Post-transcriptional Regulatory Networks Research Articles

BDNF and the maturation of posttranscriptional regulatory networks in human SH-SY5Y neuroblast differentiation.

The SH-SY5Y culture system is a convenient neuronal model with the potential to elaborate human/primate-specific transcription networks and pathways related to human cognitive disorders. While this system allows for the exploration of specialized features in the human genome, there is still significant debate about how this model should be implemented, and its appropriateness for answering complex functional questions related to human neural architecture. In view of these questions we sought to characterize the posttranscriptional regulatory structure of the two-stage ATRA differentiation, BDNF maturation protocol proposed by Encinas et al. (2000) using integrative whole-genome gene and microRNA (miRNA) expression analysis. We report that ATRA-BDNF induced significant increases in expression of key synaptic genes, brain-specific miRNA and miRNA biogenesis machinery, and in AChE activity, compared with ATRA alone. Functional annotation clustering associated BDNF more significantly with neuronal terms, and with synaptic terms not found in ATRA-only clusters. While our results support use of SH-SY5Y as a neuronal model, we advocate considered selection of the differentiation agent/s relative to the system being modeled.

Harnessing Natural Sequence Variation to Dissect Posttranscriptional Regulatory Networks in Yeast

Understanding how genomic variation influences phenotypic variation through the molecular networks of the cell is one of the central challenges of biology. Transcriptional regulation has received much attention, but equally important is the posttranscriptional regulation of mRNA stability. Here we applied a systems genetics approach to dissect posttranscriptional regulatory networks in the budding yeast Saccharomyces cerevisiae. Quantitative sequence-to-affinity models were built from high-throughput in vivo RNA binding protein (RBP) binding data for 15 yeast RBPs. Integration of these models with genome-wide mRNA expression data allowed us to estimate protein-level RBP regulatory activity for individual segregants from a genetic cross between two yeast strains. Treating these activities as a quantitative trait, we mapped trans-acting loci (activity quantitative trait loci, or aQTLs) that act via posttranscriptional regulation of transcript stability. We predicted and experimentally confirmed that a coding polymorphism at the IRA2 locus modulates Puf4p activity. Our results also indicate that Puf3p activity is modulated by distinct loci, depending on whether it acts via the 5′ or the 3′ untranslated region of its target mRNAs. Together, our results validate a general strategy for dissecting the connectivity between posttranscriptional regulators and their upstream signaling pathways.

Evolutionary patterns of Escherichia coli small RNAs and their regulatory interactions.

Most bacterial small RNAs (sRNAs) are post-transcriptional regulators of gene expression, exerting their regulatory function by base-pairing with their target mRNAs. While it has become evident that sRNAs play central regulatory roles in the cell, little is known about their evolution and the evolution of their regulatory interactions. Here we used the prokaryotic phylogenetic tree to reconstruct the evolutionary history of Escherichia coli sRNAs and their binding sites on target mRNAs. We discovered that sRNAs currently present in E. coli mainly accumulated inside the Enterobacteriales order, succeeding the appearance of other types of noncoding RNAs and concurrently with the evolution of a variant of the Hfq protein exhibiting a longer C-terminal region. Our analysis of the evolutionary ages of sRNA-mRNA interactions revealed that while all sRNAs were evolutionarily older than most of their known binding sites on mRNA targets, for quite a few sRNAs there was at least one binding site that coappeared with or preceded them. It is conceivable that the establishment of these first interactions forced selective pressure on the sRNAs, after which additional targets were acquired by fitting a binding site to the active region of the sRNA. This conjecture is supported by the appearance of many binding sites on target mRNAs only after the sRNA gain, despite the prior presence of the target gene in ancestral genomes. Our results suggest a selective mechanism that maintained the sRNAs across the phylogenetic tree, and shed light on the evolution of E. coli post-transcriptional regulatory network.

Alteration of miRNA activity via context-specific modifications of Argonaute proteins.

miRNAs are enclosed within Argonaute (Ago) proteins, the downstream effectors of small RNA-mediated gene silencing. Because miRNAs mediate extensive networks of post-transcriptional control, cells have evolved multiple strategies to control their activity with precision. A growing theme of recent years is how post-translational modifications of Ago proteins, such as prolyl hydroxylation, phosphorylation, ubiquitination, and poly-ADP-ribosylation, alter miRNA activity at global or specific levels. In this review, we discuss recent advances in Ago modifications in mammalian cells and emphasize how such alterations modulate small RNA function to coordinate appropriate downstream cellular responses. These findings provide a framework to understand how Ago protein modifications are linked to reorganization of post-transcriptional regulatory networks, enabling dynamic responses to diverse external stimuli and changing environmental conditions.

Interactions between Distant ceRNAs in Regulatory Networks

Competing endogenous RNAs (ceRNAs) were recently introduced as RNA transcripts that affect each other’s expression level through competition for their microRNA (miRNA) coregulators. This stems from the bidirectional effects between miRNAs and their target RNAs, where a change in the expression level of one target affects the level of the miRNA regulator, which in turn affects the level of other targets. By the same logic, miRNAs that share targets compete over binding to their common targets and therefore also exhibit ceRNA-like behavior. Taken together, perturbation effects could propagate in the posttranscriptional regulatory network through a path of coregulated targets and miRNAs that share targets, suggesting the existence of distant ceRNAs. Here we study the prevalence of distant ceRNAs and their effect in cellular networks. Analyzing the network of miRNA-target interactions deciphered experimentally in HEK293 cells, we show that it is a dense, intertwined network, suggesting that many nodes can act as distant ceRNAs of one another. Indeed, using gene expression data from a perturbation experiment, we demonstrate small, yet statistically significant, changes in gene expression caused by distant ceRNAs in that network. We further characterize the magnitude of the propagated perturbation effect and the parameters affecting it by mathematical modeling and simulations. Our results show that the magnitude of the effect depends on the generation and degradation rates of involved miRNAs and targets, their interaction rates, the distance between the ceRNAs and the topology of the network. Although demonstrated for a miRNA-mRNA regulatory network, our results offer what to our knowledge is a new view on various posttranscriptional cellular networks, expanding the concept of ceRNAs and implying possible distant cross talk within the network, with consequences for the interpretation of indirect effects of gene perturbation.

Widespread context dependency of microRNA-mediated regulation

Gene expression is regulated in a context-dependent, cell-type-specific manner. Condition-specific transcription is dependent on the presence of transcription factors (TFs) that can activate or inhibit its target genes (global context). Additional factors, such as chromatin structure, histone, or DNA modifications, also influence the activity of individual target genes (individual context). The role of the global and individual context for post-transcriptional regulation has not systematically been investigated on a large scale and is poorly understood. Here we show that global and individual context dependency is a pervasive feature of microRNA-mediated regulation. Our comprehensive and highly consistent data set from several high-throughput technologies (PAR-CLIP, RIP-chip, 4sU-tagging, and SILAC) provides strong evidence that context-dependent microRNA target sites (CDTS) are as frequent and functionally relevant as constitutive target sites (CTS). Furthermore, we found the global context to be insufficient to explain the CDTS, and that flanking sequence motifs provide individual context that is an equally important factor. Our results demonstrate that, similar to TF-mediated regulation, global and individual context dependency are prevalent in microRNA-mediated gene regulation, implying a much more complex post-transcriptional regulatory network than is currently known. The necessary tools to unravel post-transcriptional regulations and mechanisms need to be much more involved, and much more data will be needed for particular cell types and cellular conditions in order to understand microRNA-mediated regulation and the context-dependent post-transcriptional regulatory network.

Post-transcriptional regulatory network of epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions

Epithelial-to-mesenchymal transition (EMT) and its reverse process, mesenchymal-to-epithelial transition (MET), play important roles in embryogenesis, stem cell biology, and cancer progression. EMT can be regulated by many signaling pathways and regulatory transcriptional networks. Furthermore, post-transcriptional regulatory networks regulate EMT; these networks include the long non-coding RNA (lncRNA) and microRNA (miRNA) families. Specifically, the miR-200 family, miR-101, miR-506, and several lncRNAs have been found to regulate EMT. Recent studies have illustrated that several lncRNAs are overexpressed in various cancers and that they can promote tumor metastasis by inducing EMT. MiRNA controls EMT by regulating EMT transcription factors or other EMT regulators, suggesting that lncRNAs and miRNA are novel therapeutic targets for the treatment of cancer. Further efforts have shown that non-coding-mediated EMT regulation is closely associated with epigenetic regulation through promoter methylation (e.g., miR-200 or miR-506) and protein regulation (e.g., SET8 via miR-502). The formation of gene fusions has also been found to promote EMT in prostate cancer. In this review, we discuss the post-transcriptional regulatory network that is involved in EMT and MET and how targeting EMT and MET may provide effective therapeutics for human disease.

Integrated Analysis of Differential miRNA and mRNA Expression Profiles in Human Radioresistant and Radiosensitive Nasopharyngeal Carcinoma Cells

BackgroundThe purpose of this study was to identify miRNAs and genes involved in nasopharyngeal carcinoma (NPC) radioresistance, and explore the underlying mechanisms in the development of radioresistance.MethodsWe used microarrays to compare the differences of both miRNA and mRNA expression profiles in the radioresistant NPC CNE2-IR and radiosensitive NPC CNE2 cells, applied qRT-PCR to confirm the reliability of microarray data, adopted databases prediction and anticorrelated analysis of miRNA and mRNA expression to identify the miRNA target genes, and employed bioinformatics tools to examine the functions and pathways in which miRNA target genes are involved, and construct a miRNA-target gene regulatory network. We further investigated the roles of miRNA-23a and its target gene IL-8 in the NPC radioresistance.ResultsThe main findings were fourfold: (1) fifteen differential miRNAs and 372 differential mRNAs were identified, and the reliability of microarray data was validated for randomly selected eight miRNAs and nine genes; (2) 174 miRNA target were identified, and most of their functions and regulating pathways were related to tumor therapeutic resistance; (3) a posttranscriptional regulatory network including 375 miRNA-target gene pairs was constructed, in which the ten genes were coregulated by the six miRNAs; (4) IL-8 was a direct target of miRNA-23a, the expression levels of IL-8 were elevated in the radioresistant NPC tissues and showed inverse correlation with miRNA-23a expression, and genetic upregulation of miRNA-23a and antibody neutralization of secretory IL-8 could reduce NPC cells radioresistance.ConclusionsWe identified fifteen differential miRNAs and 372 differential mRNAs in the radioresistant NPC cells, constructed a posttranscriptional regulatory network including 375 miRNA-target gene pairs, discovered the ten target genes coregulated by the six miRNAs, and validated that downregulated miRNA-23a was involved in NPC radioresistance through directly targeting IL-8. Our data form a basis for further investigating the mechanisms of NPC radioresistance.

Trypanosome MKT1 and the RNA-binding protein ZC3H11: interactions and potential roles in post-transcriptional regulatory networks

The trypanosome zinc finger protein ZC3H11 binds to AU-rich elements in mRNAs. It is essential for survival of the mammalian-infective bloodstream form, where it stabilizes several mRNAs including some encoding chaperones, and is also required for stabilization of chaperone mRNAs during the heat-shock response in the vector-infective procyclic form. When ZC3H11 was artificially ‘tethered’ to a reporter mRNA in bloodstream forms it increased reporter expression. We here show that ZC3H11 interacts with trypanosome MKT1 and PBP1, and that domains required for both interactions are necessary for function in the bloodstream-form tethering assay. PBP1 interacts with MKT1, LSM12 and poly(A) binding protein, and localizes to granules during parasite starvation. All of these proteins are essential for bloodstream-form trypanosome survival and increase gene expression in the tethering assay. MKT1 is cytosolic and polysome associated. Using a yeast two-hybrid screen and tandem affinity purification we found that trypanosome MKT1 interacts with multiple RNA-binding proteins and other potential RNA regulators, placing it at the centre of a post-transcriptional regulatory network. A consensus interaction sequence, H(E/D/N/Q)PY, was identified. Recruitment of MKT1-containing regulatory complexes to mRNAs via sequence-specific mRNA-binding proteins could thus control several different post-transcriptional regulons.

Posttranscriptional regulatory networks: from expression profiling to integrative analysis of mRNA and microRNA data.

Protein coding RNAs are posttranscriptionally regulated by microRNAs, a class of small noncoding RNAs. Insights in messenger RNA (mRNA) and microRNA (miRNA) regulatory interactions facilitate the understanding of fine-tuning of gene expression and might allow better estimation of protein synthesis. However, in silico predictions of mRNA-microRNA interactions do not take into account the specific transcriptomic status of the biological system and are biased by false positives. One possible solution to predict rather reliable mRNA-miRNA relations in the specific biological context is to integrate real mRNA and miRNA transcriptomic data as well as in silico target predictions. This chapter addresses the workflow and methods one can apply for expression profiling and the integrative analysis of mRNA and miRNA data, as well as how to analyze and interpret results, and how to build up models of posttranscriptional regulatory networks.

Competition Between Micrornas and its Role in Post-Transcriptional Regulation

miRNAs play a fundamental role in post-transcriptional regulation. It was found that the regulation is affected by competition between miRNAs for limiting amounts of shared components, required for their biogenesis and processing. More specifically, they compete for Argonaute (Ago), the catalytic component of the RNA silencing complex. Aiming to better understand the effects of competition in this context, we introduced a mathematical model and studied the change in the expression level of target genes under a variety of conditions (1). Due to the stoichiometric nature of the miRNA-mRNA interaction, the regulation is bi-directional, namely the transcripts affect the activity of their miRNA regulators. This implies that there should be cross-talk between mRNAs that share a miRNA regulator as well as between miRNAs that share common targets, which is mediated by the shared regulators and targets, respectively. We have analyzed a recently published, experimentally-determined human miRNA-mRNA interactome and found that it is a dense, intertwined network, suggesting that the effect of an expression change in a single mRNA or miRNA could propagate along paths in the network, affecting non-adjacent regulators and targets. Through computational modeling we determined the parameters governing the magnitude of this propagation and support the model by analysis of experimental perturbation data (2). Our results offer a new view of post-transcriptional regulatory networks, expanding the concept of ceRNAs (competing endogenous RNAs), implying significant cross-talk within the network with far-reaching consequences for perturbation effects. (1) A. Loinger, Y. Shemla, I. Simon, H. Margalit and O. Biham, Competition between small RNAs: a quantitative view, Biophysical Journal 102, 1712 (2012). (2) M. Nitzan, A. Steiman-Shimony, Y. Altuvia, O. Biham and H. Margalit, Long range ceRNA relay in regulatory networks, submitted for publication (2013).

C. elegans germline stem cells and their niche

C. elegans germline stem cells are a particularly simple system for analysis of stem cell regulation. Their well-defined mesenchymal niche consists of a single cell, the Distal Tip Cell, which uses Notch signaling to maintain a pool of germline stem cells. Downstream of Notch signaling a post-transcriptional regulatory network dictates self-renewal or differentiation. The major self-renewal hub of that network is FBF, a conserved RNA-binding protein and conserved stem cell regulator. FBF represses mRNAs encoding key regulators of germline differentiation (entry into the meiotic cell cycle, sperm or oocyte specification) as well as established regulators of somatic differentiation. Transcriptional and post-transcriptional mechanisms also control totipotency in the C. elegans germline. The key C. elegans GSC regulators are conserved broadly, making this system a paradigm for stem cell regulation.

HuR-Regulated mRNAs Associated with Nuclear hnRNP A1-RNP Complexes

Post-transcriptional regulatory networks are dependent on the interplay of many RNA-binding proteins having a major role in mRNA processing events in mammals. We have been interested in the concerted action of the two RNA-binding proteins hnRNP A1 and HuR, both stable components of immunoselected hnRNP complexes and having a major nuclear localization. Specifically, we present here the application of the RNA-immunoprecipitation (RIP)-Chip technology to identify a population of nuclear transcripts associated with hnRNP A1-RNPs as isolated from the nuclear extract of either HuR WT or HuR-depleted (KO) mouse embryonic fibroblast (MEF) cells. The outcome of this analysis was a list of target genes regulated via HuR for their association (either increased or reduced) with the nuclear hnRNP A1-RNP complexes. Real time PCR analysis was applied to validate a selected number of nuclear mRNA transcripts, as well as to identify pre-spliced transcripts (in addition to their mature mRNA counterpart) within the isolated nuclear hnRNP A1-RNPs. The differentially enriched mRNAs were found to belong to GO categories relevant to biological processes anticipated for hnRNP A1 and HuR (such as transport, transcription, translation, apoptosis and cell cycle) indicating their concerted function in mRNA metabolism.

Study on Key Genes and Regulatory Networks Associated with Osteoporosis by Microarray Technology

DNA microarray data of patients with osteoporosis were compared with that of healthy people to identify key genes and thus disclose the underlying regulatory network. Microarray dataset GSE35958 was downloaded from the Gene Expression Omnibus database, including five gene chips from patients with primary osteoporosis and four from age-matching nonosteoporosis controls. Raw data were preprocessed and differentially expressed genes (DEGs) were identified by the t-test. Then, function and pathway annotations were given by gene ontology (GO) and KEGG. The regulatory network for the DEGs was established from the aspects of transcription factors and microRNAs (miRNAs). The regulators of the miRNAs were also predicted by the MATCH algorithm. A total of 274 DEGs were obtained with 47 significantly over-represented GO terms and 2 KEGG pathways. Transcriptional and post-transcriptional regulatory networks were established for the DEGs. Moreover, upstream regulators of the miRNAs were also obtained. A range of genes, which might be implicated in the development of osteoporosis were obtained in the present study. Our findings are of possible benefit for the understanding of the unsolved regulatory mechanisms, and future clinical diagnosis as well as treatment.

Phased, Secondary, Small Interfering RNAs in Posttranscriptional Regulatory Networks

Plant genomes are the source of large numbers of small RNAs, generated via a variety of genetically separable pathways. Several of these pathways converge in the production of phased, secondary, small interfering RNAs (phasiRNAs), originally designated as trans-acting small interfering RNAs or tasiRNAs. PhasiRNA biogenesis requires the involvement of microRNAs as well as the cellular machinery for the production of siRNAs. PhasiRNAs in Arabidopsis thaliana have been well described for their ability to function in trans to suppress target transcript levels. Plant genomic data from an expanding set of species have demonstrated that Arabidopsis is relatively sparing in its use of phasiRNAs, while other genomes contain hundreds or even thousands of phasiRNA-generating loci. In the dicots, targets of those phasiRNAs include several large or conserved families of genes, such as those encoding disease resistance proteins or transcription factors. Suppression of nucleotide-binding, leucine-rich repeat (NB-LRR) disease resistance genes by small RNAs is particularly unusual because of a high level of redundancy. In this review, we discuss plant phasiRNAs and the possible mechanistic significance of phasiRNA-based regulation of the NB-LRRs.

Widespread and extensive lengthening of 3' UTRs in the mammalian brain.

Remarkable advances in techniques for gene expression profiling have radically changed our knowledge of the transcriptome. Recently, the mammalian brain was reported to express many long intergenic noncoding (lincRNAs) from loci downstream from protein-coding genes. Our experimental tests failed to validate specific accumulation of lincRNA transcripts, and instead revealed strongly distal 3′ UTRs generated by alternative cleavage and polyadenylation (APA). With this perspective in mind, we analyzed deep mammalian RNA-seq data using conservative criteria, and identified 2035 mouse and 1847 human genes that utilize substantially distal novel 3′ UTRs. Each of these extends at least 500 bases past the most distal 3′ termini available in Ensembl v65, and collectively they add 6.6 Mb and 5.1 Mb to the mRNA space of mouse and human, respectively. Extensive Northern analyses validated stable accumulation of distal APA isoforms, including transcripts bearing exceptionally long 3′ UTRs (many >10 kb and some >18 kb in length). The Northern data further illustrate that the extensions we annotated were not due to unprocessed transcriptional run-off events. Global tissue comparisons revealed that APA events yielding these extensions were most prevalent in the mouse and human brain. Finally, these extensions collectively contain thousands of conserved miRNA binding sites, and these are strongly enriched for many well-studied neural miRNAs. Altogether, these new 3′ UTR annotations greatly expand the scope of post-transcriptional regulatory networks in mammals, and have particular impact on the central nervous system.

Iron Regulatory Proteins Control a Mucosal Block to Intestinal Iron Absorption

Mammalian iron metabolism is regulated systemically by the hormone hepcidin and cellularly by iron regulatory proteins (IRPs) that orchestrate a posttranscriptional regulatory network. Through ligand-inducible genetic ablation of both IRPs in the gut epithelium of adult mice, we demonstrate that IRP deficiency impairs iron absorption and promotes mucosal iron retention via a ferritin-mediated "mucosal block." We show that IRP deficiency does not interfere with intestinal sensing of body iron loading and erythropoietic iron need, but rather alters the basal expression of the iron-absorption machinery. IRPs thus secure sufficient iron transport across absorptive enterocytes by restricting the ferritin "mucosal block" and define a basal set point for iron absorption upon which IRP-independent systemic regulatory inputs are overlaid.

ELAV proteins along evolution: Back to the nucleus?

The complex interplay of post-transcriptional regulatory mechanisms mediated by RNA-binding proteins (RBP) at different steps of RNA metabolism is pivotal for the development of the nervous system and the maintenance of adult brain activities. In this review, we will focus on the highly conserved ELAV gene family encoding for neuronal-specific RBPs which are necessary for proper neuronal differentiation and important for synaptic plasticity process. In the evolution from Drosophila to man, ELAV proteins seem to have changed their biological functions in relation to their different subcellular localization. While in Drosophila, they are localized in the nuclear compartment of neuronal cells and regulate splicing and polyadenylation, in mammals, the neuronal ELAV proteins are mainly present in the cytoplasm where they participate in regulating mRNA target stability, translation and transport into neurites. However, recent data indicate that the mammalian ELAV RBPs also have nuclear activities, similarly to their fly counterpart, being them able to continuously shuttle between the cytoplasm and the nucleus. Here, we will review and comment on all the biological functions associated with neuronal ELAV proteins along evolution and will show that the post-transcriptional regulatory network mediated by these RBPs in the brain is highly complex and only at an initial stage of being fully understood. This article is part of a Special Issue entitled 'RNA and splicing regulation in neurodegeneration'.

An integrated approach to characterize transcription factor and microRNA regulatory networks involved in Schwann cell response to peripheral nerve injury

BackgroundThe regenerative response of Schwann cells after peripheral nerve injury is a critical process directly related to the pathophysiology of a number of neurodegenerative diseases. This SC injury response is dependent on an intricate gene regulatory program coordinated by a number of transcription factors and microRNAs, but the interactions among them remain largely unknown. Uncovering the transcriptional and post-transcriptional regulatory networks governing the Schwann cell injury response is a key step towards a better understanding of Schwann cell biology and may help develop novel therapies for related diseases. Performing such comprehensive network analysis requires systematic bioinformatics methods to integrate multiple genomic datasets.ResultsIn this study we present a computational pipeline to infer transcription factor and microRNA regulatory networks. Our approach combined mRNA and microRNA expression profiling data, ChIP-Seq data of transcription factors, and computational transcription factor and microRNA target prediction. Using mRNA and microRNA expression data collected in a Schwann cell injury model, we constructed a regulatory network and studied regulatory pathways involved in Schwann cell response to injury. Furthermore, we analyzed network motifs and obtained insights on cooperative regulation of transcription factors and microRNAs in Schwann cell injury recovery.ConclusionsThis work demonstrates a systematic method for gene regulatory network inference that may be used to gain new information on gene regulation by transcription factors and microRNAs.

A Compendium of Caenorhabditis elegans RNA Binding Proteins Predicts Extensive Regulation at Multiple Levels

Gene expression is regulated at multiple levels, including transcription and translation, as well as mRNA and protein stability. Although systems-level functions of transcription factors and microRNAs are rapidly being characterized, few studies have focused on the posttranscriptional gene regulation by RNA binding proteins (RBPs). RBPs are important to many aspects of gene regulation. Thus, it is essential to know which genes encode RBPs, which RBPs regulate which gene(s), and how RBP genes are themselves regulated. Here we provide a comprehensive compendium of RBPs from the nematode Caenorhabditis elegans (wRBP1.0). We predict that as many as 887 (4.4%) of C. elegans genes may encode RBPs ~250 of which likely function in a gene-specific manner. In addition, we find that RBPs, and most notably gene-specific RBPs, are themselves enriched for binding and modification by regulatory proteins, indicating the potential for extensive regulation of RBPs at many different levels. wRBP1.0 will provide a significant contribution toward the comprehensive delineation of posttranscriptional regulatory networks and will provide a resource for further studies regulation by RBPs.