Structural Analysis of microRNA-Target Interaction by Sequential Seed Mutagenesis and Stem-Loop 3' RACE

Abstract

BackgroundAs a consequence of recent RNAseq efforts, miRNAomes of diverse tissues and species are available. However, most interactions between microRNAs and regulated mRNAs are still to be deciphered. While in silico analysis of microRNAs results in prediction of hundreds of potential targets, bona-fide interactions have to be verified e.g. by luciferase reporter assays using fused target sites as well as controls incorporating mutated seed sequences. The aim of this study was the development of a straightforward approach for sequential mutation of multiple target sites within a given 3’ UTR. Methodology/Principal FindingsThe established protocol is based on Seed Mutagenesis Assembly PCR (SMAP) allowing for rapid identification of microRNA target sites. Based on the presented approach, we were able to determine the transcription factor NKX3.1 as a genuine target of miR-155. The sequential mutagenesis of multiple microRNA target sites was examined by miR-29a mediated CASP7 regulation, which revealed one of two predicted target sites as the predominant site of interaction. Since 3’ UTR sequences of non-model organisms are either lacking in databases or computationally predicted, we developed a Stem-Loop 3’ UTR RACE PCR (SLURP) for efficient generation of required 3’ UTR sequence data. The stem-loop primer allows for first strand cDNA synthesis by nested PCR amplification of the 3’ UTR. Besides other applications, the SLURP method was used to gain data on porcine CASP7 3’UTR evaluating evolutionary conservation of the studied interaction. Conclusions/SignificanceSequential seed mutation of microRNA targets based on the SMAP approach allows for rapid structural analysis of several target sites within a given 3’ UTR. The combination of both methods (SMAP and SLURP) enables targeted analysis of microRNA binding sites in hitherto unknown mRNA 3’ UTRs within a few days.

Down regulated lncRNA MEG3 eliminates mycobacteria in macrophages via autophagy

“…CCL 185), human colorectal adenocarcinoma cells HT-29 (DSMZ No. : ACC 299) and its sub clone HT-29/B6 59 were cultured as described previously 5 57 60 61 or following the protocols for nucleofection (Lonza). Total RNA from cell lines was isolated and quality controlled as described earlier 62 .…”

Hsa-miR-183-5p Modulates Cell Adhesion by Repression of ITGB1 Expression in Prostate Cancer

“…The 3 UTR of ITGB1 was amplified from human genomic DNA using the following primers: MssI-NotI-3 UTR-ITGB1 forward primer 5 -GTTTAAACGCG GCCGCAGTATGTTGAGAGTTGCTGGTGT-3 and XbaI-3 UTR-ITGB1 reverse primer 5 -TCTAGAAGCAGAAAATTGCTCGGTTCT-3 . Mutations in the putative binding site of miR-183 were introduced by Seed Mutagenesis Assembly PCR (SMAP), as described in [75], using the partially overlapping primers complementary to the putative binding site of miR-183: ITGB1mut forward primer 5 -GCTTTAAAACCTGTGTCCAGTTTTAAGAGTTACTTA ATG-3 and ITGB1mut reverse primer 5 -CATTAAGTAACTCTTAAAACTGGACACAGG TTTTAAAGC-3 . Cloning of the 3 UTR wild type and mutated sequences into pmiRGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA) was performed by MssI and XbaI digestion.…”

MicroRNAs regulating cluster of differentiation 46 (CD46) in cardioembolic and non-cardioembolic stroke

“…In addition, by using in silico prediction and biochemical methodology we have identified miR-19a, -20a, -185 and -374b to be targeting CD46 . We have utilized luciferase assay, a direct method that is used to prove the binding between miRNA:mRNA pair in various complementary binding studies [ 49 ], [ 50 ]. However, when CD46 protein expression were measured, only miR-19a and miR-20a proved to be useful to significantly modulate CD46 in HUVECs.…”