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• W2543449938 abstract "•UNR promotes melanoma invasion and metastasis•UNR coordinates novel pro-metastatic RNA regulons•UNR regulates translation elongation of Vimentin and RAC1 mRNAs•UNR controls the steady-state levels of PTEN mRNA RNA binding proteins (RBPs) modulate cancer progression through poorly understood mechanisms. Here we show that the RBP UNR/CSDE1 is overexpressed in melanoma tumors and promotes invasion and metastasis. iCLIP sequencing, RNA sequencing, and ribosome profiling combined with in silico studies unveiled sets of pro-metastatic factors coordinately regulated by UNR as part of RNA regulons. In addition to RNA steady-state levels, UNR was found to control many of its targets at the level of translation elongation/termination. Key pro-oncogenic targets of UNR included VIM and RAC1, as validated by loss- and gain-of-function studies. Our results identify UNR as an oncogenic modulator of melanoma progression, unravel the underlying molecular mechanisms, and identify potential targets for this therapeutically challenging malignancy. RNA binding proteins (RBPs) modulate cancer progression through poorly understood mechanisms. Here we show that the RBP UNR/CSDE1 is overexpressed in melanoma tumors and promotes invasion and metastasis. iCLIP sequencing, RNA sequencing, and ribosome profiling combined with in silico studies unveiled sets of pro-metastatic factors coordinately regulated by UNR as part of RNA regulons. In addition to RNA steady-state levels, UNR was found to control many of its targets at the level of translation elongation/termination. Key pro-oncogenic targets of UNR included VIM and RAC1, as validated by loss- and gain-of-function studies. Our results identify UNR as an oncogenic modulator of melanoma progression, unravel the underlying molecular mechanisms, and identify potential targets for this therapeutically challenging malignancy. RNA binding proteins (RBPs) are gaining great attention in cancer research for their potential to regulate essentially every hallmark of tumor development. Yet the molecular mechanisms that underlie these capacities are unclear, particularly in aggressive cancers such as melanoma, where RBPs remain largely uncharacterized. Our work establishes that the RBP UNR/CSDE1 is a critical modulator of melanoma metastasis, and reveals a network of UNR targets including well-known cancer drivers as well as genes not previously linked to the disease. Our data broaden the functions of UNR, unveiling a role in translation elongation/termination, and highlight the physiological relevance of RBP-mediated control of oncogenic networks in cancer. Melanoma is an increasingly prevalent cancer that remains a paradigm of genetically and histopathologically heterogeneous diseases (Schadendorf et al., 2015Schadendorf D. Fisher D.E. Garbe C. Gershenwald J.E. Grob J.J. Halpern A. Herlyn M. Marchetti M.A. McArthur G. Ribas A. et al.Melanoma.Nat. Rev. Dis. Primers. 2015; 1: 15003Crossref PubMed Scopus (337) Google Scholar). Both benign and malignant lesions can share dysplastic features that greatly complicate diagnosis. Despite great progress in dermoscopic techniques, staging is still largely defined on the basis of the thickness of the primary melanocytic lesion, although this criterion is long known to be subject to error (Thompson et al., 2004Thompson J.F. Shaw H.M. Hersey P. Scolyer R.A. The history and future of melanoma staging.J. Surg. Oncol. 2004; 86: 224-235Crossref PubMed Scopus (14) Google Scholar). Activating alterations in key signaling cascades (e.g., driven by BRAF or MEK) have resulted in the development of molecularly guided therapies, but response rates are frequently transient (Flaherty et al., 2012Flaherty K.T. Hodi F.S. Fisher D.E. From genes to drugs: targeted strategies for melanoma.Nat. Rev. Cancer. 2012; 12: 349-361Crossref PubMed Scopus (295) Google Scholar). Inhibitors of checkpoint blockers such as CTLA-4, PD1, or PDL1 are providing unprecedented durable responses, although only to a fraction of metastatic melanoma patients (Bathia and Thompson, 2016Bathia S. Thompson J.A. PD-1 blockade in melanoma: a promising start, but a long way to go.JAMA. 2016; 315: 1573-1575Crossref PubMed Scopus (7) Google Scholar). Thus, the melanoma field is in need of diagnostic and prognostic biomarkers, as well as new targets for drug development. High-throughput sequencing efforts have revealed massive heterogeneity of the melanoma transcriptome (Dutton-Regester and Hayward, 2012Dutton-Regester K. Hayward N.K. Reviewing the somatic genetics of melanoma: from current to future analytical approaches.Pigment Cell Melanoma Res. 2012; 25: 144-154Crossref PubMed Scopus (44) Google Scholar). Despite this defining property, RNA-related mechanisms remain surprisingly understudied. A comprehensive analysis of RNA binding proteins (RBPs) and their targets in the context of melanoma initiation and progression seems lacking. RBPs are particularly relevant because they can modulate every post-transcriptional step of gene expression, from splicing in the nucleus to mRNA export, localization, translation, or decay in the cytoplasm. Each single RBP can bind to hundreds of mRNAs, forming extensive networks in which functionally related genes may be co-regulated, representing “RNA regulons” (Morris et al., 2010Morris A.R. Mukherjee N. Keene J.D. Systematic analysis of posttranscriptional gene expression.Wiley Interdiscip. Rev. Syst. Biol. Med. 2010; 2: 162-180Crossref PubMed Scopus (95) Google Scholar). As a result, RBP malfunction may have dramatic consequences for cell physiology. Indeed, RBPs are emerging as critical modulators of cancerous traits, yet little is known about the underlying mechanisms and key downstream targets (Wurth and Gebauer, 2015Wurth L. Gebauer F. RNA-binding proteins, multifaceted translational regulators in cancer.Biochim. Biophys. Acta. 2015; 1849: 881-886Crossref PubMed Scopus (86) Google Scholar). Upstream-of-N-Ras (UNR), known as CSDE1 in mammals, is a conserved RBP containing five cold-shock domains (CSDs) that bind single-stranded RNA (Goroncy et al., 2010Goroncy A.K. Koshiba S. Tochio N. Tomizawa T. Inoue M. Watanabe S. Harada T. Tanaka A. Ohara O. Kigawa T. et al.The NMR solution structures of the five constituent cold-shock domains (CSD) of the human UNR (upstream of N-ras) protein.J. Struct. Funct. Genomics. 2010; 11: 181-188Crossref PubMed Scopus (11) Google Scholar, Triqueneaux et al., 1999Triqueneaux G. Velten M. Franzon P. Dautry F. Jacquemin-Sablon H. RNA binding specificity of Unr, a protein with five cold shock domains.Nucleic Acids Res. 1999; 27: 1926-1934Crossref PubMed Scopus (65) Google Scholar). UNR locates primarily in the cytoplasm, where it regulates mRNA translation and stability (reviewed in Mihailovich et al., 2010Mihailovich M. Militti C. Gabaldon T. Gebauer F. Eukaryotic cold shock domain proteins: highly versatile regulators of gene expression.Bioessays. 2010; 32: 109-118Crossref PubMed Scopus (123) Google Scholar). In Drosophila, UNR inhibits cap-dependent translation of msl-2 mRNA and modifies the structure of roX2 long non-coding RNA, both events contributing to the regulation of X chromosome dosage compensation (Abaza et al., 2006Abaza I. Coll O. Patalano S. Gebauer F. Drosophila UNR is required for translational repression of male-specific lethal 2 mRNA during regulation of X-chromosome dosage compensation.Genes Dev. 2006; 20: 380-389Crossref PubMed Scopus (59) Google Scholar, Duncan et al., 2006Duncan K. Grskovic M. Strein C. Beckmann K. Niggeweg R. Abaza I. Gebauer F. Wilm M. Hentze M.W. Sex-lethal imparts a sex-specific function to UNR by recruiting it to the msl-2 mRNA 3' UTR: translational repression for dosage compensation.Genes Dev. 2006; 20: 368-379Crossref PubMed Scopus (69) Google Scholar, Militti et al., 2014Militti C. Maenner S. Becker P.B. Gebauer F. UNR facilitates the interaction of MLE with the lncRNA roX2 during Drosophila dosage compensation.Nat. Commun. 2014; 5: 4762Crossref PubMed Scopus (19) Google Scholar). In the case of msl-2, UNR binds to the 3′ UTR together with the RBP SXL, establishing extensive intertwined interactions that explain cooperative RNA recognition (Hennig et al., 2014Hennig J. Militti C. Popowicz G.M. Wang I. Sonntag M. Geerlof A. Gabel F. Gebauer F. Sattler M. Structural basis for the assembly of the Sxl-Unr translation regulatory complex.Nature. 2014; 515: 287-290Crossref PubMed Scopus (85) Google Scholar). In the case of roX2, UNR can bind on its own. Thus, UNR binding requirements and regulatory mechanisms may vary depending on the target transcript. The versatile binding of UNR to RNA likely underlies the diverse biological roles of this protein. In mammals, UNR may either promote or inhibit apoptosis and differentiation depending on cell type (Dormoy-Raclet et al., 2007Dormoy-Raclet V. Markovits J. Malato Y. Huet S. Lagarde P. Montaudon D. Jacquemin-Sablon A. Jacquemin-Sablon H. Unr, a cytoplasmic RNA-binding protein with cold-shock domains, is involved in control of apoptosis in ES and HuH7 cells.Oncogene. 2007; 26: 2595-2605Crossref PubMed Scopus (40) Google Scholar, Elatmani et al., 2011Elatmani H. Dormoy-Raclet V. Dubus P. Dautry F. Chazaud C. Jacquemin-Sablon H. The RNA-binding protein Unr prevents mouse embryonic stem cells differentiation toward the primitive endoderm lineage.Stem Cells. 2011; 29: 1504-1516Crossref PubMed Scopus (37) Google Scholar, Horos et al., 2012Horos R. Ijspeert H. Pospisilova D. Sendtner R. Andrieu-Soler C. Taskesen E. Nieradka A. Cmejla R. Sendtner M. Touw I.P. et al.Ribosomal deficiencies in Diamond-Blackfan anemia impair translation of transcripts essential for differentiation of murine and human erythroblasts.Blood. 2012; 119: 262-272Crossref PubMed Scopus (127) Google Scholar). RNA immunoprecipitation (RIP) analysis revealed that Drosophila UNR binds to hundreds of transcripts (Mihailovic et al., 2012Mihailovic M. Wurth L. Zambelli F. Abaza I. Militti C. Mancuso F.M. Roma G. Pavesi G. Gebauer F. Widespread generation of alternative UTRs contributes to sex-specific RNA binding by UNR.RNA. 2012; 18: 53-64Crossref PubMed Scopus (19) Google Scholar). Among these, mRNAs encoding conserved regulators related to human cancer progression were identified (e.g., TGFB1, ABL1, or CTNNB1). The targets of mammalian UNR have not been described at genome-wide level. However, the small list of known targets suggests a potential role of UNR in proliferation and cancer progression. For example, UNR participates in the destabilization of FOS mRNA by binding to its coding sequence (CDS) (Chang et al., 2004Chang T.C. Yamashita A. Chen C.Y. Yamashita Y. Zhu W. Durdan S. Kahvejian A. Sonenberg N. Shyu A.B. UNR, a new partner of poly(A)-binding protein, plays a key role in translationally coupled mRNA turnover mediated by the c-fos major coding-region determinant.Genes Dev. 2004; 18: 2010-2023Crossref PubMed Scopus (123) Google Scholar). In addition, UNR regulates internal ribosome entry site (IRES)-dependent translation of the transcripts encoding the oncogene MYC, the cell-cycle kinase PITSLRE, and the apoptosis regulator APAF-1 (Evans et al., 2003Evans J.R. Mitchell S.A. Spriggs K.A. Ostrowski J. Bomsztyk K. Ostarek D. Willis A.E. Members of the poly (rC) binding protein family stimulate the activity of the c-myc internal ribosome entry segment in vitro and in vivo.Oncogene. 2003; 22: 8012-8020Crossref PubMed Scopus (186) Google Scholar, Mitchell et al., 2003Mitchell S.A. Spriggs K.A. Coldwell M.J. Jackson R.J. Willis A.E. The Apaf-1 internal ribosome entry segment attains the correct structural conformation for function via interactions with PTB and unr.Mol. Cell. 2003; 11: 757-771Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, Tinton et al., 2005Tinton S.A. Schepens B. Bruynooghe Y. Beyaert R. Cornelis S. Regulation of the cell-cycle-dependent internal ribosome entry site of the PITSLRE protein kinase: roles of Unr (upstream of N-ras) protein and phosphorylated translation initiation factor eIF-2alpha.Biochem. J. 2005; 385: 155-163Crossref PubMed Scopus (77) Google Scholar). Furthermore, UNR represses the translation from its own IRES, providing a negative feedback loop to temper the levels of UNR along the cell cycle (Schepens et al., 2007Schepens B. Tinton S.A. Bruynooghe Y. Parthoens E. Haegman M. Beyaert R. Cornelis S. A role for hnRNP C1/C2 and Unr in internal initiation of translation during mitosis.EMBO J. 2007; 26: 158-169Crossref PubMed Scopus (81) Google Scholar). Here, we have evaluated the potential role of UNR in cancer progression using melanoma as a model system. To obtain insights into a potential role of UNR in cancer progression, we first interrogated the TCGA database for alterations of CSDE1/UNR expression in a variety of human cancer samples. CSDE1 mRNA is upregulated in a high percentage of tumors, especially in skin and ovary cancers (Figure S1). We therefore focused on melanoma as a prototypical skin cancer. Because UNR negatively regulates its own translation (Schepens et al., 2007Schepens B. Tinton S.A. Bruynooghe Y. Parthoens E. Haegman M. Beyaert R. Cornelis S. A role for hnRNP C1/C2 and Unr in internal initiation of translation during mitosis.EMBO J. 2007; 26: 158-169Crossref PubMed Scopus (81) Google Scholar), RNA levels may not correlate with protein amounts. Hence, we monitored the levels of UNR protein in melanoma cell lines and tissue specimens. We found that UNR was overexpressed in a variety of melanoma cell lines compared with normal melanocytes. Importantly, we also observed high expression of UNR in primary and metastatic lesions from melanoma patients compared with benign nevi (Figures 1A and 1B ). An inducible short hairpin RNA (shRNA) system was then used to conditionally deplete UNR from a representative melanoma cell line (SK-Mel-103) characterized by its aggressive and metastatic properties (Figure 2A , left panel). A cumulative growth assay, which monitors proliferation in a confluence-independent manner, showed no differences between control and UNR-depleted cells (Figure 2A, middle panel). However, a reduction in cell number was observed when cells reached confluence (Figures 2A [right panel] and S2A). Furthermore, while control cells were able to grow as spheres, UNR-depleted cells remained tightly attached to the surface (Figure 2A, insets in right panel). These results suggest that UNR influences proliferation under stress conditions such as crowding. UNR depletion reduced the clonogenic capacity of melanoma cells and their ability to grow in soft agar (Figures 2B and 2C), results that were recapitulated in different melanoma cell lines characterized by prototypical genetic mutations (Figure S2B). Unless otherwise indicated, we henceforth used SK-Mel-103 cells. Restoring the expression of UNR in depleted cells using a cDNA resistant to shRNA inhibition rescued colony formation in soft agar, indicating that impaired colony formation upon UNR depletion is unlikely to result from off-target effects of the shRNA (Figures 2C and S2C). In addition, enforced expression of UNR in cancerous cells increased colony formation, suggesting that the dosage of UNR is important for colony growth (Figure S2C, right panel). These results suggest that UNR promotes anchorage-independent growth. This was further confirmed by growing cells in suspension. Contrary to control cells, UNR-depleted cells were unable to grow detached from the surface and underwent cell death as measured by caspase-3/7 cleavage activity (Figures 2D, S2D, and S2E), suggesting that UNR promotes resistance to anoikis. In addition, UNR depletion resulted in decreased migration of cells within collagen (Figure 2E). Anchorage-independent growth, migration, and resistance to anoikis are frequently associated with invasive and metastatic capacities of tumor cells. To evaluate these properties more directly, we cultured melanoma spheroids in Matrigel and measured their invasion index. UNR-depleted cells formed significantly smaller spheroids with dramatically reduced invasion index (Figures 2F and S2F). To investigate how general these effects were, we depleted UNR in a number of melanoma cell lines and cells from other tumor origins (breast and ovarian cancer), carrying either wild-type or mutated BRAF, a gene frequently mutated in melanoma. In all cases, depletion of UNR favored anoikis and reduced the invasive capacities of cells (Figure S2H). Conversely, overexpression of UNR augmented these traits (Figure S2H). These results indicate that UNR promotes tumorigenesis in vitro. Next, we validated the pro-tumorigenic functions of UNR in vivo, using xenograft models in mice. To this end, UNR-depleted SK-Mel-103 melanoma cells were implanted subcutaneously in immunodeficient mice. As shown in Figure 2G, UNR-depleted cells resulted in smaller tumors compared with control cells. To assess for UNR roles in surrogate models of metastasis, we labeled control and UNR-depleted cells with luciferase to monitor tumor growth and dissemination upon tail-vein injection. The results showed a striking reduction of the metastatic capacity of UNR-depleted cells to the lung, a frequent site for melanoma metastasis in mice (Figure 2H). For a more direct assessment of metastatic potential, mice were injected subcutaneously, and lymph nodes proximal and distal to the site of injection were monitored for the presence of tumor cells. Depletion of UNR from highly metastatic SK-Mel-103 cells indeed eliminated lymph node metastasis (Figure 2I). Conversely, overexpression of UNR in otherwise non-metastatic UACC-257 cells promoted migration and invasion in vitro, as well as lymph node metastasis formation in vivo (Figures 2I and S2G). Taken together, these data indicate that UNR is required to sustain the invasive and metastatic capacity of melanoma cells. To unravel the mechanisms by which UNR promotes invasion and metastasis, we intersected three types of high-throughput analysis: (1) individual-nucleotide resolution crosslinking immunoprecipitation sequencing (iCLIP-seq) to identify direct RNA targets of UNR, (2) RNA sequencing (RNA-seq), and (3) ribosome profiling to determine at which level UNR regulates the expression of its targets (Figure 3A ). To perform iCLIP-seq (Konig et al., 2011Konig J. Zarnack K. Rot G. Curk T. Kayikci M. Zupan B. Turner D.J. Luscombe N.M. Ule J. iCLIP—transcriptome-wide mapping of protein-RNA interactions with individual nucleotide resolution.J. Vis. Exp. 2011; https://doi.org/10.3791/2638Crossref PubMed Scopus (155) Google Scholar), we generated an anti-UNR antibody that immunoprecipitates endogenous UNR efficiently and with high specificity (Figure 3B [left panel] and Figure S3A). After UV crosslink of melanoma cells and partial RNA digestion, UNR-RNA complexes were immunoprecipitated and appeared as a smear over the UNR band in a protein gel (Figure 3B, right panel, red square). No complex was present in control lanes lacking UV crosslinking or immunoprecipitated with rabbit immunoglobulin G (IgG), indicating specificity (Figure 3B, lanes 1 and 2). In addition, upon extensive digestion with RNase the signal of the complex was reduced to a sharp band close to the molecular weight of UNR (Figure 3B, lane 4) that was barely detectable in UNR-depleted cells (Figure 3B, lanes 5–6). We performed two independent iCLIP experiments that showed a high correlation (Figure S3B; see Table S1 for iCLIP statistics), revealing a set of 1,532 common targets (Figure 3C and Table S2). Most of these targets are protein-coding RNAs, while only few long-non-coding RNAs were identified. Strikingly, 18.5% of the common targets overlap with Drosophila UNR targets previously identified by RIP sequencing (Mihailovic et al., 2012Mihailovic M. Wurth L. Zambelli F. Abaza I. Militti C. Mancuso F.M. Roma G. Pavesi G. Gebauer F. Widespread generation of alternative UTRs contributes to sex-specific RNA binding by UNR.RNA. 2012; 18: 53-64Crossref PubMed Scopus (19) Google Scholar), which is considerable given the strong divergence between the two species (Table S2), suggesting conserved functions for UNR. The vast majority of the remaining targets have not been previously described in the literature. Of the known targets of mammalian UNR, only PABPC1 and CSDE1 mRNAs were retrieved in our system. Binding of UNR to its own mRNA occurred preferentially at the 5′ UTR, consistent with the proposed role of UNR regulating translation from its own IRES (Schepens et al., 2007Schepens B. Tinton S.A. Bruynooghe Y. Parthoens E. Haegman M. Beyaert R. Cornelis S. A role for hnRNP C1/C2 and Unr in internal initiation of translation during mitosis.EMBO J. 2007; 26: 158-169Crossref PubMed Scopus (81) Google Scholar) (Figure 3D). However, unlike previous reports, binding to PABPC1 mRNA was detected preferentially at the 3′ UTR (Figure 3D), suggesting potential additional regulatory mechanisms of this transcript (Patel et al., 2005Patel G.P. Ma S. Bag J. The autoregulatory translational control element of poly(A)-binding protein mRNA forms a heteromeric ribonucleoprotein complex.Nucleic Acids Res. 2005; 33: 7074-7089Crossref PubMed Scopus (62) Google Scholar). Identified UNR targets included a significant fraction of ribosomal protein and histone mRNAs, among others (Table S2). Gene ontology (GO) analysis showed enrichment for ER-associated translation, extracellular components, exosome, cell junctions, focal adhesions, or melanosome, which fit well with functions of UNR in invasion and metastasis (Figure S3C and Table S3). Interestingly, of the 1,532 UNR targets, 396 were found to be linked to cancer development (Table S2). Validation of a subset of these targets by independent RNA immunoprecipitation experiments showed an 89% validation rate (Figure S3D). Thus, a high proportion (26%) of UNR targets encodes cancer-related factors previously unknown to be regulated by this RBP. iCLIP enrichment of UNR targets showed a general correlation with RNA abundance, suggesting relaxed sequence binding requirements for this protein (Figure 3E). Binding is nevertheless specific, as many highly abundant transcripts were not bound by UNR. DREME analysis identified a consensus binding motif similar to that described by in vitro SELEX experiments (Triqueneaux et al., 1999Triqueneaux G. Velten M. Franzon P. Dautry F. Jacquemin-Sablon H. RNA binding specificity of Unr, a protein with five cold shock domains.Nucleic Acids Res. 1999; 27: 1926-1934Crossref PubMed Scopus (65) Google Scholar) (Figure 3F). UNR binds mature mRNA, preferentially in the CDS and the 3′ UTR and to a lesser extent in the 5′ UTR, suggesting functions for UNR in addition to its described roles as an IRES trans-acting factor (ITAF) (Figure 3G). Overall, the binding motif is centered around the UNR iCLIP peaks; however, when different regions of the mRNA are considered separately, a strong tendency for the motif to be located in the center of the iCLIP tags is detected for CDS peaks but not for 5′ or 3′ UTR peaks, suggesting different binding modes of UNR to these regions (Figure S3E). RNA structural analysis using experimental PARS (parallel analysis of RNA structure) data shows a drop in the PARS score at UNR peaks, indicating that UNR binds single-stranded RNA (Figure S3F) (Wan et al., 2014Wan Y. Qu K. Zhang Q.C. Flynn R.A. Manor O. Ouyang Z. Zhang J. Spitale R.C. Snyder M.P. Segal E. et al.Landscape and variation of RNA secondary structure across the human transcriptome.Nature. 2014; 505: 706-709Crossref PubMed Scopus (371) Google Scholar). In addition, UNR targets show significantly decreased PARS scores compared with all transcripts, indicating a preference of UNR for unstructured mRNAs (Figure 3H). Metagene analysis of the position of UNR binding along the mRNA shows preference of binding downstream of the start and/or stop codons, as well as at the stop codon (Figure 3I). This binding profile is specific for UNR, as we did not observe it for other RBPs such as HuR or TDP43 (Figure S3G). Binding by an RBP does not necessarily imply regulation of the bound target at the tested condition. To identify iCLIP targets regulated by UNR, we first undertook RNA-seq analysis of cells where UNR had been depleted for 5 days compared with shControl pairs. To obtain complete information about coding and non-coding RNAs, we performed RNA-seq on both poly(A) RNA and total RNA after ribozero treatment, each in duplicate. The duplicates showed high correlation (Figure S4A; see also Table S1 for statistics). Because UNR binds mostly mature mRNAs, we focused our analysis on poly(A) RNA-seq. For histone mRNAs, which lack poly(A) tails, we used total RNA-seq. Evaluation of iCLIP enrichment and RNA abundance changes showed a poor correlation, indicating that only a reduced number of binding targets were regulated by UNR at the steady-state level (Figure 4A ). A total of 715 genes showed altered RNA levels, 93 of which were direct UNR targets (Figure 4B). Comparatively more mRNAs were downregulated upon UNR depletion, suggesting prevalent roles of UNR as an activator of mRNA accumulation (Figure 4B). GO analysis of regulated iCLIP targets (other than histones and ribosomal protein mRNAs) showed over-representation of cell adhesion and extracellular matrix components (Figures 4C and Table S4). We selected iCLIP targets encoding factors involved in these functions for validation and observed consistent downregulation of tumor-promoting factors upon UNR depletion, while tumor-suppressing factors were upregulated (Figure 4D). A transcript downregulated by UNR encodes the tumor suppressor PTEN, a gene whose expression is frequently reduced in melanoma. We find upregulation of PTEN both at the mRNA and protein levels upon UNR depletion (Figures 4D and S4B). Finally, we tested whether the regulation of mRNA levels by UNR was associated with a specific binding pattern of UNR along the transcript. Interestingly, a meta-analysis indicated strong positioning of UNR at the stop codon of regulated transcripts (Figure 4E). mRNAs upregulated upon UNR depletion (i.e., downregulated by UNR) showed additional binding of UNR at the start codon. In summary, UNR regulates the steady-state levels of mRNAs coding for tumor-promoting and -suppressing factors in a manner that correlates with its oncogenic properties. Given that UNR binds to a subset of melanoma transcripts, we expected this protein to be an mRNA-specific regulator. To rule out indirect effects on global translation, we analyzed de novo protein synthesis by 35S metabolic labeling as well as polysome profiling of shUNR versus shControl cells. We did not detect significant differences in global translation by any of these assays (Figure S5A). To identify genes regulated by UNR at the translation level, we performed ribosome profiling (RP), a technology that provides quantitative and positional information of ribosomes along transcripts at codon resolution (Ingolia et al., 2009Ingolia N.T. Ghaemmaghami S. Newman J.R. Weissman J.S. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.Science. 2009; 324: 218-223Crossref PubMed Scopus (2407) Google Scholar). Three independent RP experiments were performed on shUNR and shControl cells after a 5-day induction of the shRNA. These experiments showed a good correlation (Figure S5B). Metagene analysis indicated that reads mapped primarily at the CDS, ending abruptly at the stop codon and extending to some degree into the 5′ UTR, typical of RP. In addition, single-nucleotide resolution analysis revealed the triplet (codon) pace of the ribosome, attesting to the quality of our data (Figure S5C). Our coverage was nevertheless limited, as lowly abundant genes could not be quantified with statistical significance (see Table S1). For example, β-catenin (CTNNB1) mRNA appears as “not translationally regulated” by our statistical analysis. However, western blot analysis showed that the levels of CTNNB1 decrease upon UNR depletion even in the presence of proteasome inhibitors, suggesting that UNR promotes CTNNB1 translation (Figure S5D, left panel). The details of our RP analysis are described in Supplemental Experimental Procedures (see also Figure S5E). In brief, we performed three types of analyses: (1) we considered all transcripts and normalized the number of ribosome protected fragments (RPFs) with their RNA levels (translational efficiency or “TE” group); (2) we excluded transcripts changing at the steady-state level and quantified normalized RPFs (“RPF” group); and (3) we analyzed the distribution of ribosomes along the mRNA, even for transcripts that did not change at the TE or RPF levels (“Distribution” group). Altogether, the analysis revealed 451 genes regulated at the translation level, of which 127 are UNR iCLIP targets (Figure 5A ; Tables S5 and S6). GO analysis of UNR targets (excluding histone and ribosomal protein mRNAs) showed enrichment in categories related to the extracellular matrix, among others (Figure 5B and Table S5). A summary of all changes observed upon UNR depletion is shown in Figure 5C. In this graph, mRNA abundance change is plotted against changes in the number of RPFs. Quadrants a and c are the most populated, and show genes for which there is a positive correlation between mRNA abundance and translation, while almost no changes are detected in the anti-correlation quadrants d and b. These results nicely illustrate that our RP data reproduce events observed at the RNA-seq level. However, most UNR direct targets (orange crosses) fall outside these quadrants and are present in the green, pink, and white areas. The green regions represent genes that change at the mRNA steady-state level without significant differences in RPF reads. Transcripts in these regions show differences in amount that are not" @default.
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• W2543449938 date "2016-11-01" @default.
• W2543449938 modified "2023-10-18" @default.
• W2543449938 title "UNR/CSDE1 Drives a Post-transcriptional Program to Promote Melanoma Invasion and Metastasis" @default.
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• W2543449938 doi "https://doi.org/10.1016/j.ccell.2016.10.004" @default.
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