Search

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., The ribosomal protein RPL10 is frequently mutated in T-cell acute lymphoblastic leukemia (T-ALL). Here, the authors show that it promotes proliferation of T-ALL cells by upregulating the serine biosynthesis enzyme phosphoserine phosphatase which in turn modulates serine and glycine metabolism.

Systematic identification of noncoding regulatory elements has, to date, mainly relied on large-scale reporter assays that do not reproduce endogenous conditions. We present two distinct CRISPR-Cas9 genetic screens to identify and characterize functional enhancers in their native context. Our strategy is to target Cas9 to transcription factor binding sites in enhancer regions. We identified several functional enhancer elements and characterized the role of two of them in mediating p53 (TP53) and ERα (ESR1) gene regulation. Moreover, we show that a genomic CRISPR-Cas9 tiling screen can precisely map functional domains within enhancer elements. Our approach expands the utility of CRISPR-Cas9 to elucidate the functions of the noncoding genome.

The health of an organism is intricately linked to its gut microbiome. However, the mechanisms by which the microbiome affect the host gene regulation are still not well established. A new study by Tuorto et al (2018) shows that queuine, a nitrogenous base obtained from the gut microbiota, is used to modify tRNAs and affects cellular behavior. Dietary queuine is required for proper protein synthesis, and its depletion activates cellular stress responses in vitro and in vivo .

Introduction: Regulation of gene expression involves a variety of mechanisms driven by a complex regulatory network of factors. Control of transcription is an important step in gene expression regulation, which integrates the function of cis-acting and trans-acting elements. Among cis-regulatory elements, enhancer RNA (eRNA)-producing domains recently emerged as widespread and potent regulators of transcription and cell fate decision. Thus, manipulation of eRNA levels becomes a novel and appealing avenue for the design of new therapeutic treatments.Areas covered: In this review, we focus on eRNA-producing domains. We describe mechanisms involved in their cell-type specific selection and activation as well as their epigenetic features. In addition, we present their function and the growing evidences of their deregulation in human diseases. Finally, we discuss eRNAs as potential therapeutic targets.Expert opinion: As key factors in the control of transcription, eRNAs appear to possess a great potential for the establishment of new therapy options. However, thorough testing as well as providing the genetic toolbox to target eRNAs will be needed to fully assess the practical and clinical possibilities.

Long non-coding RNA (lncRNA) genes have recently been discovered as key regulators of developmental, physiological, and pathological processes in humans. Recent studies indicate that lncRNAs regulate every step of gene expression, and their aberrant expression can be found in the majority of cancer types. Particularly, lncRNAs were found to function in tumor development and metastasis, which is the major cause of cancer-related death. Thus, exploring key roles of lncRNAs in metastasis is predicted to enhance our knowledge of metastasis, and uncover novel therapeutic targets and biomarkers of this process. In this review, we discuss the molecular mechanisms of lncRNAs in gene expression regulation and their function in metastasis.

Summary 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.

Background— Diabetes mellitus impairs endothelial cell (EC) function and postischemic reparative neovascularization by molecular mechanisms that are not fully understood. microRNAs negatively regulate the expression of target genes mainly by interaction in their 3′ untranslated region. Methods and Results— We found that microRNA-503 (miR-503) expression in ECs is upregulated in culture conditions mimicking diabetes mellitus (high D-glucose) and ischemia-associated starvation (low growth factors). Under normal culture conditions, lentivirus-mediated miR-503–forced expression inhibited EC proliferation, migration, and network formation on Matrigel (comparisons versus lentivirus.GFP control). Conversely, blocking miR-503 activity by either adenovirus-mediated transfer of a miR-503 decoy ( Ad.decoymiR-503 ) or by antimiR-503 (antisense oligonucleotide) improved the functional capacities of ECs cultured under high D-glucose/low growth factors. We identified CCNE1 and cdc25A as direct miR-503 targets which are downregulated by high glucose/low growth factors in ECs. Next, we obtained evidence that miR-503 expression is increased in ischemic limb muscles of streptozotocin-diabetic mice and in ECs enriched from these muscles. Moreover, Ad.decoymiR-503 delivery to the ischemic adductor of diabetic mice corrected diabetes mellitus–induced impairment of postischemic angiogenesis and blood flow recovery. We finally investigated miR-503 and target gene expression in muscular specimens from the amputated ischemic legs of diabetic patients. As controls, calf biopsies of nondiabetic and nonischemic patients undergoing saphenous vein stripping were used. In diabetic muscles, miR-503 expression was remarkably higher, and it inversely correlated with cdc25 protein expression. Plasma miR-503 levels were also elevated in the diabetic individuals. Conclusions— Our data suggest miR-503 as a possible therapeutic target in diabetic patients with critical limb ischemia.

MicroRNAs (miRNAs) are single-stranded, noncoding RNAs that are important in many biological processes1,2. Although the oncogenic and tumour-suppressive functions of several miRNAs have been characterized, the role of miRNAs in mediating tumour metastasis was addressed only recently3 and still remains largely unexplored4,5. To identify potential metastasis-promoting miRNAs, we set up a genetic screen using a non-metastatic, human breast tumour cell line that was transduced with a miRNA-expression library and subjected to a trans-well migration assay. We found that human miR-373 and miR-520c stimulated cancer cell migration and invasion in vitro and in vivo, and that certain cancer cell lines depend on endogenous miR-373 activity to migrate efficiently. Mechanistically, the migration phenotype of miR-373 and miR-520c can be explained by suppression of CD44. We found significant upregulation of miR-373 in clinical breast cancer metastasis samples that correlated inversely with CD44 expression. Taken together, our findings indicate that miRNAs are involved in tumour migration and invasion, and implicate miR-373 and miR-520c as metastasis-promoting miRNAs.

SummaryEndogenous small RNAs (miRNAs) regulate gene expression by mechanisms conserved across metazoans. While the number of verified human miRNAs is still expanding, only few have been functionally annotated. To perform genetic screens for novel functions of miRNAs, we developed a library of vectors expressing the majority of cloned human miRNAs and created corresponding DNA barcode arrays. In a screen for miRNAs that cooperate with oncogenes in cellular transformation, we identified miR-372 and miR-373, each permitting proliferation and tumorigenesis of primary human cells that harbor both oncogenic RAS and active wild-type p53. These miRNAs neutralize p53-mediated CDK inhibition, possibly through direct inhibition of the expression of the tumor-suppressor LATS2. We provide evidence that these miRNAs are potential novel oncogenes participating in the development of human testicular germ cell tumors by numbing the p53 pathway, thus allowing tumorigenic growth in the presence of wild-type p53.

Activating point mutations of the small GTPase Ras are present in about 30% of all human tumors. Constitutively active Ras induces growth factor independent cell proliferation and cell survival. Oncogenic Ras appears to be essential for tumor progression and maintenance. Several therapeutic agents have been developed to inhibit Ras, such as FTIs and antisense oligonucleotides. A new tool for blocking oncogenes in cancer cells has emerged with the discovery that RNA interference can specifically silence expression of endogenous human genes. The therapeutic potential of a RNAi-mediating vector was recently demonstrated by the stable suppression of oncogenic K-Ras in tumor cells.

Most human tumors harbor multiple genetic alterations, including dominant mutant oncogenes. It is often not clear which of these oncogenes are continuously required and which, when inactivated, may inhibit tumorigenesis. Recently, we developed a vector that mediates suppression of gene expression through RNA interference. Here, we use a retroviral version of this vector to specifically and stably inhibit expression of only the oncogenic K-RASV12 allele in human tumor cells. Loss of expression of K-RASV12 leads to loss of anchorage-independent growth and tumorigenicity. These results indicate that viral delivery of small interfering RNAs can be used for tumor-specific gene therapy to reverse the oncogenic phenotype of cancer cells.

Research into the molecular basis of cancer has a central tenet. Cancer arises from genetic alterations that disconnect growth and differentiation signaling pathways from the machinery that regulates cellular proliferation. In multi-cellular eukaryotes, proliferation is regulated by external signals, such as the availability of growth factors and nutrients and by internal signals, such as those sensing cellular integrity. Cellular stress created either by lack of mitogens or damage to cellular components, such as DNA, stimulates responses that enforce temporal or permanent withdrawal from the cell cycle. Although these stress responses stem from different sources and activate distinct pathways, they converge on the same components of the cell cycle machinery in the G1 phase of the cell cycle. This review will highlight and compare aspects of the G1 arrest in response to stress generated either by lack of mitogens or damage to DNA.

Telomeres consist of DNA tandem repeats that recruit the multiprotein complex shelterin to build a chromatin structure that protects chromosome ends. Although cancer formation is linked to alterations in telomere homeostasis, there is little understanding of how shelterin function is limited in cancer cells. Using a small-scale screening approach, we identified miR-155 as a key regulator in breast cancer cell expression of the shelterin component TERF1 (TRF1). miR-155 targeted a conserved sequence motif in the 3′UTR of TRF1, resulting in its translational repression. miR-155 was upregulated commonly in breast cancer specimens, as associated with reduced TRF1 protein expression, metastasis-free survival, and relapse-free survival in estrogen receptor–positive cases. Modulating miR-155 expression in cells altered TRF1 levels and TRF1 abundance at telomeres. Compromising TRF1 expression by elevating miR-155 increased telomere fragility and altered the structure of metaphase chromosomes. In contrast, reducing miR-155 levels improved telomere function and genomic stability. These results implied that miR-155 upregulation antagonizes telomere integrity in breast cancer cells, increasing genomic instability linked to poor clinical outcome in estrogen receptor–positive disease. Our work argued that miRNA-dependent regulation of shelterin function has a clinically significant impact on telomere function, suggesting the existence of “telo-miRNAs” that have an impact on cancer and aging. Cancer Res; 74(15); 4145–56. ©2014 AACR.

The use of alternative polyadenylation sites produces mRNA isoforms with different 3′ untranslated regions. A recent report in Science (Sandberg et al., 2008) suggests that alternative polyadenylation is connected to microRNA-mediated regulation of gene expression as part of a global program for cellular proliferation.

EP is a DNA element found in regulatory regions of viral and cellular genes. While being a key functional element in viral enhancers, EP has no intrinsic enhancer activity but can stimulate or silence transcription in a context-dependent manner. The EP element is bound by RFX1, which belongs to a novel, evolutionarily conserved protein family. In an attempt to decipher the mechanism by which EP regulates transcription, the intrinsic transcriptional activity of RFX1 was investigated. A functional dissection of RFX1, by analysis of deletion mutants and chimeric proteins, identified several regions with independent transcriptional activity. An activation domain containing a glutamine-rich region is found in the N-terminal half of RFX1, while a region with repressor activity overlaps the C-terminal dimerization domain. In RFX1 these activities were mutually neutralized, producing a nearly inactive transcription factor. This neutralization effect was reproduced by fusing RFX1 sequences to a heterologous DNA-binding domain. We propose that relief of self-neutralization may allow RFX1 to act as a dual-function regulator via its activation and repression domains, accounting for the context-dependent activity of EP.

The 3' end of most protein-coding genes and long non-coding RNAs is cleaved and polyadenylated. Recent discoveries have revealed that a large proportion of these genes contains more than one polyadenylation site. Therefore, alternative polyadenylation (APA) is a widespread phenomenon, generating mRNAs with alternative 3' ends. APA contributes to the complexity of the transcriptome by generating isoforms that differ either in their coding sequence or in their 3' untranslated regions (UTRs), thereby potentially regulating the function, stability, localization and translation efficiency of target RNAs. Here, we review our current understanding of the polyadenylation process and the latest progress in the identification of APA events, mechanisms that regulate poly(A) site selection, and biological processes and diseases resulting from APA.

Heterozygous germline mutations in breast cancer 1 (BRCA1) strongly predispose women to breast cancer. BRCA1 plays an important role in DNA double-strand break (DSB) repair via homologous recombination (HR), which is important for tumor suppression. Although BRCA1-deficient cells are highly sensitive to treatment with DSB-inducing agents through their HR deficiency (HRD), BRCA1-associated tumors display heterogeneous responses to platinum drugs and poly(ADP-ribose) polymerase (PARP) inhibitors in clinical trials. It is unclear whether all pathogenic BRCA1 mutations have similar effects on the response to therapy. Here, we have investigated mammary tumorigenesis and therapy sensitivity in mice carrying the Brca1(185stop) and Brca1(5382stop) alleles, which respectively mimic the 2 most common BRCA1 founder mutations, BRCA1(185delAG) and BRCA1(5382insC). Both the Brca1(185stop) and Brca1(5382stop) mutations predisposed animals to mammary tumors, but Brca1(185stop) tumors responded markedly worse to HRD-targeted therapy than did Brca1(5382stop) tumors. Mice expressing Brca1(185stop) mutations also developed therapy resistance more rapidly than did mice expressing Brca1(5382stop). We determined that both murine Brca1(185stop) tumors and human BRCA1(185delAG) breast cancer cells expressed a really interesting new gene domain-less (RING-less) BRCA1 protein that mediated resistance to HRD-targeted therapies. Together, these results suggest that expression of RING-less BRCA1 may serve as a marker to predict poor response to DSB-inducing therapy in human cancer patients.

DNA sequences, that control expression of the spliced leader (SL) RNA gene in the parasitic protozoan Leishmania amazonensis, were mapped by block substitution mutagenesis. In the absence of a functional in vitro system for transcription, no promoter elements have yet been identified in this organism. We therefore developed an alternative in vivo approach, in which the SL RNA gene was tagged and then subjected to a series of linker scanning mutations. Each tagged and mutated SL RNA construct was introduced into parasite cells via the pX transfection vector, and was examined for expression of the tagged SL RNA followed by characterization of its transcriptional start site. The replacement of a critical DNA element was expected to prevent expression of the tagged SL RNA. We found that the putative SL RNA promoter is complex and includes two elements: one is located upstream to the coding region, between positions -30 to -70; and the other is located between -10 to +10, and includes transcribed sequences. In addition to the functional relationship between the SL RNA and vertebrate U snRNAs, we found structural similarities in their regulatory elements, which may possibly indicate a common evolutionary ancestry for these molecules.

Transforming growth factor (TGF)-β is one of the main fibrogenic cytokines that drives the pathophysiology of progressive renal scarring. MicroRNAs (miRNAs) are endogenous non-coding RNAs that post-transcriptionally regulate gene expression. We examined the role of TGF-β-induced expression of miR-21, miRNAs in cell culture models and miRNA expression in relevant models of renal disease. In vitro, TGF-β changed expression of miR-21, miR-214, and miR-145 in rat mesangial cells (CRL-2753) and miR-214, miR-21, miR-30c, miR-200b, and miR-200c during induction of epithelial-mesenchymal transition in rat tubular epithelial cells (NRK52E). miR-214 expression was robustly modulated in both cell types, whereas in tubular epithelial cells miR-21 was increased and miR-200b and miR-200c were decreased by 58% and 48%, respectively, in response to TGF-β. TGF-β receptor-1 was found to be a target of miR-200b/c and was down-regulated after overexpression of miR-200c. To assess the differential expression of these miRNAs in vivo, we used the anti-Thy1.1 mesangial glomerulonephritis model and the unilateral ureteral obstruction model in which TGF-β plays a role and also a genetic model of hypertension, the stroke-prone spontaneously hypertensive rat with and without salt loading. The expressions of miR-214 and miR-21 were significantly increased in all in vivo models, showing a possible miRNA signature of renal damage despite differing causes.

When stem cells and multipotent progenitors differentiate, they undergo fate restriction, enabling a single fate and blocking differentiation along alternative routes. We herein present a mechanism whereby such unequivocal commitment is achieved, based on microRNA (miRNA)-dependent repression of an alternative cell fate. We show that the commitment of monocyte RAW264.7 progenitors to active macrophage differentiation involves rapid up-regulation of miR-155 expression, which leads to the suppression of the alternative pathway, namely RANK ligand-induced osteoclastogenesis, by repressing the expression of MITF, a transcription factor essential for osteoclast differentiation. A temporal asymmetry, whereby miR-155 expression precedes and overrides the activation of the osteoclast transcriptional program, provides the means for coherent macrophage differentiation, even in the presence of osteoclastogenic signals. Based on these findings, we propose that miRNA may provide a general mechanism for the unequivocal commitment underlying stem cell differentiation.

Several biological functions in mammals are regulated in a circadian fashion. The molecular mechanisms orchestrating these circadian rhythms have been unravelled. The biological clock, with its core transcriptional unit Bmal1/CLOCK, is composed of several self-sustaining feedback loops. In this study, we describe another mechanism impinging on the core components of the circadian clock. Using a forward genetic screen, we identified the miR-192/194 cluster as a potent inhibitor of the entire Period gene family. In accordance, the exogenous expression of miR-192/194 leads to an altered circadian rhythm. Thus, our results have uncovered a new mechanism for the control of the circadian clock at the post-transcriptional level.

Activated oncogenic signaling is central to the development of nearly all forms of cancer, including the most common class of primary brain tumor, glioma. Research over the last two decades has revealed the particular importance of the Akt pathway, and its molecular antagonist PTEN (phosphatase and tensin homolog), in the process of gliomagenesis. Recent studies have also demonstrated that microRNAs (miRNAs) may be responsible for the modulation of cancer-implicated genes in tumors. Here we report the identification miR-26a as a direct regulator of PTEN expression. We also show that miR-26a is frequently amplified at the DNA level in human glioma, most often in association with monoallelic PTEN loss. Finally, we demonstrate that miR-26a-mediated PTEN repression in a murine glioma model both enhances de novo tumor formation and precludes loss of heterozygosity and the PTEN locus. Our results document a new epigenetic mechanism for PTEN regulation in glioma and further highlight dysregulation of Akt signaling as crucial to the development of these tumors.

Melanoma is the most aggressive skin cancer once metastasis begins; therefore, it is important to characterize the molecular players involved in melanoma dissemination. The chemokine receptor CXCR4 and the membrane-bound metalloproteinase MT1-MMP are expressed on melanoma cells and represent candidate molecules for the control of metastasis. Using human melanoma transfectants that either overexpress or silence CXCR4 or MT1-MMP, or that have a combination of overexpression and interference of these proteins, we show that CXCR4 and MT1-MMP coordinate their activities at different steps along melanoma cell metastasis into the lungs. Results from in vivo xenograft mouse models of melanoma lung colonization and mice survival and short-term, homing nested polymerase chain reaction experiments from lung samples indicated that CXCR4 is required at early phases of melanoma cell arrival in the lungs. In contrast, MT1-MMP is not needed for these initial steps but promotes subsequent invasion and dissemination of the tumor with CXCR4. Investigation of potential cross talk between CXCR4 and MT1-MMP revealed that MT1-MMP accumulates intracellularly after melanoma cell stimulation with the CXCR4 ligand CXCL12, and that this process involves the activation of the Rac-Erk1/2 pathway. Subsequent to cell contact with specific basement membrane proteins, MT1-MMP redistributes to the cell membrane in a phosphatidylinositol 3-kinase-dependent manner. These results suggest that combination therapies that target CXCR4 and MT1-MMP should improve the limitations of the current therapies for metastatic melanoma. Copyright © American Society for Investigative Pathology.

Elucidation of regulatory roles played by microRNAs (miRs) in various biological networks is one of the greatest challenges of present molecular and computational biology. The integrated analysis of gene expression data and 3′-UTR sequences holds great promise for being an effective means to systematically delineate active miRs in different biological processes. Applying such an integrated analysis, we uncovered a striking relationship between 3′-UTR AU content and gene response in numerous microarray datasets. We show that this relationship is secondary to a general bias that links gene response and probe AU content and reflects the fact that in the majority of current arrays probes are selected from target transcript 3′-UTRs. Therefore, removal of this bias, which is in order in any analysis of microarray datasets, is of crucial importance when integrating expression data and 3′-UTR sequences to identify regulatory elements embedded in this region. We developed visualization and normalization schemes for the detection and removal of such AU biases and demonstrate that their application to microarray data significantly enhances the computational identification of active miRs. Our results substantiate that, after removal of AU biases, mRNA expression profiles contain ample information which allows in silico detection of miRs that are active in physiological conditions., Author Summary MicroRNAs are a novel class of genes that encodes for short RNA molecules recognized to play key roles in the regulation of many biological networks. MicroRNAs, predicted to collectively target more than 30% of all human protein-coding genes, suppress gene expression by binding to regulatory elements usually embedded in the 3′-UTRs of their target mRNAs. Despite intensive efforts in recent years, biological functions carried out by microRNAs have been characterized for only a small number of these genes, making elucidation of their roles one of the greatest challenges of biology today. Bioinformatics analyses can significantly help meet this challenge. In particular, the integrated analysis of microarray mRNA expression data and 3′-UTR sequences holds great promise for systematic dissection of regulatory networks controlled by microRNAs. Applying such integrated analysis to numerous microarray datasets, we disclosed a major technical bias that hampers the identification of active microRNAs from mRNA expression profiles. We developed visualization and normalization schemes for detection and removal of the bias and demonstrate that their application to microarray data significantly enhances the identification of active microRNAs. Given the broad use of microarrays and the ever-growing interest in microRNAs, we anticipate that the methods we introduced will be widely adopted.

SummaryMicroRNAs (miRNAs) are inhibitors of gene expression capable of controlling processes in normal development and cancer. In mammals, miRNAs use a seed sequence of 6–8 nucleotides (nt) to associate with 3′ untranslated regions (3′UTRs) of mRNAs and inhibit their expression. Intriguingly, occasionally not only the miRNA-targeting site but also sequences in its vicinity are highly conserved throughout evolution. We therefore hypothesized that conserved regions in mRNAs may serve as docking platforms for modulators of miRNA activity. Here we demonstrate that the expression of dead end 1 (Dnd1), an evolutionary conserved RNA-binding protein (RBP), counteracts the function of several miRNAs in human cells and in primordial germ cells of zebrafish by binding mRNAs and prohibiting miRNAs from associating with their target sites. These effects of Dnd1 are mediated through uridine-rich regions present in the miRNA-targeted mRNAs. Thus, our data unravel a novel role of Dnd1 in protecting certain mRNAs from miRNA-mediated repression.

MicroRNAs (miRNAs) are small RNA molecules controlling messenger RNA (mRNA) and protein abundance. Since their discovery, research has been aimed at identifying the functions and target genes of miRNAs. A number of computer algorithms have been developed capable of predicting putative targets for any given miRNA. However, they might predict many false-positive targets and on top of that some true targets could be missed. This reflects the incomplete knowledge we still have concerning the rules governing true and effective miRNA-mRNA interactions. To experimentally identify miRNA-target genes, we have recently developed a genetic approach and employed it on p27(Kip1), a hapo-insufficient tumor suppressor and cell cycle inhibitor. Here we review the difficulties interpreting the data from available computer algorithms, and critically address the pros and cons of our genetic screening method. Additionally, we focus on the different ways in which p27 is managed, argue how miRNAs could be involved in the regulation of p27 in both normal and malignant conditions, and discuss possible use of this knowledge for cancer therapy.

MicroRNAs (miRNAs) are potent post-transcriptional regulators of protein coding genes. Patterns of misexpression of miRNAs in cancer suggest key functions of miRNAs in tumorigenesis. However, current bioinformatics tools do not entirely support the identification and characterization of the mode of action of such miRNAs. Here, we used a novel functional genetic approach and identified miR-221 and miR-222 (miR-221&222) as potent regulators of p27(Kip1), a cell cycle inhibitor and tumor suppressor. Using miRNA inhibitors, we demonstrate that certain cancer cell lines require high activity of miR-221&222 to maintain low p27(Kip1) levels and continuous proliferation. Interestingly, high levels of miR-221&222 appear in glioblastomas and correlate with low levels of p27(Kip1) protein. Thus, deregulated expression of miR-221&222 promotes cancerous growth by inhibiting the expression of p27(Kip1).

With the human genome fully sequenced, the need to obtain functional knowledge of many genes became apparent. Amongst them is a large set of recently discovered genes with powerful promise: the microRNA family. Accumulating data assigned functions in a wide array of biological processes to some miRNAs. Here we review the main approaches used to identify and validate miRNAs and describe ways to discover their functions. As potential effectors of many cellular pathways, mis-expression of miRNAs has been implicated in human cancer.

Activation of tumor suppressor p53 in response to genotoxic stress imposes cellular growth arrest or apoptosis. We identified Cdc6, a licensing factor of the prereplication complex, as a novel target of the p53 pathway. We show that activation of p53 by DNA damage results in enhanced Cdc6 destruction by the anaphase-promoting complex. This destruction is triggered by inhibition of CDK2-mediated CDC6 phosphorylation at serine 54. Conversely, suppression of p53 expression results in stabilization of Cdc6. We demonstrate that loss of p53 results in more replicating cells, an effect that can be reversed by reducing Cdc6 protein levels. Collectively, our data suggest that initiation of DNA replication is regulated by p53 through Cdc6 protein stability.

Long noncoding RNAs (lncRNAs) are a novel class of regulators that play crucial roles in development and disease. Here we highlight the findings by Huang and colleagues that a regulatory lncRNA (treRNA) acts as a scaffold for a new ribonucleic protein complex that inhibits translation of E‐cadherin and promotes cell invasion. This work underscores the potential importance of lncRNAs in cancer metastasis. Genome‐wide transcriptome sequencing efforts have led to the discovery of thousands of long noncoding RNAs (lncRNAs). LncRNAs are polyadenylated transcripts, typically more than 200 base pairs in length, that show polymerase II transcription initiation and elongation marks (Ulitsky and Bartel, 2013). Little is known about the biological roles of lncRNAs, but several potential mechanisms for the action of nuclear and cytoplasmic lncRNAs have been proposed. Nuclear lncRNAs mainly regulate the transcription of target genes by recruiting chromatin‐modifying complexes to specific regions (Rinn and Chang, 2012), while cytoplasmic lncRNAs may interact with other RNAs, serve as molecular decoys for microRNAs and RNA‐binding proteins, or function as cytoplasmic scaffolds of RNA–protein complexes. Interestingly, cytoplasmic lncRNAs may also interact with target mRNAs and protein complexes …

Rev-Erbα and Rev-Erbβ are nuclear receptors that regulate the expression of genes involved in the control of circadian rhythm1,2, metabolism3,4, and inflammatory responses5. Rev-Erbs function as transcriptional repressors by recruiting NCoR/HDAC3 co-repressor complexes to Rev-Erb response elements in enhancers and promoters of target genes6-8, but the molecular basis for cell-specific programs of repression is not known. Here, we present evidence that in macrophages, Rev-Erbs regulate target gene expression by inhibiting the functions of distal enhancers that are selected by macrophage lineage-determining factors, thereby establishing a macrophage-specific program of repression. Remarkably, the repressive functions of Rev-Erbs are associated with their ability to inhibit the transcription of enhancer-derived RNAs (eRNAs). Furthermore, targeted degradation of eRNAs at two enhancers subject to negative regulation by Rev-Erbs resulted in reduced expression of nearby mRNAs, implying a direct role of these eRNAs in enhancer function. By precisely defining eRNA start sites using a method that quantifies nascent 5′ ends (5′-GRO-Seq), we show that transfer of full enhancer activity to a target promoter requires both the sequences mediating transcription factor binding and the specific sequences encoding the eRNA transcript. These studies provide evidence for direct roles of eRNAs in contributing to enhancer functions and suggest that Rev-Erbs act to suppress gene expression at a distance by repressing eRNA transcription.

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control mechanism that eliminates unwanted proteins from the endoplasmic reticulum (ER) through a ubiquitin-dependent proteasomal degradation pathway. gp78 is a previously described ER membrane-anchored ubiquitin ligase (E3) involved in ubiquitination of ER proteins. AAA ATPase (ATPase associated with various cellular activities) p97/valosin-containing protein (VCP) subsequently dislodges the ubiquitinated proteins from the ER and chaperones them to the cytosol, where they undergo proteasomal degradation. We now report that gp78 physically interacts with p97/VCP and enhances p97/VCP-polyubiquitin association. The enhanced association correlates with decreases in ER stress-induced accumulation of polyubiquitinated proteins. This effect is abolished when the p97/VCP-interacting domain of gp78 is removed. Further, using ERAD substrate CD3delta, gp78 consistently enhances p97/VCP-CD3delta binding and facilitates CD3delta degradation. Moreover, inhibition of endogenous gp78 expression by RNA interference markedly increases the levels of total polyubiquitinated proteins, including CD3delta, and abrogates VCP-CD3delta interactions. The gp78 mutant with deletion of its p97/VCP-interacting domain fails to increase CD3delta degradation and leads to accumulation of polyubiquitinated CD3delta, suggesting a failure in delivering ubiquitinated CD3delta for degradation. These data suggest that gp78-p97/VCP interaction may represent one way of coupling ubiquitination with retrotranslocation and degradation of ERAD substrates.

Genetic evidence is mounting that survivin plays a crucial role in mitosis, but its exact role in human cell division remains elusive. We show that mammalian cells lacking survivin are unable to align their chromosomes, fail to recruit Aurora B to kinetochores and become polyploid at a very high frequency. Survivin-depleted cells enter mitosis with normal kinetics, but are delayed in prometaphase in a BubR1/Mad2-dependent fashion. Nonetheless, these cells exit mitosis prior to completion of chromosome congression and without sister chromatid segregation, indicating that the spindle assembly checkpoint is not fully functional. Indeed, in survivin-depleted cells, BubR1 and Mad2 are prematurely displaced from kinetochores, yet no tension is generated at kinetochores. Importantly, these cells fail to respond to drugs that prevent tension, but do arrest in mitosis after depolymerization of the mitotic spindle. This demonstrates that survivin is not required for initial checkpoint activation, or for sustained checkpoint activation by loss of microtubules. However, stable association of BubR1 to kinetochores and sustained checkpoint signalling in response to lack of tension crucially depend on survivin.

In the past year, the genetic research of mammalian cells in vitro has gained the advantages of RNA interference (RNAi), a process found in worms and plants by which double stranded RNAs mediate selective gene inactivation through mRNA destruction. Recently, two papers have shown that genes could be suppressed in vivo in mammals by RNAi, which has potential implications for both therapeutics and research.

Introduction of double-stranded RNAs into cells can suppress gene expression by mechanisms such as mRNA degradation or inhibition of translation. In mammalian cells, these two responses intersect, a feature that was recently used for the development of novel tools for stable and specific gene inactivation. These new tools were successfully applied to inhibit tumorigenicity and viral replication. Future development of appropriate in vivo delivery systems may make this technology useful for disease therapy.

Background mRNA levels in cells are determined by the relative rates of RNA production and degradation. Yet, to date, most analyses of gene expression profiles were focused on mechanisms which regulate transcription, while the role of mRNA stability in modulating transcriptional networks was to a large extent overlooked. In particular, kinetic waves in transcriptional responses are usually interpreted as resulting from sequential activation of transcription factors. Results In this study, we examined on a global scale the role of mRNA stability in shaping the kinetics of gene response. Analyzing numerous expression datasets we revealed a striking global anti-correlation between rapidity of induction and mRNA stability, fitting the prediction of a kinetic mathematical model. In contrast, the relationship between kinetics and stability was less significant when gene suppression was analyzed. Frequently, mRNAs that are stable under standard conditions were very rapidly down-regulated following stimulation. Such effect cannot be explained even by a complete shut-off of transcription, and therefore indicates intense modulation of RNA stability. Conclusion Taken together, our results demonstrate the key role of mRNA stability in determining induction kinetics in mammalian transcriptional networks.

Microsomal triglyceride transfer protein (MTP) is an endoplasmic reticulum (ER)–resident lipid transfer protein involved in the biosynthesis and lipid loading of apolipoprotein B. MTP was recently suggested to directly regulate the biosynthesis of the MHC I–like, lipid antigen presenting molecule CD1d, based on coprecipitation experiments and lipid loading assays. However, we found that the major impact of MTP deficiency occurred distal to the ER and Golgi compartments. Thus, although the rates of CD1d biosynthesis, glycosylation maturation, and internalization from the cell surface were preserved, the late but essential stage of recycling from lysosome to plasma membrane was profoundly impaired. Likewise, functional experiments indicated defects of CD1d-mediated lipid presentation in the lysosome but not in the secretory pathway. These intriguing findings suggest a novel, unexpected role of MTP at a late stage of CD1d trafficking in the lysosomal compartment.

Tumour growth and metabolic adaptation may restrict the availability of certain amino acids for protein synthesis. It has recently been shown that certain types of cancer cells depend on glycine, glutamine, leucine and serine metabolism to proliferate and survive. In addition, successful therapies using L-asparaginase-induced asparagine deprivation have been developed for acute lymphoblastic leukaemia. However, a tailored detection system for measuring restrictive amino acids in each tumour is currently not available. Here we harness ribosome profiling for sensing restrictive amino acids, and develop diricore, a procedure for differential ribosome measurements of codon reading. We first demonstrate the functionality and constraints of diricore using metabolic inhibitors and nutrient deprivation assays. Notably, treatment with L-asparaginase elicited both specific diricore signals at asparagine codons and high levels of asparagine synthetase (ASNS). We then applied diricore to kidney cancer and discover signals indicating restrictive proline. As for asparagine, this observation was linked to high levels of PYCR1, a key enzyme in proline production, suggesting a compensatory mechanism allowing tumour expansion. Indeed, PYCR1 is induced by shortage of proline precursors, and its suppression attenuated kidney cancer cell proliferation when proline was limiting. High PYCR1 is frequently observed in invasive breast carcinoma. In an in vivo model system of this tumour, we also uncover signals indicating restrictive proline. We further show that CRISPR-mediated knockout of PYCR1 impedes tumorigenic growth in this system. Thus, diricore has the potential to reveal unknown amino acid deficiencies, vulnerabilities that can be used to target key metabolic pathways for cancer treatment.

Previously, we demonstrated the establishment of synthetic lethality screening in cultured somatic human cells, or mouse embryo fibroblasts (MEFs), for chemicals or mutant genes synergistically lethal with a mutated gene of interest. Here, we show in MEFs that the usage of RNA interference-based genetic suppressor elements encoding short hairpin RNAs (shRNAs) enables for genetic synthetic lethality screening at a frequency much higher than that achieved before with short truncated sense and antisense RNAs. These findings open up the possibility of using in mammalian cells genome-wide shRNA libraries for genetic synthetic lethality screening at the multi-gene level.

DNA damage causes stabilization of p53, leading to G1 arrest through induction of p21cip1. As this process requires transcription, several hours are needed to exert this response. We show that DNA damage causes an immediate and p53-independent G1 arrest, caused by rapid proteolysis of cyclin D1. Degradation is mediated through a previously unrecognized destruction box in cyclin D1 and leads to a release of p21cip1 from CDK4 to inhibit CDK2. Interference with cyclin D1 degradation prevents initiation of G1 arrest and renders cells more susceptible to DNA damage, indicating that cyclin D1 degradation is an essential component of the cellular response to genotoxic stress. Thus, induction of G1 arrest in response to DNA damage is minimally a two step process: a fast p53-independent initiation of G1 arrest mediated by cyclin D1 proteolysis and a slower maintenance of arrest resulting from increased p53 stability.