c-Myc is one of the major human proto-oncogenes and is often associated with tumor aggression and poor clinical outcome. Paradoxically, Myc was also reported as a suppressor of cell motility, invasiveness, and metastasis. Among the direct targets of Myc are many components of the protein synthesis machinery whose induction results in an overall increase in protein synthesis that empowers tumor cell growth. At present, it is largely unknown whether beyond the global enhancement of protein synthesis, Myc activation results in translation modulation of specific genes. Here, we measured Myc-induced global changes in gene expression at the transcription, translation, and protein levels and uncovered extensive transcript-specific regulation of protein translation. Particularly, we detected a broad coordination between regulation of transcription and translation upon modulation of Myc activity and showed the connection of these responses to mTOR signaling to enhance oncogenic transformation and to the TGFβ pathway to modulate cell migration and invasiveness. Our results elucidate novel facets of Myc-induced cellular responses and provide a more comprehensive view of the consequences of its activation in cancer cells.

Alternative cleavage and polyadenylation (APA) is emerging as an important layer of gene regulation. Factors controlling APA are largely unknown. We developed a reporter-based RNAi screen for APA and identified PABPN1 as a regulator of this process. Genome-wide analysis of APA in human cells showed that loss of PABPN1 resulted in extensive 3' untranslated region shortening. Messenger RNA transcription, stability analyses, and in vitro cleavage assays indicated enhanced usage of proximal cleavage sites (CSs) as the underlying mechanism. Using Cyclin D1 as a test case, we demonstrated that enhanced usage of proximal CSs compromises microRNA-mediated repression. Triplet-repeat expansion in PABPN1 (trePABPN1) causes autosomal-dominant oculopharyngeal muscular dystrophy (OPMD). The expression of trePABPN1 in both a mouse model of OPMD and human cells elicited broad induction of proximal CS usage, linked to binding to endogenous PABPN1 and its sequestration in nuclear aggregates. Our results elucidate a novel function for PABPN1 as a suppressor of APA.

Transcription and translation are two main pillars of gene expression. Due to the different timings, spots of action, and mechanisms of regulation, these processes are mainly regarded as distinct and generally uncoupled, despite serving a common purpose. Here, we sought for a possible connection between transcription and translation. Employing an unbiased screen of multiple human promoters, we identified a positive effect of TATA box on translation and a general coupling between mRNA expression and translational efficiency. Using a CRISPR-Cas9-mediated approach, genome-wide analyses, and in vitro experiments, we show that the rate of transcription regulates the efficiency of translation. Furthermore, we demonstrate that m6A modification of mRNAs is co-transcriptional and depends upon the dynamics of the transcribing RNAPII. Suboptimal transcription rates lead to elevated m6A content, which may result in reduced translation. This study uncovers a general and widespread link between transcription and translation that is governed by epigenetic modification of mRNAs.

Background: Gene expression in human mitochondria has various idiosyncratic features. One of these was recently revealed as the unprecedented recruitment of a mitochondrially-encoded tRNA as a structural component of the large mitoribosomal subunit. In porcine particles this is mt-tRNA Phe whilst in humans it is mt-tRNA Val. We have previously shown that when a mutation in mt-tRNA Val causes very low steady state levels, there is preferential recruitment of mt-tRNA Phe. We have investigated whether this altered mitoribosome affects intra-organellar protein synthesis. Methods: By using mitoribosomal profiling we have revealed aspects of mitoribosome behaviour with its template mt-mRNA under both normal conditions as well as those where the mitoribosome has incorporated mt-tRNA Phe. Results: Analysis of the mitoribosome residency on transcripts under control conditions reveals that although mitochondria employ only 22 mt-tRNAs for protein synthesis, the use of non-canonical wobble base pairs at codon position 3 does not cause any measurable difference in mitoribosome occupancy irrespective of the codon. Comparison of the profile of aberrant mt-tRNA Phe containing mitoribosomes with those of controls that integrate mt-tRNA Val revealed that the impaired translation seen in the latter was not due to stalling on triplets encoding either of these amino acids. The alterations in mitoribosome interactions with start codons was not directly attributable to the either the use of non-cognate initiation codons or the presence or absence of 5' leader sequences, except in the two bicistronic RNA units, RNA7 and RNA14 where the initiation sites are internal. Conclusions: These data report the power of mitoribosomal profiling in helping to understand the subtleties of mammalian mitochondrial protein synthesis. Analysis of profiles from the mutant mt-tRNA Val cell line suggest that despite mt-tRNA Phe being preferred in the porcine mitoribosome, its integration into the human counterpart results in a suboptimal structure that modifies its interaction with mt-mRNAs.

The use of alternative promoters, splicing, and cleavage and polyadenylation (APA) generates mRNA isoforms that expand the diversity and complexity of the transcriptome. Here, we uncovered thousands of previously undescribed 5' uncapped and polyadenylated transcripts (5' UPTs). We show that these transcripts resist exonucleases due to a highly structured RNA and N6-methyladenosine modification at their 5' termini. 5' UPTs appear downstream of APA sites within their host genes and are induced upon APA activation. Strong enrichment in polysomal RNA fractions indicates 5' UPT translational potential. Indeed, APA promotes downstream translation initiation, non-canonical protein output, and consistent changes to peptide presentation at the cell surface. Lastly, we demonstrate the biological importance of 5' UPTs using Bcl2, a prominent anti-apoptotic gene whose entire coding sequence is a 5' UPT generated from 5' UTR-embedded APA sites. Thus, APA is not only accountable for terminating transcripts, but also for generating downstream uncapped RNAs with translation potential and biological impact.

By restoring tryptophan, indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors aim to reactivate anti-tumor T cells. However, a phase III trial assessing their clinical benefit failed, prompting us to revisit the role of IDO1 in tumor cells under T cell attack. We show here that IDO1 inhibition leads to an adverse protection of melanoma cells to T cell-derived interferon-gamma (IFNγ). RNA sequencing and ribosome profiling shows that IFNγ shuts down general protein translation, which is reversed by IDO1 inhibition. Impaired translation is accompanied by an amino acid deprivation-dependent stress response driving activating transcription factor-4 (ATF4)high/microphtalmia-associated transcription factor (MITF)low transcriptomic signatures, also in patient melanomas. Single-cell sequencing analysis reveals that MITF downregulation upon immune checkpoint blockade treatment predicts improved patient outcome. Conversely, MITF restoration in cultured melanoma cells causes T cell resistance. These results highlight the critical role of tryptophan and MITF in the melanoma response to T cell-derived IFNγ and uncover an unexpected negative consequence of IDO1 inhibition.

Elevated production of collagen-rich extracellular matrix is a hallmark of cancer-associated fibroblasts (CAFs) and a central driver of cancer aggressiveness. Here we find that proline, a highly abundant amino acid in collagen proteins, is newly synthesized from glutamine in CAFs to make tumour collagen in breast cancer xenografts. PYCR1 is a key enzyme for proline synthesis and highly expressed in the stroma of breast cancer patients and in CAFs. Reducing PYCR1 levels in CAFs is sufficient to reduce tumour collagen production, tumour growth and metastatic spread in vivo and cancer cell proliferation in vitro. Both collagen and glutamine-derived proline synthesis in CAFs are epigenetically upregulated by increased pyruvate dehydrogenase-derived acetyl-CoA levels. PYCR1 is a cancer cell vulnerability and potential target for therapy; therefore, our work provides evidence that targeting PYCR1 may have the additional benefit of halting the production of a pro-tumorigenic extracellular matrix. Our work unveils new roles for CAF metabolism to support pro-tumorigenic collagen production.

Translocations involving the Mixed Lineage Leukemia (MLL) gene generate in-frame fusions of MLL with more than 50 different partner genes (PGs). Common to all MLL translocations is the exchange not only of coding regions, but also of MLL and PG 3'-untranslated regions (3'UTRs). As a result, the MLL-PG fusion is normally highly expressed and considered the main driver of leukemia development, whereas the function of the PG-MLL fusions in leukemic disease is unclear. As 3'UTRs have been recognized as determinant regions for regulation of gene expression, we hypothesized that loss of the MLL 3'UTR could have a role in generating high MLL-PG levels and leukemia development. Here, we first tested the MLL-PG and PG-MLL mRNA levels in different leukemic cells and tumours and uncovered differential expression that indicates strong repression by the MLL-3'UTR. Reporter assays confirmed that the 3'UTR of MLL, but not of its main PGs, harbours a region that imposes a strong gene silencing effect. Gene suppression by the MLL 3'UTR was largely microRNA independent and did not affect mRNA stability, but inhibited transcription. This effect can at least partially be attributed to a tighter interaction of the MLL 3'UTR with RNA polymerase II than PG 3'UTRs, affecting its phosphorylation state. Altogether, our findings indicate that MLL translocations relieve oncogenic MLL-PG fusions from the repressive MLL 3'UTR, contributing to higher activity of these genes and leukaemia development.

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.

CD103+ dendritic cells (DC) are crucial for regulation of intestinal tolerance in humans. However, upon infection of the lamina propria this tolerogenic response is converted to an inflammatory response. Here we show that immunoglobulin A (IgA) immune complexes (IgA-IC), which are present after bacterial infection of the lamina propria, are important for the induction of inflammation by the human CD103+SIRPα+ DC subset. IgA-IC, by recognition through FcαRI, selectively amplify the production of proinflammatory cytokines TNF, IL-1β and IL-23 by human CD103+ DCs. These cells then enhance inflammation by promoting Th17 responses and activating human intestinal innate lymphoid cells 3. Moreover, FcαRI-induced cytokine production is orchestrated via upregulation of cytokine translation and caspase-1 activation, which is dependent on glycolytic reprogramming mediated by kinases Syk, PI3K and TBK1-IKKε. Our data suggest that the formation of IgA-IC in the human intestine provides an environmental cue for the conversion of a tolerogenic to an inflammatory response.

Impairment of mitochondrial protein homeostasis disrupts mitochondrial function and causes human diseases and aging, but the molecular mechanisms of protein synthesis and quality control in mammalian mitochondria are not fully understood. Here we demonstrate in human cells that misincorporation of an arginine analog, canavanine, during mitochondrial protein synthesis, induced aberrant translation products and destabilized the mtDNA-encoded proteome, leading to loss of mitochondrial respiratory chain complexes. Furthermore, in the presence of a high concentration of canavanine, mitoribosome stalling could be demonstrated. The stalling did not, however, occur at arginine codons, but downstream of those codons. In particular, two adjacent arginines induced the most prominent downstream stalling effect, with the distance between the arginine codons and the stalling peak corresponding roughly to the length of the ribosomal exit tunnel. These results suggest that misincorporated canavanine disrupted the proper folding of the hydrophobic nascent polypeptides within the exit tunnel or while being inserted into the inner mitochondrial membrane. The canavanine treatment provides a model system for studying the consequences of mitoribosome stalling and the responses to misfolded proteins exiting the mitochondrial ribosome.

Cell growth and proliferation are tightly connected to ensure that appropriately sized daughter cells are generated following mitosis. Energy stress blocks cell growth and proliferation, a critical response for survival under extreme conditions. Excessive oncogenic stress leads to p53 activation and the induction of senescence, an irreversible state of cell-cycle arrest and a critical component in the suppression of tumorigenesis. Nutrient-sensing and mitogenic cues converge on a major signaling node, which regulates the activity of the mTOR kinase. Although transcriptional responses to energy and oncogenic stresses have been examined by many gene-expression experiments, a global exploration of the modulation of mRNA translation in response to these conditions is lacking.

Oncogene-induced senescence (OIS), provoked in response to oncogenic activation, is considered an important tumor suppressor mechanism. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nt without a protein-coding capacity. Functional studies showed that deregulated lncRNA expression promote tumorigenesis and metastasis and that lncRNAs may exhibit tumor-suppressive and oncogenic function. Here, we first identified lncRNAs that were differentially expressed between senescent and non-senescent human fibroblast cells. Using RNA interference, we performed a loss-function screen targeting the differentially expressed lncRNAs, and identified lncRNA-OIS1 (lncRNA#32, AC008063.3 or ENSG00000233397) as a lncRNA required for OIS. Knockdown of lncRNA-OIS1 triggered bypass of senescence, higher proliferation rate, lower abundance of the cell-cycle inhibitor CDKN1A and high expression of cell-cycle-associated genes. Subcellular inspection of lncRNA-OIS1 indicated nuclear and cytosolic localization in both normal culture conditions as well as following oncogene induction. Interestingly, silencing lncRNA-OIS1 diminished the senescent-associated induction of a nearby gene (Dipeptidyl Peptidase 4, DPP4) with established role in tumor suppression. Intriguingly, similar to lncRNA-OIS1, silencing DPP4 caused senescence bypass, and ectopic expression of DPP4 in lncRNA-OIS1 knockdown cells restored the senescent phenotype. Thus, our data indicate that lncRNA-OIS1 links oncogenic induction and senescence with the activation of the tumor suppressor DPP4.

Immunotherapies targeting cancer-specific neoantigens have revolutionized the treatment of cancer patients. Recent evidence suggests that epigenetic therapies synergize with immunotherapies, mediated by the de-repression of endogenous retroviral element (ERV)-encoded promoters, and the initiation of transcription. Here, we use deep RNA sequencing from cancer cell lines treated with DNA methyltransferase inhibitor (DNMTi) and/or Histone deacetylase inhibitor (HDACi), to assemble a de novo transcriptome and identify several thousand ERV-derived, treatment-induced novel polyadenylated transcripts (TINPATs). Using immunopeptidomics, we demonstrate the human leukocyte antigen (HLA) presentation of 45 spectra-validated treatment-induced neopeptides (t-neopeptides) arising from TINPATs. We illustrate the potential of the identified t-neopeptides to elicit a T-cell response to effectively target cancer cells. We further verify the presence of t-neopeptides in AML patient samples after in vivo treatment with the DNMT inhibitor Decitabine. Our findings highlight the potential of ERV-derived neoantigens in epigenetic and immune therapies.