetase domains. Nuclear EPRS1 interacts with poly(ADP-ribose) polymerase 1 (PARP1), a DNA-damage sensor that directs poly(ADP-ribosyl)ation (PARylation) of proteins. EPRS1 is a critical regulator of PARP1 activity as shown by markedly reduced ADP-ribosylation in EPRS1 knockdown cells. Moreover, EPRS1 and PARP1 knockdown comparably alter the expression of multiple tumor-related genes, inhibit DNA-damage repair, reduce tumor cell survival, and diminish tumor sphere formation by breast cancer cells. EPRS1-mediated regulation of PARP1 activity provides a mechanistic link between PTEN loss in breast cancer cells, PARP1 activation, and cell survival and tumor growth. Targeting the noncanonical activity of EPRS1, without inhibiting canonical tRNA ligase activity, provides a therapeutic approach potentially supplementing existing PARP1 inhibitors.

Cell surface CD55 traffics to the nucleus leading to cisplatin resistance and stemness by inducing PRC2 and H3K27 trimethylation on chromatin in ovarian cancer.

BACKGROUND: Platinum resistance is the primary cause of poor survival in ovarian cancer (OC) patients. Targeted therapies and biomarkers of chemoresistance are critical for the treatment of OC patients. Our previous studies identified cell surface CD55, a member of the complement regulatory proteins, drives chemoresistance and maintenance of cancer stem cells (CSCs). CSCs are implicated in tumor recurrence and metastasis in multiple cancers. METHODS: Protein localization assays including immunofluorescence and subcellular fractionation were used to identify CD55 at the cell surface and nucleus of cancer cells. Protein half-life determinations were used to compare cell surface and nuclear CD55 stability. CD55 deletion mutants were generated and introduced into cancer cells to identify the nuclear trafficking code, cisplatin sensitivity, and stem cell frequency that were assayed using in vitro and in vivo models. Detection of CD55 binding proteins was analyzed by immunoprecipitation followed by mass spectrometry. Target pathways activated by CD55 were identified by RNA sequencing. RESULTS: CD55 localizes to the nucleus of a subset of OC specimens, ascites from chemoresistant patients, and enriched in chemoresistant OC cells. We determined that nuclear CD55 is glycosylated and derived from the cell surface pool of CD55. Nuclear localization is driven by a trafficking code containing the serine/threonine (S/T) domain of CD55. Nuclear CD55 is necessary for cisplatin resistance, stemness, and cell proliferation in OC cells. CD55 S/T domain is necessary for nuclear entry and inducing chemoresistance to cisplatin in both in vitro and in vivo models. Deletion of the CD55 S/T domain is sufficient to sensitize chemoresistant OC cells to cisplatin. In the nucleus, CD55 binds and attenuates the epigenetic regulator and tumor suppressor ZMYND8 with a parallel increase in H3K27 trimethylation and members of the Polycomb Repressive Complex 2. CONCLUSIONS: For the first time, we show CD55 localizes to the nucleus in OC and promotes CSC and chemoresistance. Our studies identify a therapeutic mechanism for treating platinum resistant ovarian cancer by blocking CD55 nuclear entry.

Aminoacyl-tRNA synthetase interactions in SARS-CoV-2 infection.

Aminoacyl-tRNA synthetases (aaRSs) are ancient enzymes that serve a foundational role in the efficient and accurate translation of genetic information from messenger RNA to proteins. These proteins play critical, non-canonical functions in a multitude of cellular processes. Multiple viruses are known to hijack the functions of aaRSs for proviral outcomes, while cells modify antiviral responses through non-canonical functions of certain synthetases. Recent findings have revealed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronaviral disease 19 (COVID-19), utilizes canonical and non-canonical functions of aaRSs, establishing a complex interplay of viral proteins, cellular factors and host aaRSs. In a striking example, an unconventional multi-aaRS complex consisting of glutamyl-prolyl-, lysyl-, arginyl- and methionyl-tRNA synthetases interact with a previously unknown RNA-element in the 3'-end of SARS-CoV-2 genomic and subgenomic RNAs. This review aims to highlight the aaRS-SARS-CoV-2 interactions identified to date, with possible implications for the biology of host aaRSs in SARS-CoV-2 infection.

Translational control of murine adiponectin expression by an upstream open reading frame element.

Adiponectin, an adipocyte-specific secretory protein encoded by the ADIPOQ gene has a causal role in insulin resistance. Anti-diabetic drugs increase plasma adiponectin by a poorly understood, post-transcriptional mechanism enhancing insulin sensitivity. Deletion analysis of a reporter bearing the mouse Adipoq mRNA 5'-leader identified an inhibitory cis-regulatory sequence. The 5'-leader harbours two potential upstream open reading frames (uORFs) overlapping the principal downstream ORF. Mutation of the uORF ATGs increased reporter translation ~3-fold, indicative of a functional uORF. uORFs are common in mammalian mRNAs; however, only a select group resist translational repression by the integrated stress response (ISR). Thapsigargin (TG), which induces endoplasmic reticulum (ER) stress and the ISR, enhanced expression of a reporter bearing the Adipoq 5'-leader; polysome profiling verified translation-stimulation. TG-stimulated translation was absent in cells defective in Ser51 phosphorylation of eukaryotic initiation factor 2α (eIF2α), required for the ISR. To determine its role in expression and function of endogenous adiponectin, the upstream uORF was disrupted by CRISPR-Cas9-mediated mutagenesis of differentiated mouse 3T3-L1 adipocytes. uORF disruption in adipocytes increased adiponectin expression, triacylglycerol accumulation, and glucose uptake, and inhibited paracrine muscle and liver cell expression of gluconeogenic enzymes, establishing an important role of the uORF in adiponectin-mediated responses to stress.

Interplay between PTB and miR-1285 at the p53 3'UTR modulates the levels of p53 and its isoform Δ40p53α.

p53 and its translational isoform Δ40p53 are involved in many important cellular functions like cell cycle, cell proliferation, differentiation and metabolism. Expression of both the isoforms can be regulated at different steps. In this study, we explored the role of 3'UTR in regulating the expression of these two translational isoforms. We report that the trans acting factor, Polypyrimidine Tract Binding protein (PTB), also interacts specifically with 3'UTR of p53 mRNA and positively regulates expression of p53 isoforms. Our results suggest that there is interplay between miRNAs and PTB at the 3'UTR under normal and stress conditions like DNA damage. Interestingly, PTB showed some overlapping binding regions in the p53 3'UTR with miR-1285. In fact, knockdown of miR-1285 as well as expression of p53 3'UTR with mutated miR-1285 binding sites resulted in enhanced association of PTB with the 3'UTR, which provides mechanistic insights of this interplay. Taken together, the results provide a plausible molecular basis of how the interplay between miRNAs and the PTB protein at the 3'UTR can play pivotal role in fine tuning the expression of the two p53 isoforms.

Stress-mediated translational control in cancer cells.

Tumor cells are continually subjected to diverse stress conditions of the tumor microenvironment, including hypoxia, nutrient deprivation, and oxidative or genotoxic stress. Tumor cells must evolve adaptive mechanisms to survive these conditions to ultimately drive tumor progression. Tight control of mRNA translation is critical for this response and the adaptation of tumor cells to such stress forms. This proceeds though a translational reprogramming process which restrains overall translation activity to preserve energy and nutrients, but which also stimulates the selective synthesis of major stress adaptor proteins. Here we present the different regulatory signaling pathways which coordinate mRNA translation in the response to different stress forms, including those regulating eIF2α, mTORC1 and eEF2K, and we explain how tumor cells hijack these pathways for survival under stress. Finally, mechanisms for selective mRNA translation under stress, including the utilization of upstream open reading frames (uORFs) and internal ribosome entry sites (IRESes) are discussed in the context of cell stress. This article is part of a Special Issue entitled: Translation and Cancer.

Host-like RNA Elements Regulate Virus Translation.

Viruses are obligate, intracellular parasites that co-opt host cell machineries for propagation. Critical among these machineries are those that translate RNA into protein and their mechanisms of control. Most regulatory mechanisms effectuate their activity by targeting sequence or structural features at the RNA termini, i.e., at the 5' or 3' ends, including the untranslated regions (UTRs). Translation of most eukaryotic mRNAs is initiated by 5' cap-dependent scanning. In contrast, many viruses initiate translation at internal RNA regions at internal ribosome entry sites (IRESs). Eukaryotic mRNAs often contain upstream open reading frames (uORFs) that permit condition-dependent control of downstream major ORFs. To offset genome compression and increase coding capacity, some viruses take advantage of out-of-frame overlapping uORFs (oORFs). Lacking the essential machinery of protein synthesis, for example, ribosomes and other translation factors, all viruses utilize the host apparatus to generate virus protein. In addition, some viruses exhibit RNA elements that bind host regulatory factors that are not essential components of the translation machinery. SARS-CoV-2 is a paradigm example of a virus taking advantage of multiple features of eukaryotic host translation control: the virus mimics the established human GAIT regulatory element and co-opts four host aminoacyl tRNA synthetases to form a stimulatory binding complex. Utilizing discontinuous transcription, the elements are present and identical in all SARS-CoV-2 subgenomic RNAs (and the genomic RNA). Thus, the virus exhibits a post-transcriptional regulon that improves upon analogous eukaryotic regulons, in which a family of functionally related mRNA targets contain elements that are structurally similar but lacking sequence identity. This "thrifty" virus strategy can be exploited against the virus since targeting the element can suppress the expression of all subgenomic RNAs as well as the genomic RNA. Other 3' end viral elements include 3'-cap-independent translation elements (3'-CITEs) and 3'-tRNA-like structures. Elucidation of virus translation control elements, their binding proteins, and their mechanisms can lead to novel therapeutic approaches to reduce virus replication and pathogenicity.

Homozygous EPRS1 missense variant causing hypomyelinating leukodystrophy-15 alters variant-distal mRNA m6A site accessibility.

Hypomyelinating leukodystrophy (HLD) is an autosomal recessive disorder characterized by defective central nervous system myelination. Exome sequencing of two siblings with severe cognitive and motor impairment and progressive hypomyelination characteristic of HLD revealed homozygosity for a missense single-nucleotide variant (SNV) in EPRS1 (c.4444 C > A; p.Pro1482Thr), encoding glutamyl-prolyl-tRNA synthetase, consistent with HLD15. Patient lymphoblastoid cell lines express markedly reduced EPRS1 protein due to dual defects in nuclear export and cytoplasmic translation of variant EPRS1 mRNA. Variant mRNA exhibits reduced METTL3 methyltransferase-mediated writing of N6-methyladenosine (m6A) and reduced reading by YTHDC1 and YTHDF1/3 required for efficient mRNA nuclear export and translation, respectively. In contrast to current models, the variant does not alter the sequence of m6A target sites, but instead reduces their accessibility for modification. The defect was rescued by antisense morpholinos predicted to expose m6A sites on target EPRS1 mRNA, or by m6A modification of the mRNA by METTL3-dCas13b, a targeted RNA methylation editor. Our bioinformatic analysis predicts widespread occurrence of SNVs associated with human health and disease that similarly alter accessibility of distal mRNA m6A sites. These results reveal a new RNA-dependent etiologic mechanism by which SNVs can influence gene expression and disease, consequently generating opportunities for personalized, RNA-based therapeutics targeting these disorders.

A viral pan-end RNA element and host complex define a SARS-CoV-2 regulon.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, generates multiple protein-coding, subgenomic RNAs (sgRNAs) from a longer genomic RNA, all bearing identical termini with poorly understood roles in regulating viral gene expression. Insulin and interferon-gamma, two host-derived, stress-related agents, and virus spike protein, induce binding of glutamyl-prolyl-tRNA synthetase (EPRS1), within an unconventional, tetra-aminoacyl-tRNA synthetase complex, to the sgRNA 3'-end thereby enhancing sgRNA expression. We identify an EPRS1-binding sarbecoviral pan-end activating RNA (SPEAR) element in the 3'-end of viral RNAs driving agonist-induction. Translation of another co-terminal 3'-end feature, ORF10, is necessary for SPEAR-mediated induction, independent of Orf10 protein expression. The SPEAR element enhances viral programmed ribosomal frameshifting, thereby expanding its functionality. By co-opting noncanonical activities of a family of essential host proteins, the virus establishes a post-transcriptional regulon stimulating global viral RNA translation. A SPEAR-targeting strategy markedly reduces SARS-CoV-2 titer, suggesting a pan-sarbecoviral therapeutic modality.

Annexin A2 and PSF proteins interact with p53 IRES and regulate translation of p53 mRNA.

p53 mRNA has been shown to be translated into two isoforms, full-length p53 (FL-p53) and a truncated isoform ΔN-p53, which modulates the functions of FL-p53 and also has independent functions. Previously, we have shown that translation of p53 and ΔN-p53 can be initiated at Internal Ribosome Entry Sites (IRES). These two IRESs were shown to regulate the translation of p53 and ΔN-p53 in a distinct cell-cycle phase-dependent manner. Earlier observations from our laboratory also suggest that the structural integrity of the p53 RNA is critical for IRES function and is compromised by mutations that affect the structure as well as RNA protein interactions. In the current study, using RNA affinity approach we have identified Annexin A2 and PTB associated Splicing Factor (PSF/SFPQ) as novel ITAFs for p53 IRESs. We have showed that the purified Annexin A2 and PSF proteins specifically bind to p53 IRES elements. Interestingly, in the presence of calcium ions Annexin A2 showed increased binding with p53 IRES. Immunopulldown experiments suggest that these two proteins associate with p53 mRNA ex vivo as well. Partial knockdown of Annexin A2 and PSF showed decrease in p53 IRES activity and reduced levels of both the p53 isoforms. More importantly the interplay between Annexin A2, PSF and PTB proteins for binding to p53mRNA appears to play a crucial role in IRES function. Taken together, our observations suggest pivotal role of two new trans-acting factors in regulating the p53-IRES function, which in turn influences the synthesis of p53 isoforms.

Analysis of nested alternate open reading frames and their encoded proteins.

Transcriptional and post-transcriptional mechanisms diversify the proteome beyond gene number, while maintaining a sequence relationship between original and altered proteins. A new mechanism breaks this paradigm, generating novel proteins by translating alternative open reading frames (Alt-ORFs) within canonical host mRNAs. Uniquely, 'alt-proteins' lack sequence homology with host ORF-derived proteins. We show global amino acid frequencies, and consequent biochemical characteristics of Alt-ORFs nested within host ORFs (nAlt-ORFs), are genetically-driven, and predicted by summation of frequencies of hundreds of encompassing host codon-pairs. Analysis of 101 human nAlt-ORFs of length ≥150 codons confirms the theoretical predictions, revealing an extraordinarily high median isoelectric point (pI) of 11.68, due to anomalous charged amino acid levels. Also, nAlt-ORF proteins exhibit a >2-fold preference for reading frame 2 versus 3, predicted mitochondrial and nuclear localization, and elevated codon adaptation index indicative of natural selection. Our results provide a theoretical and conceptual framework for exploration of these largely unannotated, but potentially significant, alternative ORFs and their encoded proteins.

Effect of mutations on the p53 IRES RNA structure: implications for de-regulation of the synthesis of p53 isoforms.

Earlier we have demonstrated the presence of internal ribosome entry site (IRES) within tumor suppressor p53 mRNA. Here we have mapped the putative secondary structure of p53- IRES RNA using information from chemical probing and nuclease mapping experiments. Additionally, the secondary structure of the IRES element of the wild-type RNA was compared with cancer-derived silent mutant p53 RNAs. These mutations might result in the conformational alterations of p53-IRES RNAs. The results also indicate decreased IRES activities of the mutants as compared to wild-type RNA. Further, it was observed that some of the cytoplasmic trans-acting factors, critical for enhancing IRES function, were unable to bind mutant RNAs as efficiently as to wild-type. Our results suggest that hnRNP C1/C2 binds to p53-IRES and siRNA mediated partial silencing of hnRNP C1/C2 showed appreciable decrease in IRES function and consequent decrease in the level of the corresponding p53 isoform. Interestingly mutant p53 IRES showed lesser binding with hnRNP C1/C2 protein. Finally, upon doxorubicin treatment, the mutant RNAs were unable to show enhanced p53 synthesis to similar extent compared to wild type. Taken together, these observations suggest that mutations occurring in the p53 IRES might have profound implications for de-regulation of its expression and activity.

The zinc-binding domain of mammalian prolyl-tRNA synthetase is indispensable for catalytic activity and organism viability.

Aminoacyl-tRNA synthetases (AARS) participate in decoding the genome by catalyzing conjugation of amino acids to their cognate tRNAs. During evolution, biochemical and environmental conditions markedly influenced the sequence and structure of the 20 AARSs, revealing adaptations dictating canonical and orthogonal activities. Here, we investigate the function of the appended Zn2+-binding domain (ZBD) in the bifunctional AARS, glutamyl-prolyl-tRNA synthetase (GluProRS). We developed GluProRS mutant mice by CRISPR-Cas9 with a deletion of 29 C-terminal amino acids, including two of four Zn2+-coordinating cysteines. Homozygous ZBD mutant mice die before embryonic day 12.5, but heterozygous mice are healthy. ZBD disruption profoundly reduces GluProRS canonical function by dual mechanisms: it induces rapid proteasomal degradation of the protein and inhibits ProRS aminoacylation activity, likely by sub-optimal positioning of ATP in the spatially adjacent catalytic domain. Collectively, our studies reveal the ZBD as a critical determinant of ProRS activity and GluProRS stability in vitro and in vivo.

MYCN amplified neuroblastoma requires the mRNA translation regulator eEF2 kinase to adapt to nutrient deprivation.

MYC family proteins are implicated in many human cancers, but their therapeutic targeting has proven challenging. MYCN amplification in childhood neuroblastoma (NB) is associated with aggressive disease and high mortality. Novel and effective therapeutic strategies are therefore urgently needed for these tumors. MYC-driven oncogenic transformation impairs cell survival under nutrient deprivation (ND), a characteristic stress condition within the tumor microenvironment. We recently identified eukaryotic Elongation Factor 2 Kinase (eEF2K) as a pivotal mediator of the adaptive response of tumor cells to ND. We therefore hypothesized that eEF2K facilitates the adaptation of MYCN amplified NB to ND, and that inhibiting this pathway can impair MYCN-driven NB progression. To test our hypothesis, we first analyzed publicly available genomic databases and tissue microarrays for eEF2K expression in NB, and for links between eEF2K, MYCN, and clinical outcome in NB. Effects of eEF2K inhibition were evaluated on survival of MYCN amplified versus non-amplified NB cell lines under ND. Finally, NB xenograft mouse models were used to confirm in vitro observations. Our results indicate that high eEF2K expression and activity are strongly predictive of poor outcome in NB, and correlates significantly with MYCN amplification. Inhibition of eEF2K markedly decreases survival of MYCN amplified NB cell lines in vitro under ND. Growth of MYCN amplified NB xenografts is markedly impaired by eEF2K knockdown, particularly under caloric restriction. In summary, eEF2K protects MYCN overexpressing NB cells from ND in vitro and in vivo, highlighting this kinase as a critical mediator of the adaptive response of MYCN amplified NB cells to metabolic stress.

Influence of metabolic stress on translation of p53 isoforms.

p53 and its isoforms are integral in modulating transcriptional gene expression programs and maintaining cellular homeostasis. We recently reported that glucose deprivation/caloric restriction induced translational control of p53 mRNA by scaffold/matrix attachment region binding-protein 1 (SMAR1), adding a cytoplasmic role of SMAR1 to its traditional nuclear role as a transcription factor.

Translational Activation of HIF1α by YB-1 Promotes Sarcoma Metastasis.

Metastatic dissemination is the leading cause of death in cancer patients, which is particularly evident for high-risk sarcomas such as Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma. Previous research identified a crucial role for YB-1 in the epithelial-to-mesenchymal transition (EMT) and metastasis of epithelial malignancies. Based on clinical data and two distinct animal models, we now report that YB-1 is also a major metastatic driver in high-risk sarcomas. Our data establish YB-1 as a critical regulator of hypoxia-inducible factor 1α (HIF1α) expression in sarcoma cells. YB-1 enhances HIF1α protein expression by directly binding to and activating translation of HIF1A messages. This leads to HIF1α-mediated sarcoma cell invasion and enhanced metastatic capacity in vivo, highlighting a translationally regulated YB-1-HIF1α axis in sarcoma metastasis.

HCV-like IRESs sequester eIF3: advantage virus.

In a recent Nature paper, Hashem et al. attempted to probe deeper into the elusive role of eIF3 in translation initiation of viruses with hepatitis C virus-like internal ribosome entry sites (IRESs), but instead uncovered a surprising role of these IRESs in displacing eIF3 from the 40S subunit, favoring viral translation.