Mathematical modeling of combinatorial regulation suggests that apparent positive regulation of targets by miRNA could be an artifact resulting from competition for mRNA

MicroRNAs bind to and regulate the abundance and activity of target messenger RNA through sequestration, enhanced degradation, and suppression of translation. Although miRNA have a predominantly negative effect on the target protein concentration, several reports have demonstrated a positive effect of miRNA, i.e., increase in target protein concentration on miRNA overexpression and decrease in target concentration on miRNA repression. miRNA–target pair-specific effects such as protection of mRNA degradation owing to miRNA binding can explain some of these effects. However, considering such pairs in isolation might be an oversimplification of the RNA biology, as it is known that one miRNA interacts with several targets, and conversely target mRNA are subject to regulation by several miRNAs. We formulate a mathematical model of this combinatorial regulation of targets by multiple miRNA. Through mathematical analysis and numerical simulations of this model, we show that miRNA that individually have a negative effect on their targets may exhibit an apparently positive net effect when the concentration of one miRNA is experimentally perturbed by repression/overexpression in such a multi-miRNA multitarget situation. We show that this apparent unexpected effect is due to competition and will not be observed when miRNA interact noncompetitively with the target mRNA. This result suggests that some of the observed unusual positive effects of miRNA may be due to the combinatorial complexity of the system rather than due to any inherently unusual positive effect of the miRNA on its target.     

Comparative analysis of human intronless proteins

The availability of the complete genome sequences of Homo sapiens together with those of taxonomically diverse organisms provides an opportunity to carry out cross-species comparison. Comparisons of protein sequences from different organisms are significant source of information as these could help in answering questions regarding the fraction of proteins that are shared by humans and organisms representing the three domains of life, viz., archaea, bacteria, and eukaryota. In the present study, a comparative analysis of the proteins encoded by intronless genes in humans was undertaken. We identified 1125 human intronless proteins that are solely present in eukaryotic lineage. More than two-thirds of these eukaryotic specific proteins appear to be mammalia specific while a small fraction of proteins are conserved in bilateria and coelomata, indicating that diversification of these proteins occurred after the divergence of the major lineages of the eukaryotic crown group. A large fraction of mammalia specific proteins are enriched in proteins responsible for transport and binding, cell envelope, and housekeeping function particularly translation. Another 228 intronless proteins are observed that do not exhibit homology to any of the proteins in the database. The distribution of human intronless proteins suggests that lineage specific expansion is one of the most important sources of organizational diversity in crown-group eukaryotes. The presence of these eukaryotic as well as human specific intronless proteins provides the foundation for rapid analysis of some of the basic processes involved in human genome.     

A spectroscopic analysis of the interaction between the human regulatory proteins RACK1 and Ki-1/57

Ki-1/57 is a 57-kDa cytoplasmic and nuclear protein associated with protein kinase activity and is hyper-phosphorylated on Ser/Thr residues upon cellular activation. In previous studies we identified the receptor of activated kinase-1 (RACK1), a signaling adaptor protein that binds activated PKC, as a protein that interacts with Ki-1/57. Here we demonstrate that the far-UV circular dichroism spectrum of the WD repeat-containing RACK1 protein shows an unusual positive ellipticity at 229 nm, which in other proteins of the WD family has been attributed to surface tryptophans that are quenchable by N-bromosuccinimide (NBS). As well as NBS, in vitro binding of 6xHis-Ki-1/57(122-413) and 6xHis-Ki-1/57(264-413) can also quench the positive ellipticity of the RACK1 spectrum. We generated a model of RACK1 by homology modeling using a G protein beta subunit as template. Our model suggests the family-typical seven-bladed beta-propeller, with an aromatic cluster around the central tunnel that contains four Trp residues (17, 83, 150, 170), which are likely involved in the interaction with Ki-1/57.     

A molecular dynamics study of a miRNA:mRNA interaction

In this paper we present a methodology to evaluate the binding free energy of a miRNA:mRNA complex through molecular dynamics (MD)–thermodynamic integration (TI) simulations. We applied our method to the Caenorhabditis elegans let-7 miRNA:lin-41 mRNA complex—a validated miRNA:mRNA interaction—in order to estimate the energetic stability of the structure. To make the miRNA:mRNA simulation possible and realistic, the methodology introduces specific solutions to overcome some of the general challenges of nucleic acid simulations and binding free energy computations that have been discussed widely in many previous research reports. The main features of the proposed methodology are: (1) positioning of the restraints imposed on the simulations in order to guarantee complex stability; (2) optimal sampling of the phase space to achieve satisfactory accuracy in the binding energy value; (3) determination of a suitable trade-off between computational costs and accuracy of binding free energy computation by the assessment of the scalability characteristics of the parallel simulations required for the TI. The experiments carried out demonstrate that MD simulations are a viable strategy for the study of miRNA binding characteristics, opening the way to the development of new computational target prediction methods based on three-dimensional structure information.     

Functional analysis of KSRP interaction with the AU-rich element of interleukin-8 and identification of inflammatory mRNA targets

mRNA stability is a major determinant of inflammatory gene expression. Rapid degradation of interleukin-8 (IL-8) mRNA is imposed by a bipartite AU-rich element (ARE) in the 3′ untranslated region (R. Winzen et al., Mol. Cell. Biol. 24:4835-4847, 2004). Small interfering RNA-mediated knockdown of the ARE-binding protein KSRP resulted in stabilization of IL-8 mRNA or of a β-globin reporter mRNA containing the IL-8 ARE. Rapid deadenylation was impaired, indicating a crucial role for KSRP in this step of mRNA degradation. The two IL-8 ARE domains both contribute to interaction with KSRP, corresponding to the importance of both domains for rapid degradation. Exposure to the inflammatory cytokine IL-1 has been shown to stabilize IL-8 mRNA through p38 mitogen-activated protein (MAP) kinase and MK2. IL-1 treatment impaired the interaction of KSRP with the IL-8 ARE in a manner dependent on p38 MAP kinase but apparently independent of MK2. Instead, evidence that TTP, a target of MK2, can also destabilize the IL-8 ARE reporter mRNA is presented. In a comprehensive approach to identify mRNAs controlled by KSRP, two criteria were evaluated by microarray analysis of (i) association of mRNAs with KSRP in pulldown assays and (ii) increased amounts in KSRP knockdown cells. According to both criteria, a group of 100 mRNAs is controlled by KSRP, many of which are unstable and encode proteins involved in inflammation. These results indicate that KSRP functions as a limiting factor in inflammatory gene expression.     

Differential representation of liver proteins in obese human subjects suggests novel biomarkers and promising targets for drug development in obesity

The proteome of liver biopsies from human obese (O) subjects has been compared to those of nonobese (NO) subjects using two-dimensional gel electrophoresis (2-DE). Differentially represented proteins were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS)-based peptide mass fingerprinting (PMF) and nanoflow-liquid chromatography coupled to electrospray-tandem mass spectrometry (nLC-ESI-MS/MS). Overall, 61 gene products common to all of the liver biopsies were identified within 65 spots, among which 25 ones were differently represented between O and NO subjects. In particular, over-representation of short-chain acyl-CoA dehydrogenase, Δ(3,5)-Δ(2,4)dienoyl-CoA isomerase, acetyl-CoA acetyltransferase, glyoxylate reductase/hydroxypyruvate reductase, fructose-biphosphate aldolase B, peroxiredoxin I, protein DJ-1, catalase, α- and β-hemoglobin subunits, 3-mercaptopyruvate S-transferase, calreticulin, aminoacylase 1, phenazine biosynthesis-like domain-containing protein and a form of fatty acid-binding protein, together with downrepresentation of glutamate dehydrogenase, glutathione S-transferase A1, S-adenosylmethionine synthase 1A and a form of apolipoprotein A-I, was associated with the obesity condition. Some of these metabolic enzymes and antioxidant proteins have already been identified as putative diagnostic markers of liver dysfunction in animal models of steatosis or obesity, suggesting additional investigations on their role in these syndromes. Their differential representation in human liver was suggestive of their consideration as obesity human biomarkers and for the development of novel antiobesity drugs.     

Comparative analysis of the interaction of various estrogens with the estrogen-receptor system of the uterus

Equilibrium binding of labeled estron, estradiol, estriol and DES was studied in uterine cytosol of immature Wistar rats. The dissociation kinetics of the ligand complexes with specific high affinity sites suggested the homogeneity of estrogen receptors in rat uterine cytosol. The feasibility of intracellular regulation of estrogen action in target cells both at the receptor and post-receptor levels is discussed.     

Inference of miRNA targets using evolutionary conservation and pathway analysis

Background

MicroRNAs have emerged as important regulatory genes in a variety of cellular processes and, in recent years, hundreds of such genes have been discovered in animals. In contrast, functional annotations are available only for a very small fraction of these miRNAs, and even in these cases only partially.

Results

We developed a general Bayesian method for the inference of miRNA target sites, in which, for each miRNA, we explicitly model the evolution of orthologous target sites in a set of related species. Using this method we predict target sites for all known miRNAs in flies, worms, fish, and mammals. By comparing our predictions in fly with a reference set of experimentally tested miRNA-mRNA interactions we show that our general method performs at least as well as the most accurate methods available to date, including ones specifically tailored for target prediction in fly. An important novel feature of our model is that it explicitly infers the phylogenetic distribution of functional target sites, independently for each miRNA. This allows us to infer species-specific and clade-specific miRNA targeting. We also show that, in long human 3' UTRs, miRNA target sites occur preferentially near the start and near the end of the 3' UTR. To characterize miRNA function beyond the predicted lists of targets we further present a method to infer significant associations between the sets of targets predicted for individual miRNAs and specific biochemical pathways, in particular those of the KEGG pathway database. We show that this approach retrieves several known functional miRNA-mRNA associations, and predicts novel functions for known miRNAs in cell growth and in development.

Conclusion

We have presented a Bayesian target prediction algorithm without any tunable parameters, that can be applied to sequences from any clade of species. The algorithm automatically infers the phylogenetic distribution of functional sites for each miRNA, and assigns a posterior probability to each putative target site. The results presented here indicate that our general method achieves very good performance in predicting miRNA target sites, providing at the same time insights into the evolution of target sites for individual miRNAs. Moreover, by combining our predictions with pathway analysis, we propose functions of specific miRNAs in nervous system development, inter-cellular communication and cell growth. The complete target site predictions as well as the miRNA/pathway associations are accessible on the ElMMo web server.     

Nascent RNA antagonizes the interaction of a set of regulatory proteins with chromatin

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Analysis of tetramethylrhodamine-labeled actin polymerization and interaction with actin regulatory proteins

The hydrolysis of ATP accompanying actin polymerization destabilizes the filament, controls actin assembly dynamics in motile processes, and allows the specific binding of regulatory proteins to ATP- or ADP-actin. However, the relationship between the structural changes linked to ATP hydrolysis and the functional properties of actin is not understood. Labeling of actin Cys374 by tetramethylrhodamine (TMR) has been reported to make actin non-polymerizable and enabled the crystal structures of ADP-actin and 5'-adenylyl beta,gamma-imidodiphosphate-actin to be solved. TMR-actin has also been used to solve the structure of actin in complex with the formin homology 2 domain of mammalian Dia1. To understand how the covalent modification of actin by TMR may affect the structural changes linked to ATP hydrolysis and to evaluate the functional relevance of crystal structures of TMR-actin in complex with actin-binding proteins, we have analyzed the assembly properties of TMR-actin and its interaction with regulatory proteins. We show that TMR-actin polymerized in very short filaments that were destabilized by ATP hydrolysis. The critical concentrations for assembly of TMR-actin in ATP and ADP were only an order of magnitude higher than those for unlabeled actin. The functional interactions of actin with capping proteins, formin, actin-depolymerizing factor/cofilin, and the VCA-Arp2/3 filament branching machinery were profoundly altered by TMR labeling. The data suggest that TMR labeling hinders the intramolecular movements of actin that allow its specific adaptative recognition by regulatory proteins and that determine its function in the ATP- or ADP-bound state.     

Glucokinase and glucokinase regulatory proteins as molecular targets for novel antidiabetic drugs

Molecular Biology - The impairment of glucose homeostasis leads to hyperglycemia and type-2 diabetes mellitus. Glucokinase (GK), an enzyme that catalyzes the conversion of glucose to...     

GW182 proteins cause PABP dissociation from silenced miRNA targets in the absence of deadenylation

GW182 family proteins interact with Argonaute proteins and are required for the translational repression, deadenylation and decay of miRNA targets. To elicit these effects, GW182 proteins interact with poly(A)‐binding protein (PABP) and the CCR4–NOT deadenylase complex. Although the mechanism of miRNA target deadenylation is relatively well understood, how GW182 proteins repress translation is not known. Here, we demonstrate that GW182 proteins decrease the association of eIF4E, eIF4G and PABP with miRNA targets. eIF4E association is restored in cells in which miRNA targets are deadenylated, but decapping is inhibited. In these cells, eIF4G binding is not restored, indicating that eIF4G dissociates as a consequence of deadenylation. In contrast, PABP dissociates from silenced targets in the absence of deadenylation. PABP dissociation requires the interaction of GW182 proteins with the CCR4–NOT complex. Accordingly, NOT1 and POP2 cause dissociation of PABP from bound mRNAs in the absence of deadenylation. Our findings indicate that the recruitment of the CCR4–NOT complex by GW182 proteins releases PABP from the mRNA poly(A) tail, thereby disrupting mRNA circularization and facilitating translational repression and deadenylation.