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Most mammalian genes often feature alternative polyadenylation (APA) sites and hence diverse 3'UTR lengths. Proliferating cells were reported to favor APA sites that result in shorter 3'UTRs. One consequence of such shortening is escape of mRNAs from targeting by microRNAs (miRNAs) whose binding sites are eliminated. Such a mechanism might provide proliferation-related genes with an expression gain during normal or cancerous proliferation. Notably, miRNA sites tend to be more active when located near both ends of the 3'UTR compared to those located more centrally. Accordingly, miRNA sites located near the center of the full 3'UTR might become more active upon 3'UTR shortening. To address this conjecture we performed 3' sequencing to determine the 3' ends of all human UTRs in several cell lines. Remarkably, we found that conserved miRNA binding sites are preferentially enriched immediately upstream to APA sites, and this enrichment is more prominent in pro-differentiation/anti-proliferative genes. Binding sites of the miR17-92 cluster, upregulated in rapidly proliferating cells, are particularly enriched just upstream to APA sites, presumably conferring stronger inhibitory activity upon shortening. Thus 3'UTR shortening appears not only to enable escape from inhibition of growth promoting genes but also to potentiate repression of anti-proliferative genes.

p53 is a transcription factor that governs numerous stress response pathways within the cell. Maintaining the right levels of p53 is crucial for cell survival and proper cellular homeostasis. The tight regulation of p53 involves many cellular components, most notably its major negative regulators Mdm2 and Mdm4, which maintain p53 protein amount and activity in tight check. microRNAs (miRNAs) are small non-coding RNAs that target specific mRNAs to translational arrest and degradation. miRNAs are also key components of the normal p53 pathway, joining forces with Mdm2 and Mdm4 to maintain proper p53 activity. Here we review the current knowledge of miRNAs targeting Mdm2 and Mdm4, and their importance in different tissues and in pathological states such as cancer. In addition, we address the role of Alu sequences-highly abundant retroelements spread throughout the human genome, and their impact on gene regulation via the miRNA machinery. Alus occupy a significant portion of genes' 3'UTR, and as such they have the potential to impact mRNA regulation. Since Alus are primate-specific, they introduce a new regulatory layer into primate genomes. Alus can influence and alter gene regulation, creating primate-specific cancer-preventive regulatory mechanisms to sustain the transition to longer life span in primates. We review the possible influence of Alu sequences on miRNA functionality in general and specifically within the p53 network., (© The Author (2014). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.)

The p53 pathway is pivotal in tumor suppression. Cellular p53 activity is subject to tight regulation, in which the two related proteins Mdm2 and Mdm4 have major roles. The delicate interplay between the levels of Mdm2, Mdm4 and p53 is crucial for maintaining proper cellular homeostasis. microRNAs (miRNAs) are short non-coding RNAs that downregulate the level and translatability of specific target mRNAs. We report that miR-661, a primate-specific miRNA, can target both Mdm2 and Mdm4 mRNA in a cell type-dependent manner. miR-661 interacts with Mdm2 and Mdm4 RNA within living cells. The inhibitory effect of miR-661 is more prevalent on Mdm2 than on Mdm4. Interestingly, the predicted miR-661 targets in both mRNAs reside mainly within Alu elements, suggesting a primate-specific mechanism for regulatory diversification during evolution. Downregulation of Mdm2 and Mdm4 by miR-661 augments p53 activity and inhibits cell cycle progression in p53-proficient cells. Correspondingly, low miR-661 expression correlates with bad outcome in breast cancers that typically express wild-type p53. In contrast, the miR-661 locus tends to be amplified in tumors harboring p53 mutations, and miR-661 promotes migration of cells derived from such tumors. Thus, miR-661 may either suppress or promote cancer aggressiveness, depending on p53 status.

Motivation: The massive spread of repetitive elements in the human genome presents a substantial challenge to the organism, as such elements may accidentally contain seemingly functional motifs. A striking example is offered by the roughly one million copies of Alu repeats in the genome, of which ∼0.5% reside within genes' untranslated regions (UTRs), presenting ∼30 000 novel potential targets for highly conserved microRNAs (miRNAs). Here, we examine the functionality of miRNA targets within Alu elements in 3'UTRs in the human genome., Results: Using a comprehensive dataset of miRNA overexpression assays, we show that mRNAs with miRNA targets within Alus are significantly less responsive to the miRNA effects compared with mRNAs that have the same targets outside Alus. Using Ago2-binding mRNA profiling, we confirm that the miRNA machinery avoids miRNA targets within Alus, as opposed to the highly efficient binding of targets outside Alus. We propose three features that prevent potential miRNA sites within Alus from being recognized by the miRNA machinery: (i) Alu repeats that contain miRNA targets and genuine functional miRNA targets appear to reside in distinct mutually exclusive territories within 3'UTRs; (ii) Alus have tight secondary structure that may limit access to the miRNA machinery; and (iii) A-to-I editing of Alu-derived mRNA sequences may divert miRNA targets. The combination of these features is proposed to allow toleration of Alu insertions into mRNAs. Nonetheless, a subset of miRNA targets within Alus appears not to possess any of the aforementioned features, and thus may represent cases where Alu insertion in the genome has introduced novel functional miRNA targets., Supplementary Information: Supplementary data are available at Bioinformatics online.

Gene expression comprises multiple stages, from transcription to protein degradation. Although much is known about the regulation of each stage separately, an understanding of the regulatory coupling between the different stages is only beginning to emerge. For example, there is a clear crosstalk between translation and transcription, and the localization and stability of an mRNA in the cytoplasm could already be determined during transcription in the nucleus. We review a diversity of mechanisms discovered in recent years that couple the different stages of gene expression. We then speculate on the functional and evolutionary significance of this coupling and suggest certain systems-level functionalities that might be optimized via the various coupling modes. In particular, we hypothesize that coupling is often an economic strategy that allows biological systems to respond robustly and precisely to genetic and environmental perturbations., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

MicroRNAs (miRs) are considered major contributors to the evolution of animal morphological complexity. Multiple bursts of novel miR families were documented throughout animal evolution, yet, their evolutionary origins are not understood. Here, we discuss two alternative genomic sources for novel miR families, namely, transposable elements, which were previously described, and a newly proposed origin: CpG islands. We show that these two origins are evolutionarily distinct and that they correspond to marked differences in several functional and genomic characteristics. Together, our results shed light on the intriguing origin of one of the major constituents of regulatory networks in animals, miRs.

Epidermal growth factor (EGF) stimulates cells by launching gene expression programs that are frequently deregulated in cancer. MicroRNAs, which attenuate gene expression by binding complementary regions in messenger RNAs, are broadly implicated in cancer. Using genome-wide approaches, we showed that EGF stimulation initiates a coordinated transcriptional program of microRNAs and transcription factors. The earliest event involved a decrease in the abundance of a subset of 23 microRNAs. This step permitted rapid induction of oncogenic transcription factors, such as c-FOS, encoded by immediate early genes. In line with roles as suppressors of EGF receptor (EGFR) signaling, we report that the abundance of this early subset of microRNAs is decreased in breast and in brain tumors driven by the EGFR or the closely related HER2. These findings identify specific microRNAs as attenuators of growth factor signaling and oncogenesis.

MicroRNAs (miRNAs) appear to be key players in the maintenance of genomic integrity. Recent evidence implies that cancers often avoid miRNA-mediated regulation, and global repression of miRNAs is associated with increased tumorigenicity. Here we suggest that miRNAs are directly involved in the maintenance of genomic integrity through global repression of transposable elements (TEs), whose expression and transposition are well-documented causes of genomic instability in mammalian somatic tissues. Hence, one outcome of the tumor's ability to avoid miRNA-mediated regulation might be the enhancement of genomic instability and mutability due to derepression of TEs. We outline possible mechanisms underlying TE repression by miRNAs, including post-transcriptional silencing and transcriptional silencing through DNA and histone methylation. This hypothesis calls into consideration the need to study the role of miRNAs and the RNAi machinery in the nucleus, and specifically their impact on the maintenance of genomic integrity in the context of cancer., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Aberrant oncogene activation induces cellular senescence, an irreversible growth arrest that acts as a barrier against tumorigenesis. To identify microRNAs (miRNAs) involved in oncogene-induced senescence, we examined the expression of miRNAs in primary human TIG3 fibroblasts after constitutive activation of B-RAF. Among the regulated miRNAs, both miR-34a and miR-146a were strongly induced during senescence. Although members of the miR-34 family are known to be transcriptionally regulated by p53, we find that miR-34a is regulated independently of p53 during oncogene-induced senescence. Instead, upregulation of miR-34a is mediated by the ETS family transcription factor, ELK1. During senescence, miR-34a targets the important proto-oncogene MYC and our data suggest that miR-34a thereby coordinately controls a set of cell cycle regulators. Hence, in addition to its integration in the p53 pathway, we show that alternative cancer-related pathways regulate miR-34a, emphasising its significance as a tumour suppressor.

miRNAs function as a critical regulatory layer in development, differentiation, and the maintenance of cell fate. Depletion of miRNAs from embryonic stem cells impairs their differentiation capacity. Total elimination of miRNAs leads to premature senescence in normal cells and tissues through activation of the DNA-damage checkpoint, whereas ablation of miRNAs in cancer cell lines results in an opposite effect, enhancing their tumorigenic potential. Here we compile evidence from the literature that point at miRNAs as key players in the maintenance of genomic integrity and proper cell fate. There is an apparent gap between our understanding of the subtle way by which miRNAs modulate protein levels, and their profound impact on cell fate. We propose that examining miRNAs in the context of the regulatory transcriptional and post-transcriptional networks they are embedded in may provide a broader view of their role in controlling cell fate.

Normal cell growth is governed by a complicated biological system, featuring multiple levels of control, often deregulated in cancers. The role of microRNAs (miRNAs) in the control of gene expression is now increasingly appreciated, yet their involvement in controlling cell proliferation is still not well understood. Here we investigated the mammalian cell proliferation control network consisting of transcriptional regulators, E2F and p53, their targets and a family of 15 miRNAs. Indicative of their significance, expression of these miRNAs is downregulated in senescent cells and in breast cancers harboring wild-type p53. These miRNAs are repressed by p53 in an E2F1-mediated manner. Furthermore, we show that these miRNAs silence antiproliferative genes, which themselves are E2F1 targets. Thus, miRNAs and transcriptional regulators appear to cooperate in the framework of a multi-gene transcriptional and post-transcriptional feed-forward loop. Finally, we show that, similarly to p53 inactivation, overexpression of representative miRNAs promotes proliferation and delays senescence, manifesting the detrimental phenotypic consequence of perturbations in this circuit. Taken together, these findings position miRNAs as novel key players in the mammalian cellular proliferation network.

microRNAs (miRs) are small RNAs that regulate gene expression at the posttranscriptional level. It is anticipated that, in combination with transcription factors (TFs), they span a regulatory network that controls thousands of mammalian genes. Here we set out to uncover local and global architectural features of the mammalian miR regulatory network. Using evolutionarily conserved potential binding sites of miRs in human targets, and conserved binding sites of TFs in promoters, we uncovered two regulation networks. The first depicts combinatorial interactions between pairs of miRs with many shared targets. The network reveals several levels of hierarchy, whereby a few miRs interact with many other lowly connected miR partners. We revealed hundreds of "target hubs" genes, each potentially subject to massive regulation by dozens of miRs. Interestingly, many of these target hub genes are transcription regulators and they are often related to various developmental processes. The second network consists of miR-TF pairs that coregulate large sets of common targets. We discovered that the network consists of several recurring motifs. Most notably, in a significant fraction of the miR-TF coregulators the TF appears to regulate the miR, or to be regulated by the miR, forming a diversity of feed-forward loops. Together these findings provide new insights on the architecture of the combined transcriptional-post transcriptional regulatory network.

Purpose: The aim of this study was to investigate the role of p53 in regulating micro-RNA (miRNA) expression due to its function as a transcription factor. In addition, p53 may also affect other cellular mRNA gene expression at the translational level either via its mediated miRNAs or due to its RNA-binding function., Experimental Design: The possible interaction between p53 and miRNAs in regulating gene expression was investigated using human colon cancer HCT-116 (wt-p53) and HCT-116 (null-p53) cell lines. The effect of p53 on the expression of miRNAs was investigated using miRNA expression array and real-time quantitative reverse transcription-PCR analysis., Results: Our investigation indicated that the expression levels of a number of miRNAs were affected by wt-p53. Down-regulation of wt-p53 via small interfering RNA abolished the effect of wt-p53 in regulating miRNAs in HCT-116 (wt-p53) cells. Global sequence analysis revealed that over 46% of the 326 miRNA putative promoters contain potential p53-binding sites, suggesting that some of these miRNAs were potentially regulated directly by wt-p53. In addition, the expression levels of steady-state total mRNAs and actively translated mRNA transcripts were quantified by high-density microarray gene expression analysis. The results indicated that nearly 200 cellular mRNA transcripts were regulated at the posttranscriptional level, and sequence analysis revealed that some of these mRNAs may be potential targets of miRNAs, including translation initiation factor eIF-5A, eIF-4A, and protein phosphatase 1., Conclusion: To the best of our knowledge, this is the first report demonstrating that wt-p53 and miRNAs interact in influencing gene expression and providing insights of how p53 regulates genes at multiple levels via unique mechanisms.