Dataset Information

Improved microRNA suppression by WPRE-linked tough decoy microRNA sponges.

ABSTRACT: Our genes are post-transcriptionally regulated by microRNAs (miRNAs) inducing translational suppression and degradation of targeted mRNAs. Strategies to inhibit miRNAs in a spatiotemporal manner in a desired cell type or tissue, or at a desired developmental stage, can be crucial for understanding miRNA function and for pushing forward miRNA suppression as a feasible rationale for genetic treatment of disease. For such purposes, RNA polymerase II (RNA Pol II)-transcribed tough decoy (TuD) miRNA inhibitors are particularly attractive. Here, we demonstrate augmented miRNA suppression capacity of TuD RNA hairpins linked to the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). This effect is position-dependent and evident only when the WPRE is positioned upstream of the TuD. In accordance, inclusion of the WPRE does not change nuclear export, translation, total levels of TuD-containing RNA transcripts, or cytoplasmic P-body localization, suggesting that previously reported WPRE functions are negligible for improved TuD function. Notably, deletion analysis of TuD-fused WPRE unveils truncated WPRE variants resulting in optimized miRNA suppression. Together, our findings add to the guidelines for production of WPRE-supported anti-miRNA TuDs.

SUBMITTER: Hollensen AK

PROVIDER: S-EPMC5513069 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Improved microRNA suppression by WPRE-linked tough decoy microRNA sponges.

Hollensen Anne Kruse AK Thomsen Rune R Bak Rasmus O RO Petersen Charlotte Christie CC Ermegaard Eva R ER Aagaard Lars L Damgaard Christian Kroun CK Mikkelsen Jacob Giehm JG

RNA (New York, N.Y.) 20170509 8

Our genes are post-transcriptionally regulated by microRNAs (miRNAs) inducing translational suppression and degradation of targeted mRNAs. Strategies to inhibit miRNAs in a spatiotemporal manner in a desired cell type or tissue, or at a desired developmental stage, can be crucial for understanding miRNA function and for pushing forward miRNA suppression as a feasible rationale for genetic treatment of disease. For such purposes, RNA polymerase II (RNA Pol II)-transcribed tough decoy (TuD) miRNA ...[more]

PMID: 28487381

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Project description:BackgroundIn humans, the heterochronic cascade composed of the RNA-binding protein LIN28 and its major target, the let-7 family of microRNAs (miRNAs), is highly regulated during human erythroid ontogeny. Additionally, down-regulation of the let-7 miRNAs in cultured adult CD34(+) cells or the over-expression of LIN28 in cultured erythrocytes from pediatric patients with HbSS genotype causes increased levels of fetal hemoglobin (HbF) in the range of 19-40% of the total. Therefore, we hypothesized that focused targeting of individual let-7 miRNA family members would exhibit regulatory effect on HbF expression in human adult erythroblasts.MethodsThe expression levels of mature let-7 family members were measured by RT-qPCR in purified cell populations sorted from peripheral blood. To study the effects of let-7 miRNAs upon globin expression, a lentiviral construct that incorporated the tough decoy (TuD) design to target let-7a or let-7b was compared with empty vector controls. Transductions were performed in CD34(+) cells from adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Downstream analyses included RT-qPCR, Western blot and HPLC for the characterization of adult and fetal hemoglobins.ResultsThe expression of individual let-7 miRNA family members in adult peripheral blood cell populations demonstrated that let-7a and let-7b miRNAs are expressed at much higher levels than the other let-7 family members in purified adult human blood cell subsets with expression being predominantly in reticulocytes. Therefore, we focused this study upon the targeted inhibition of let-7a and let-7b with the TuD design to explore its effects upon developmentally-timed erythroid genes. Let-7a-TuD transductions significantly increased gamma-globin mRNA expression and HbF to an average of 38%. Let-7a-TuD also significantly decreased the mRNA expression of some ontogeny-regulated erythroid genes, namely CA1 and GCNT2. In addition, the erythroid-related transcription factors BCL11A and HMGA2 were down- and up-regulated, respectively, by let-7a-TuD, while ZBTB7A, KLF1 and SOX6 remained unchanged.ConclusionsOverall, our data demonstrate that let-7 miRNAs are differentially expressed in human hematopoietic cells, and that targeted inhibition of the highly-expressed species of this family is sufficient for developmentally-specific changes in gamma-globin expression and HbF levels.

Project description:BackgroundA key component in protein structure prediction is a scoring or discriminatory function that can distinguish near-native conformations from misfolded ones. Various types of scoring functions have been developed to accomplish this goal, but their performance is not adequate to solve the structure selection problem. In addition, there is poor correlation between the scores and the accuracy of the generated conformations.ResultsWe present a simple and nonparametric formula to estimate the accuracy of predicted conformations (or decoys). This scoring function, called the density score function, evaluates decoy conformations by performing an all-against-all Calpha RMSD (Root Mean Square Deviation) calculation in a given decoy set. We tested the density score function on 83 decoy sets grouped by their generation methods (4state_reduced, fisa, fisa_casp3, lmds, lattice_ssfit, semfold and Rosetta). The density scores have correlations as high as 0.9 with the Calpha RMSDs of the decoy conformations, measured relative to the experimental conformation for each decoy. We previously developed a residue-specific all-atom probability discriminatory function (RAPDF), which compiles statistics from a database of experimentally determined conformations, to aid in structure selection. Here, we present a decoy-dependent discriminatory function called self-RAPDF, where we compiled the atom-atom contact probabilities from all the conformations in a decoy set instead of using an ensemble of native conformations, with a weighting scheme based on the density scores. The self-RAPDF has a higher correlation with Calpha RMSD than RAPDF for 76/83 decoy sets, and selects better near-native conformations for 62/83 decoy sets. Self-RAPDF may be useful not only for selecting near-native conformations from decoy sets, but also for fold simulations and protein structure refinement.ConclusionsBoth the density score and the self-RAPDF functions are decoy-dependent scoring functions for improved protein structure selection. Their success indicates that information from the ensemble of decoy conformations can be used to derive statistical probabilities and facilitate the identification of near-native structures.

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Improved microRNA suppression by WPRE-linked tough decoy microRNA sponges.

Publications

Improved microRNA suppression by WPRE-linked tough decoy microRNA sponges.

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