TY - JOUR

T1 - Improved microRNA suppression by WPRE-linked Tough Decoy microRNA sponges

AU - Hollensen, Anne Kruse

AU - Thomsen, Rune

AU - Bak, Rasmus O

AU - Petersen, Charlotte Christie

AU - Ermegaard, Eva Reumert

AU - Aagaard, Lars

AU - Damgaard, Christian Kroun

AU - Mikkelsen, Jacob Giehm

N1 - Published by Cold Spring Harbor Laboratory Press for the RNA Society.

PY - 2017/8

Y1 - 2017/8

N2 - Our genes are posttranscriptionally 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 (RNApolII)-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 posttranscriptional 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.

AB - Our genes are posttranscriptionally 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 (RNApolII)-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 posttranscriptional 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.

KW - Journal Article

U2 - 10.1261/rna.061192.117

DO - 10.1261/rna.061192.117

M3 - Journal article

C2 - 28487381

VL - 23

SP - 1247

EP - 1258

JO - RNA

JF - RNA

SN - 1355-8382

IS - 8

ER -