Andrew M. Thomson

A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes.

We present rna22, a method for identifying microRNA binding sites and their corresponding heteroduplexes. Rna22 does not rely upon cross-species conservation, is resilient to noise, and, unlike previous methods, it first finds putative microRNA binding sites in the sequence of interest, then identifies the targeting microRNA. Computationally, we show that rna22 identifies most of the currently known heteroduplexes. Experimentally, with luciferase assays, we demonstrate average repressions of 30% or more for 168 of 226 tested targets. The analysis suggests that some microRNAs may have as many as a few thousand targets, and that between 74% and 92% of the gene transcripts in four model genomes are likely under microRNA control through their untranslated and amino acid coding regions. We also extended the methods key idea to a low-error microRNA-precursor-discovery scheme; our studies suggest that the number of microRNA precursors in mammalian genomes likely ranges in the tens of thousands.

MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation

MicroRNAs (miRNAs) are short RNAs that direct messenger RNA degradation or disrupt mRNA translation in a sequence-dependent manner. For more than a decade, attempts to study the interaction of miRNAs with their targets were confined to the 3′ untranslated regions of mRNAs, fuelling an underlying assumption that these regions are the principal recipients of miRNA activity. Here we focus on the mouse Nanog, Oct4 (also known as Pou5f1) and Sox2 genes and demonstrate the existence of many naturally occurring miRNA targets in their amino acid coding sequence (CDS). Some of the mouse targets analysed do not contain the miRNA seed, whereas others span exon–exon junctions or are not conserved in the human and rhesus genomes. miR-134, miR-296 and miR-470, upregulated on retinoic-acid-induced differentiation of mouse embryonic stem cells, target the CDS of each transcription factor in various combinations, leading to transcriptional and morphological changes characteristic of differentiating mouse embryonic stem cells, and resulting in a new phenotype. Silent mutations at the predicted targets abolish miRNA activity, prevent the downregulation of the corresponding genes and delay the induced phenotype. Our findings demonstrate the abundance of CDS-located miRNA targets, some of which can be species-specific, and support an augmented model whereby animal miRNAs exercise their control on mRNAs through targets that can reside beyond the 3′ untranslated region.

MicroRNA-134 modulates the differentiation of mouse embryonic stem cells, where it causes post-transcriptional attenuation of Nanog and LRH1.

Hundreds of microRNAs (miRNAs) are expressed in mammalian cells, where they aid in modulating gene expression by mediating mRNA transcript cleavage and/or regulation of translation rate. Functional studies to date have demonstrated that several of these miRNAs are important during development. However, the role of miRNAs in the regulation of stem cell growth and differentiation is not well understood. We show herein that microRNA (miR)‐134 levels are maximally elevated at day 4 after retinoic acid‐induced differentiation or day 2 after N2B27‐induced differentiation of mouse embryonic stem cells (mESCs), but this change is not observed during embryoid body differentiation. The elevation of miR‐134 levels alone in mESCs enhances differentiation toward ectodermal lineages, an effect that is blocked by a miR‐134 antagonist. The promotion of mESC differentiation by miR‐134 is due, in part, to its direct translational attenuation of Nanog and LRH1, both of which are known positive regulators of Oct4/POU5F1 and mESC growth. Together, the data demonstrate that miR‐134 alone can enhance the differentiation of mESCs to ectodermal lineages and establish a functional role for miR‐134 in modulating mESC differentiation through its potential to target and regulate multiple mRNAs.

IRON-REGULATORY PROTEINS, IRON-RESPONSIVE ELEMENTS AND FERRITIN MRNA TRANSLATION

Iron plays a central role in the metabolism of all cells. This is evident by its major contribution to many diverse functions, such as DNA replication, bacterial pathogenicity, photosynthesis, oxidative stress control and cell proliferation. In mammalian systems, control of intracellular iron homeostasis is largely due to posttranscriptional regulation of binding by iron-regulatory RNA-binding proteins (IRPs) to iron-responsive elements (IREs) within ferritin and transferrin receptor (TfR) mRNAs. the TfR transports iron into cells and the iron is subsequently stored within ferritin. IRP binding is under tight control so that it responds to changes in intracellular iron requirements in a coordinate manner by differentially regulating ferritin mRNA translational efficiency and TfR mRNA stability. Several different stimuli, as well as intracellular iron levels and oxidative stress, are capable of regulating these RNA-protein interactions. In this mini-review, we shall concentrate on the mechanisms underlying modulation of the interaction of IRPs and the ferritin IRE and its role in regulating ferritin gene expression.

mRNA Stability and the Control of Gene Expression: Implications for Human Disease

Regulation of gene expression is essential for the homeostasis of an organism, playing a pivotal role in cellular proliferation, differentiation, and response to specific stimuli. Multiple studies over the last two decades have demonstrated that the modulation of mRNA stability plays an important role in regulating gene expression. The stability of a given mRNA transcript is determined by the presence of sequences within an mRNA known as cis-elements, which can be bound by trans-acting RNA-binding proteins to inhibit or enhance mRNA decay. These cis-trans interactions are subject to a control by a wide variety of factors including hypoxia, hormones, and cytokines. In this review, we describe mRNA biosynthesis and degradation, and detail the cis-elements and RNA-binding proteins known to affect mRNA turnover. We present recent examples in which dysregulation of mRNA stability has been associated with human diseases including cancer, inflammatory disease, and Alzheimers disease.

Thyrotropin-releasing Hormone and Epidermal Growth Factor Regulate Iron-regulatory Protein Binding in Pituitary Cells via Protein Kinase C-dependent and -independent Signaling Pathways

Intracellular iron homeostasis is regulated, in part, by interactions between iron-regulatory proteins (IRP1 and IRP2) and iron-responsive elements (IREs) in ferritin and transferrin receptor mRNAs. In addition to iron, cellular oxidative stress induced by H2O2, nitric oxide, and hypoxia, and hormonal activation by thyroid hormone and erythropoeitin have each been shown to regulate IRP binding to IREs. Hormonal signals, in particular mediated through protein kinase C (PKC), play a central role in the modulation of IRP/IRE interactions since phorbol esters were shown to activate IRP binding (Eisenstein, R. S., Tuazon, P. T., Schalinske, K. L., Anderson, S. A., and Traugh, J. A. (1993) J. Biol. Chem. 268, 27363–27370). In pituitary thyrotrophs (TtT97), we found that thyrotropin releasing hormone (TRH) and epidermal growth factor (EGF) increased IRP binding to a ferritin IRE, dependent on PKC and mitogen-activated protein kinase (MAPK) activity. In contrast, TRH and EGF decreased IRP binding in pituitary lactotrophs (GH3), despite activation of PKC and MAPK. IRP1 and IRP2 levels remained constant and IRP2 binding was predominant throughout. TRH and EGF markedly decreased IRP binding in MAPK kinase inhibitor-treated GH3 cells, whereas, they increased IRP binding in phosphatase inhibitor-treated GH3 cells. IRE-dependent CAT reporter translational expression closely reflected IRP binding to the ferritin IRE in both GH3 and TtT97 cells. Interestingly, ferritin protein levels were regulated similarly by TRH in both cell lines. These data link two different cell receptor systems to common signaling pathways that regulate IRP binding and ferritin expression. Remarkably, for TRH and EGF, these effects may be PKC-dependent or -independent determined by the cell type.

The Acute Box cis-Element in Human Heavy Ferritin mRNA 5′-Untranslated Region Is a Unique Translation Enhancer That Binds Poly(C)-binding Proteins

Intracellular levels of the light (L) and heavy (H) ferritin subunits are regulated by iron at the level of message translation via a modulated interaction between the iron regulatory proteins (IRP1 and IRP2) and a 5′-untranslated region. Iron-responsive element (IRE). Here we show that iron and interleukin-1β (IL-1β) act synergistically to increase H- and L-ferritin expression in hepatoma cells. A GC-rich cis-element, the acute box (AB), located downstream of the IRE in the H-ferritin mRNA 5′-untranslated region, conferred a substantial increase in basal and IL-1β-stimulated translation over a similar time course to the induction of endogenous ferritin. A scrambled version of the AB was unresponsive to IL-1. Targeted mutation of the AB altered translation; reverse orientation and a deletion of the AB abolished the wild-type stem-loop structure and abrogated translational enhancement, whereas a conservative structural mutant had little effect. Labeled AB transcripts formed specific complexes with hepatoma cell extracts that contained the poly(C)-binding proteins, iso-αCP1 and -αCP2, which have well defined roles as translation regulators. Iron influx increased the association of αCP1 with ferritin mRNA and decreased the αCP2-ferritin mRNA interaction, whereas IL-1β reduced the association of αCP1 and αCP2 with H-ferritin mRNA. In summary, the H-ferritin mRNA AB is a key cis-acting translation enhancer that augments H-subunit expression in Hep3B and HepG2 hepatoma cells, in concert with the IRE. The regulated association of H-ferritin mRNA with the poly(C)-binding proteins suggests a novel role for these proteins in ferritin translation and iron homeostasis in human liver.

Cholesterol esters regulate apoB48 secretion in CaCo2 cells

In this study, we investigated the effect of atorvastatin, an HMG-CoA reductase inhibitor and CL277082, an ACAT inhibitor, on apolipoprotein B48 synthesis, degradation and secretion in transformed human intestinal enterocytes (CaCo2 cells). Cells were incubated with atorvastatin or CL277082 in the absence or presence of sterol containing media and pulsed with [S35]-methionine and chased with unlabelled methionine. Concomitantly, the effect of atorvastatin and CL277082 on the relative amount of apoB48 protein in cells and media was also quantified by western blotting using an apoB antibody and enhanced chemiluminescence. Suppression of cholesterol synthesis with atorvastatin did not attenuate the production or secretion of apoB48 from CaCo2 cells under basal conditions. On the other hand, suppression of cholesterol biosynthesis with atorvastatin under stimulatory conditions accelerated the degradation of apoB48 in cells without affecting its synthesis or secretion. There was no effect of exogenous sterols on apoB48 secretion. Taken together, neither endogenous nor exogenous cholesterol appears to acutely modulate apoB48 secretion from intestinal cells. In contrast, inhibition of cholesterol esterification with ACAT inhibitor significantly attenuated apoB48 secretion under basal and stimulatory conditions by a mechanism which enhanced apoB48 degradation. Collectively, our results suggest that in CaCo2 cells, newly synthesized cholesterol ester may be an immediate regulator apoB48 secretion.

Polyunsaturated fatty acids downregulate the low density lipoprotein receptor of human HepG2 cells

The aim of the study was to investigate the effect of different fatty acids on the low density lipoprotein (LDL) receptor of cultured human liver HepG2 cells. Previous studies investigating the effect of fatty acids on LDL expression have reported conflicting findings and are limited to measurements of LDL receptor binding activity. Therefore, this study is unique in that the relative effects of different fatty acids on the LDL receptor were investigated at three different stages of expression: 1) functional cellular LDL binding activity, 2) amount of LDL receptor protein and 3) LDL receptor mRNA level. The HepG2 cells were incubated for 24 hr with either 100 &mgr;M palmitic, oleic, linoleic or eicosapentaenoic acid (EPA). The measurement of LDL receptor binding activity was with colloidal gold-LDL conjugates, cellular LDL receptor protein was by western blotting and LDL receptor mRNA by Southern blotting of reverse-transcribed, polymerase chain reaction-amplified cDNA. The LDL receptor binding activity, protein and mRNA levels decreased as the degree of unsaturation of the fatty acids increased (palmitic acid greater-than-or-equal oleic acid > linoleic acid > EPA) and the inverse relationship held whether or not cholesterol was included in the culture media. The relative differences were very similar for the three stages of expression indicating that modulation of the LDL receptor by the fatty acids occurred at the level of gene transcription. The increased susceptibility to oxidation of the polyunsaturated fatty acids was unlikely to be a factor in the effect because EPA and linoleic acid (250 &mgr;M) still downregulated the LDL receptor in the presence of the antioxidant vitamin E (50 &mgr;M). In conclusion, the polyunsaturates, linoleic acid and EPA, effectively downregulated the LDL receptor of HepG2 cells compared to palmitic acid. The effects of these fatty acids were observed at the level of LDL receptor binding activity, protein and mRNA, strongly suggesting that the fatty acid effects were at the level of gene transcription.

Divergent LIN28-mRNA associations result in translational suppression upon the initiation of differentiation

LIN28 function is fundamental to the activity and behavior of human embryonic stem cells (hESCs) and induced pluripotent stem cells. Its main roles in these cell types are the regulation of translational efficiency and let-7 miRNA maturation. However, LIN28-associated mRNA cargo shifting and resultant regulation of translational efficiency upon the initiation of differentiation remain unknown. An RNA-immunoprecipitation and microarray analysis protocol, eRIP, that has high specificity and sensitivity was developed to test endogenous LIN28-associated mRNA cargo shifting. A combined eRIP and polysome analysis of early stage differentiation of hESCs with two distinct differentiation cues revealed close similarities between the dynamics of LIN28 association and translational modulation of genes involved in the Wnt signaling, cell cycle, RNA metabolism and proteasomal pathways. Our data demonstrate that change in translational efficiency is a major contributor to early stages of differentiation of hESCs, in which LIN28 plays a central role. This implies that eRIP analysis of LIN28-associated RNA cargoes may be used for rapid functional quality control of pluripotent stem cells under manufacture for therapeutic applications.