G-Quadruplexes in RNA Biology: Recent Advances and Future Directions.

RNA G-quadruplexes (RG4s) are four-stranded structures known to control gene expression mechanisms, from transcription to protein synthesis, and DNA-related processes. Their potential impact on RNA biology allows these structures to shape cellular processes relevant to disease development, making their targeting for therapeutic purposes an attractive option. We review here the current knowledge on RG4s, focusing on the latest breakthroughs supporting the notion of transient structures that fluctuate dynamically in cellulo, their interplay with RNA modifications, their role in cell compartmentalization, and their deregulation impacting the host immune response. We emphasize RG4-binding proteins as determinants of their transient conformation and effectors of their biological functions.

QUADRatlas: the RNA G-quadruplex and RG4-binding proteins database.

RNA G-quadruplexes (RG4s) are non-canonical, disease-associated post-transcriptional regulators of gene expression whose functions are driven by RNA-binding proteins (RBPs). Being able to explore transcriptome-wide RG4 formation and interaction with RBPs is thus paramount to understanding how they are regulated and exploiting them as potential therapeutic targets. Towards this goal, we present QUADRatlas (https://rg4db.cibio.unitn.it), a database of experimentally-derived and computationally predicted RG4s in the human transcriptome, enriched with biological function and disease associations. As RBPs are key to their function, we mined known interactions of RG4s with such proteins, complemented with an extensive RBP binding sites dataset. Users can thus intersect RG4s with their potential regulators and effectors, enabling the formulation of novel hypotheses on RG4 regulation, function and pathogenicity. To support this capability, we provide analysis tools for predicting whether an RBP can bind RG4s, RG4 enrichment in a gene set, and de novo RG4 prediction. Genome-browser and table views allow exploring, filtering, and downloading the data quickly for individual genes and in batch. QUADRatlas is a significant step forward in our ability to understand the biology of RG4s, offering unmatched data content and enabling the integrated analysis of RG4s and their interactions with RBPs.

RNA G-quadruplexes: emerging mechanisms in disease.

RNA G-quadruplexes (G4s) are formed by G-rich RNA sequences in protein-coding (mRNA) and non-coding (ncRNA) transcripts that fold into a four-stranded conformation. Experimental studies and bioinformatic predictions support the view that these structures are involved in different cellular functions associated to both DNA processes (telomere elongation, recombination and transcription) and RNA post-transcriptional mechanisms (including pre-mRNA processing, mRNA turnover, targeting and translation). An increasing number of different diseases have been associated with the inappropriate regulation of RNA G4s exemplifying the potential importance of these structures on human health. Here, we review the different molecular mechanisms underlying the link between RNA G4s and human diseases by proposing several overlapping models of deregulation emerging from recent research, including (i) sequestration of RNA-binding proteins, (ii) aberrant expression or localization of RNA G4-binding proteins, (iii) repeat associated non-AUG (RAN) translation, (iv) mRNA translational blockade and (v) disabling of protein-RNA G4 complexes. This review also provides a comprehensive survey of the functional RNA G4 and their mechanisms of action. Finally, we highlight future directions for research aimed at improving our understanding on RNA G4-mediated regulatory mechanisms linked to diseases.

Essential role for the interaction between hnRNP H/F and a G quadruplex in maintaining p53 pre-mRNA 3'-end processing and function during DNA damage.

Following DNA damage, mRNA 3'-end formation is inhibited, contributing to repression of mRNA synthesis. Here we investigated how DNA-damaged cells accomplish p53 mRNA 3'-end formation when normal mechanisms of pre-mRNA 3'-end processing regulation are inhibited. The underlying mechanism involves the interaction between a G-quadruplex structure located downstream from the p53 cleavage site and hnRNP H/F. Importantly, this interaction is critical for p53 expression and contributes to p53-mediated apoptosis. Our results uncover the existence of a specific rescue mechanism of 3'-end processing regulation allowing stress-induced p53 accumulation and function in apoptosis.

A novel cytoprotective function for the DNA repair protein Ku in regulating p53 mRNA translation and function.

Ku heterodimer is a DNA binding protein with a prominent role in DNA repair. Here, we investigate whether and how Ku impacts the DNA damage response by acting as a post-transcriptional regulator of gene expression. We show that Ku represses p53 protein synthesis and p53-mediated apoptosis by binding to a bulged stem-loop structure within the p53 5' UTR However, Ku-mediated translational repression of the p53 mRNA is relieved after genotoxic stress. The underlying mechanism involves Ku acetylation which disrupts Ku-p53 mRNA interactions. These results suggest that Ku-mediated repression of p53 mRNA translation constitutes a novel mechanism linking DNA repair and mRNA translation.

Stabilization of the G-quadruplex at the VEGF IRES represses cap-independent translation.

The activation of translation contributes to malignant transformation and is an emerging target for cancer therapies. RNA G-quadruplex structures are general inhibitors of cap-dependent mRNA translation and were recently shown to be targeted for oncoprotein translational activation. In contrast however, the G-quadruplex within the 5'UTR of the human vascular endothelial growth factor A (VEGF) has been shown to be essential for IRES-mediated translation. Since VEGF has a pivotal role in tumor angiogenesis and is a major target of anti-tumoral therapies, we investigated the structure/function relationship of the VEGF G-quadruplex and defined whether it could have a therapeutic potential. We found that the G-quadruplex within the VEGF IRES is dispensable for cap-independent function and activation in stress conditions. However, stabilization of the VEGF G-quadruplex by increasing the G-stretches length or by replacing it with the one of NRAS results in strong inhibition of IRES-mediated translation of VEGF. We also demonstrate that G-quadruplex ligands stabilize the VEGF G-quadruplex and inhibit cap-independent translation in vitro. Importantly, the amount of human VEGF mRNA associated with polysomes decreases in the presence of a highly selective stabilizing G-quadruplex ligand, resulting in reduced VEGF protein expression. Together, our results uncover the existence of functionally silent G-quadruplex structures that are susceptible to conversion into efficient repressors of cap-independent mRNA translation. These findings have implications for the in vivo applications of G-quadruplex-targeting compounds and for anti-angiogenic therapies.

VEGF-A mRNA processing, stability and translation: a paradigm for intricate regulation of gene expression at the post-transcriptional level.

Vascular Endothelial Growth Factor A (VEGF-A) is a potent secreted mitogen crucial for physiological and pathological angiogenesis. Post-transcriptional regulation of VEGF-A occurs at multiple levels. Firstly, alternative splicing gives rise to different transcript variants encoding diverse isoforms that exhibit distinct biological properties with regard to receptor binding and extra-cellular localization. Secondly, VEGF-A mRNA stability is regulated by effectors such as hypoxia or growth factors through the binding of stabilizing and destabilizing proteins at AU-rich elements located in the 3'-untranslated region. Thirdly, translation of VEGF-A mRNA is a controlled process involving alternative initiation codons, internal ribosome entry sites (IRESs), an upstream open reading frame (uORF), miRNA targeting and a riboswitch in the 3' untranslated region. These different levels of regulation cooperate for the crucial fine-tuning of the expression of VEGF-A variants. This review will be focused on our current knowledge of the complex post-transcriptional regulatory switches that modulate the cellular VEGF-A level, a paradigmatic model of post-transcriptional regulation.

Int6/eIF3e is essential for proliferation and survival of human glioblastoma cells.

Glioblastomas (GBM) are very aggressive and malignant brain tumors, with frequent relapses despite an appropriate treatment combining surgery, chemotherapy and radiotherapy. In GBM, hypoxia is a characteristic feature and activation of Hypoxia Inducible Factors (HIF-1α and HIF-2α) has been associated with resistance to anti-cancer therapeutics. Int6, also named eIF3e, is the "e" subunit of the translation initiation factor eIF3, and was identified as novel regulator of HIF-2α. Eukaryotic initiation factors (eIFs) are key factors regulating total protein synthesis, which controls cell growth, size and proliferation. The functional significance of Int6 and the effect of Int6/EIF3E gene silencing on human brain GBM has not yet been described and its role on the HIFs is unknown in glioma cells. In the present study, we show that Int6/eIF3e suppression affects cell proliferation, cell cycle and apoptosis of various GBM cells. We highlight that Int6 inhibition induces a diminution of proliferation through cell cycle arrest and increased apoptosis. Surprisingly, these phenotypes are independent of global cell translation inhibition and are accompanied by decreased HIF expression when Int6 is silenced. In conclusion, we demonstrate here that Int6/eIF3e is essential for proliferation and survival of GBM cells, presumably through modulation of the HIFs.

The shaping of mRNA translation plasticity by RNA G-quadruplexes in cancer progression and therapy resistance.

Translational reprogramming in response to oncogenic signaling or microenvironmental stress factors shapes the proteome of cancer cells, enabling adaptation and phenotypic changes underlying cell plasticity, tumor progression and response to cancer therapy. Among the mechanisms regulating translation are RNA G-quadruplexes (RG4s), non-canonical four-stranded structures whose conformational modulation by small molecule ligands and RNA-binding proteins affects the expression of cancer proteins. Here, we discuss the role of RG4s in the regulation of mRNA translation by focusing on paradigmatic examples showing their contribution to adaptive mechanisms of mRNA translation in cancer.

PD-L1 at the crossroad between RNA metabolism and immunosuppression.

Programmed death ligand-1 (PD-L1) is a key component of tumor immunosuppression. The uneven therapeutic results of PD-L1 therapy have stimulated intensive studies to better understand the mechanisms underlying altered PD-L1 expression in cancer cells, and to determine whether, beyond its immune function, PD-L1 might have intracellular functions promoting tumor progression and resistance to treatments. In this Opinion, we focus on paradigmatic examples highlighting the central role of PD-L1 in post-transcriptional regulation, with PD-L1 being both a target and an effector of molecular mechanisms featured prominently in RNA research, such as RNA methylation, phase separation and RNA G-quadruplex structures, in order to highlight vulnerabilities on which future anti-PD-L1 therapies could be built.

Molecular mechanisms of eukaryotic pre-mRNA 3' end processing regulation.

Messenger RNA (mRNA) 3' end formation is a nuclear process through which all eukaryotic primary transcripts are endonucleolytically cleaved and most of them acquire a poly(A) tail. This process, which consists in the recognition of defined poly(A) signals of the pre-mRNAs by a large cleavage/polyadenylation machinery, plays a critical role in gene expression. Indeed, the poly(A) tail of a mature mRNA is essential for its functions, including stability, translocation to the cytoplasm and translation. In addition, this process serves as a bridge in the network connecting the different transcription, capping, splicing and export machineries. It also participates in the quantitative and qualitative regulation of gene expression in a variety of biological processes through the selection of single or alternative poly(A) signals in transcription units. A large number of protein factors associates with this machinery to regulate the efficiency and specificity of this process and to mediate its interaction with other nuclear events. Here, we review the eukaryotic 3' end processing machineries as well as the comprehensive set of regulatory factors and discuss the different molecular mechanisms of 3' end processing regulation by proposing several overlapping models of regulation.

Analysis of mRNA Translation by Polysome Profiling.

mRNA translation is a key step in gene expression that allows the cell to qualitatively and quantitatively modulate the cell's proteome according to intra- or extracellular signals. Polysome profiling is the most comprehensive technique to study both the translation state of mRNAs and the protein machinery associated with the mRNAs being translated. Here we describe the procedure commonly used in our laboratory to gain insights into the molecular mechanisms underlying translation regulation under pathophysiological conditions.

Translational Regulation by hnRNP H/F Is Essential for the Proliferation and Survival of Glioblastoma.

Deregulation of mRNA translation is a widespread characteristic of glioblastoma (GBM), aggressive malignant brain tumors that are resistant to conventional therapies. RNA-binding proteins (RBPs) play a critical role in translational regulation, yet the mechanisms and impact of these regulations on cancer development, progression and response to therapy remain to be fully understood. Here, we showed that hnRNP H/F RBPs are potent regulators of translation through several mechanisms that converge to modulate the expression and/or the activity of translation initiation factors. Among these, hnRNP H/F regulate the phosphorylation of eIF4E and its translational targets by controlling RNA splicing of the A-Raf kinase mRNA, which in turn modulates the MEK-ERK/MAPK signaling pathway. The underlying mechanism involves RNA G-quadruplex (RG4s), RNA structures whose modulation phenocopies hnRNP H/F translation regulation in GBM cells. Our results highlighted that hnRNP H/F are essential for key functional pathways regulating proliferation and survival of GBM, highlighting its targeting as a promising strategy for improving therapeutic outcomes.

A physical and functional link between splicing factors promotes pre-mRNA 3' end processing.

Polypyrimidine tract-binding protein (PTB) is a splicing regulator that also plays a positive role in pre-mRNA 3' end processing when bound upstream of the polyadenylation signal (pA signal). Here, we address the mechanism of PTB stimulatory function in mRNA 3' end formation. We identify PTB as the protein factor whose binding to the human beta-globin (HBB) 3' UTR is abrogated by a 3' end processing-inactivating mutation. We show that PTB promotes both in vitro 3' end cleavage and polyadenylation and recruits directly the splicing factor hnRNP H to G-rich sequences associated with several pA signals. Increased binding of hnRNP H results in stimulation of polyadenylation through a direct interaction with poly(A) polymerase. Therefore, our results provide evidence of a concerted regulation of pA signal recognition by splicing factors bound to auxiliary polyadenylation sequence elements.

The NMD Pathway Regulates GABARAPL1 mRNA during the EMT.

EMT is a reversible cellular process that is linked to gene expression reprogramming, which allows for epithelial cells to undergo a phenotypic switch to acquire mesenchymal properties. EMT is associated with cancer progression and cancer therapeutic resistance and it is known that, during the EMT, many stress response pathways, such as autophagy and NMD, are dysregulated. Therefore, our goal was to study the regulation of ATG8 family members (GABARAP, GABARAPL1, LC3B) by the NMD and to identify molecular links between these two cellular processes that are involved in tumor development and metastasis formation. IHC experiments, which were conducted in a cohort of patients presenting lung adenocarcinomas, showed high GABARAPL1 and low UPF1 levels in EMT+ tumors. We observed increased levels of GABARAPL1 correlated with decreased levels of NMD factors in A549 cells in vitro. We then confirmed that GABARAPL1 mRNA was indeed targeted by the NMD in a 3'UTR-dependent manner and we identified four overlapping binding sites for UPF1 and eIF4A3 that are potentially involved in the recognition of this transcript by the NMD pathway. Our study suggests that 3'UTR-dependent NMD might be an important mechanism that is involved in the induction of autophagy and could represent a promising target in the development of new anti-cancer therapies.

Activation of the integrated stress response confers vulnerability to mitoribosome-targeting antibiotics in melanoma.

The ability to adapt to environmental stress, including therapeutic insult, contributes to tumor evolution and drug resistance. In suboptimal conditions, the integrated stress response (ISR) promotes survival by dampening cytosolic translation. We show that ISR-dependent survival also relies on a concomitant up-regulation of mitochondrial protein synthesis, a vulnerability that can be exploited using mitoribosome-targeting antibiotics. Accordingly, such agents sensitized to MAPK inhibition, thus preventing the development of resistance in BRAFV600E melanoma models. Additionally, this treatment compromised the growth of melanomas that exhibited elevated ISR activity and resistance to both immunotherapy and targeted therapy. In keeping with this, pharmacological inactivation of ISR, or silencing of ATF4, rescued the antitumoral response to the tetracyclines. Moreover, a melanoma patient exposed to doxycycline experienced complete and long-lasting response of a treatment-resistant lesion. Our study indicates that the repurposing of mitoribosome-targeting antibiotics offers a rational salvage strategy for targeted therapy in BRAF mutant melanoma and a therapeutic option for NRAS-driven and immunotherapy-resistant tumors.

The c.5242C>A BRCA1 missense variant induces exon skipping by increasing splicing repressors binding.

Several unclassified variants (UV) of BRCA1 can be deleterious by affecting normal pre-mRNA splicing. Here, we investigated the consequences at the mRNA level of the frequently encountered c.5242C>A UV in BRCA1 exon 18. We show that the c.5242C>A variant induces skipping of exon 18 in UV carriers and in vitro. This alteration predicted to disrupt the first BRCT domain of BRCA1. We show that two splicing repressors, hnRNP A1 and hnRNP H/F, display a significant preference toward binding with the mutated exon 18 and assemble into a protein complex. Sequence analysis of the region surrounding the c.5242C>A change reveals the presence of hnRNP A1 and hnRNP H/F binding sites, which are modified by several UVs. Mutation of these sites alters the RNA binding ability of both splicing regulators. In conclusion, our work supports the model of the pathogenicity of the c.5242C>A BRCA1 variant that induces exon skipping by creating a sequence with silencer properties. We propose that other UVs in exon 18 interfere with splicing complex assembly by perturbing the binding of hnRNP A1 and hnRNP H/F to their respective cis-elements. RNA analysis is therefore necessary for the assessment of the consequences of UVs on splicing of disease-associated genes and to enable adequate genetic counseling for breast/ovarian cancer families.

Translation reprogramming by eIF3 linked to glioblastoma resistance.

Intrinsic resistance to current therapies, leading to dismal clinical outcomes, is a hallmark of glioblastoma multiforme (GBM), the most common and aggressive brain tumor. Understanding the underlying mechanisms of such malignancy is, therefore, an urgent medical need. Deregulation of the protein translation machinery has been shown to contribute to cancer initiation and progression, in part by driving selective translational control of specific mRNA transcripts involved in distinct cancer cell behaviors. Here, we focus on eIF3, a multimeric complex with a known role in the initiation of translation and that is frequently deregulated in cancer. Our results show that the deregulated expression of eIF3e, the e subunit of eIF3, in specific GBM regions could impinge on selective protein synthesis impacting the GBM outcome. In particular, eIF3e restricts the expression of proteins involved in the response to cellular stress and increases the expression of key functional regulators of cell stemness. Such a translation program can therefore serve as a double-edged sword promoting GBM tumor growth and resistance to radiation.

hnRNP H/F drive RNA G-quadruplex-mediated translation linked to genomic instability and therapy resistance in glioblastoma.

RNA G-quadruplexes (RG4s) are four-stranded structures known to control mRNA translation of cancer relevant genes. RG4 formation is pervasive in vitro but not in cellulo, indicating the existence of poorly characterized molecular machinery that remodels RG4s and maintains them unfolded. Here, we performed a quantitative proteomic screen to identify cytosolic proteins that interact with a canonical RG4 in its folded and unfolded conformation. Our results identified hnRNP H/F as important components of the cytoplasmic machinery modulating the structural integrity of RG4s, revealed their function in RG4-mediated translation and uncovered the underlying molecular mechanism impacting the cellular stress response linked to the outcome of glioblastoma.

Microtubule-Driven Stress Granule Dynamics Regulate Inhibitory Immune Checkpoint Expression in T Cells.

Despite the clinical success of blocking inhibitory immune checkpoint receptors such as programmed cell death-1 (PD-1) in cancer, the mechanisms controlling the expression of these receptors have not been fully elucidated. Here, we identify a post-transcriptional mechanism regulating PD-1 expression in T cells. Upon activation, the PDCD1 mRNA and ribonucleoprotein complexes coalesce into stress granules that require microtubules and the kinesin 1 molecular motor to proceed to translation. Hence, PD-1 expression is highly sensitive to microtubule or stress granule inhibitors targeting this pathway. Evidence from healthy donors and cancer patients reveals a common regulation for the translation of CTLA4, LAG3, TIM3, TIGIT, and BTLA but not of the stimulatory co-receptors OX40, GITR, and 4-1BB mRNAs. In patients, disproportionality analysis of immune-related adverse events for currently used microtubule drugs unveils a significantly higher risk of autoimmunity. Our findings reveal a fundamental mechanism of immunoregulation with great importance in cancer immunotherapy.