LightSwitch™ 3´UTR Reporter GoClone® Collection

Post transcriptional regulation through miRNA-3´UTR interactions

Gene regulation studies have historically focused on events that influence transcription, such as the binding of transcription factors to promoters. More recently, much research has centered on the presence or absence of histone modifications that alter the chromatin structure, which influences DNA accessibility and its ability to be transcribed. However, post-transcriptional events play an equally important role in gene regulation.

MicroRNAs (miRNAs) are small, non-coding RNA molecules of approximately 22 nucleotides that play a large role as post-transcriptional regulators. miRNAs are thought to repress expression by targeting the 3´UTR (untranslated) region of mRNA transcripts. The interaction of a miRNA with a 3´UTR results in either increased degradation of the target transcript or inhibition of translation.

While the human genome is thought to encode only about 1000 miRNAs, they are believed to target ~60% of mRNA transcripts. A given miRNA may have multiple 3´UTR targets, and a given 3´UTR may be targeted by multiple miRNAs. Therefore, there is considerable interest in studying miRNA-3´UTR interactions.

LightSwitch products for the study of 3´UTR-miRNA interactions

LightSwitch 3´UTR Reporter constructs are ideal for performing miRNA target validation, as well as assessing the functional impact of miRNA-3´UTR interactions. In combination with our miRNA Mimics and Inhibitors, which are co-transfected with the 3´UTR Reporter constructs (Figure 1), you have everything needed to validate miRNA targets, measure RNA stability, translation efficiency and the functional impact of miRNAs on a gene-by-gene basis. We also offer over 900 optimized LightSwitch Synthetic miRNA Target Reporter constructs, which can provide increased response relative to the endogenous sequences cloned into the LightSwitch 3´UTR Reporter constructs.

Figure 1: Luciferase activity used for target validation of miRNAs on a specific human 3´UTR.

1. Transfect a 3´UTR Reporter construct into your cell line.
2. A constitutive promoter drives the production of a hybrid luciferase-human 3´UTR transcript.
3. Total luciferase output depends on the effect of the 3´UTR on transcript stability and/or translational efficiency.
4. A synthetic microRNA or microRNA inhibitor can be co-transfected with the 3´UTR reporter vector to study its impact.

The 3´UTR activity can be measured to quantitively characterize:

• The impact of the UTR on post-transcriptional gene regulation
• The impact of a miRNA or siRNA on the regulation of transcript stability or translation efficiency
• The targets of a miRNA or siRNA (Figure 2)
• The effect of sequence variants on UTR function
• The impact of miRNA inhibitors or stimuli

Figure 2: Predicted mir-122 targets were validated using constructs from the LightSwitch 3´UTR Reporter Collection.

Putative 3´UTR targets of miR-122 that had been predicted using the miRanda algorithm were co-transfected with either 20 nM of miR-122 mimic or a non-targeting control miRNA in K-562 cells. The graph above shows the four strongest responders from this experiment along with two non-responding controls.

Applications

• Understand the mechanism by which a gene is induced or repressed.
• Measure the impact of the 3´UTR on post-transcriptional gene regulation.
• Validate the targets of a miRNA or siRNA.
• Measure the effects of sequence variants on 3´UTR or miRNA function.

The LightSwitch 3´UTR Reporter Collection includes over 12,000 human 3´UTRs that have been cloned into the pLightSwitch_3UTR reporter vector, which utilizes the novel, engineered RenSP luciferase gene. LightSwitch 3´UTR Reporter constructs can be used with the optimized reagents found in the LightSwitch Luciferase Assay Kit to measure the impact of a 3´UTR on post-transcriptional regulation.

In addition to the over 12,000 endogenous human 3´UTR clones that make up the LightSwitch 3´UTR Reporter Collection, we offer over 900 LightSwitch Synthetic miRNA Target Reporter constructs. These constructs, which have also been cloned into the pLightSwitch_3UTR vector, contain optimized sequence repeats, so can provide better sensitivity and a stronger response than their native counterparts.

Both the LightSwitch 3´UTR Reporter constructs and the LightSwitch Synthetic miRNA Target Reporter constructs were designed to be co-transfected with our collections of LightSwitch miRNA Mimics and Inhibitors to study the effects of over-expression of a miRNA of interest, or to knock down the expression of endogenous human miRNAs in living cells.

The following publications cite the use of and/or provide additional information about the LightSwitch Luciferase Assay System:

3´UTRs & miRNAs

Myocardial MiR-30 downregulation triggered by doxorubicin drives alterations in β-adrenergic signaling and enhances apoptosis Roca-Alonso et al. (2015) Cell Death and Disease
Microenvironment-induced downregulation of miR-193b drives ovarian cancer metastasis Mitra et al. (2015) Oncogene
EGF induces microRNAs that target suppressors of cell migration: miR-15b targets MTSS1 in breast cancer Kedmi et al. (2015) Science Signaling
MicroRNA-31 Promotes Skin Wound Healing by Enhancing Keratinocyte Proliferation and Migration Li et al. (2015) Journal of Investigative Dermatology
Identification of microRNA-mRNA dysregulations in paroxysmal atrial fibrillation Chiang et al. (2015) International Journal of Cardiology
Metformin and Rapamycin Reduce Pancreatic Cancer Growth in Obese Prediabetic Mice by Distinct MicroRNA-Regulated Mechanisms Cifarelli et al. (2015) Diabetes
Identification and Computational Analysis of Gene Regulatory Elements Taher et al. (2015) Cold Spring Harb Protocols
miR-155 augments CD8⁺ T-cell antitumor activity in lymphoreplete hosts by enhancing responsiveness to homeostatic γ_c cytokines Ji et al. (2014) PNAS
PLK1 Inhibition Down-regulates Polycomb Group Protein BMI1 via Modulation of the miR-200c/141 Cluster Dimri et al. (2014) Journal of Biological Chemistry
Mir-660 is downregulated in lung cancer patients and its replacement inhibits lung tumorigenesis by targeting MDM2-p53 interaction Fortunato et al. (2014) Cell Death and Disease
miR30a inhibits LOX expression and anaplastic thyroid cancer progression Boufraqech et al. (2014) Cancer Research
MicroRNA silencing for cancer therapy targeted to the tumour microenvironment Cheng et al. (2014) Nature
TRPM3 and miR-204 Establish a Regulatory Circuit that Controls Oncogenic Autophagy in Clear Cell Renal Cell Carcinoma Hall et al. (2014) Cancer Cell
miR-24 limits aortic vascular inflammation and murine abdominal aneurysm development Maegdefessel et al. (2014) Nature Communications
miR-154^* and miR-379 in the DLK1-DIO3 MicroRNA Mega-Cluster Regulate Epithelial to Mesenchymal Transition and Bone Metastasis of Prostate Cancer Gururajan et al. (2014) Clinical Cancer Research
MiR-29b replacement inhibits proteasomes and disrupts aggresome+autophagosome formation to enhance the antimyeloma benefit of bortezomib Jagannathan et al. (2014) Leukemia
The Regulatory Function of miR-200c on Inflammatory and Cell-Cycle Associated Genes in SK-LMS-1, A Leiomyosarcoma Cell Line Chuang et al. (2014) Reproductive Sciences
Integrative post-genome-wide association analysis of CDKN2A and TP53 SNPs and risk of esophageal adenocarcinoma Buas et al. (2014) Carcinogenesis
Kaposi’s Sarcoma-Associated Herpesvirus Downregulates Transforming Growth Factor β2 To Promote Enhanced Stability of Capillary-Like Tube Formation DiMaio et al. (2014) Journal of Virology
MicroRNA-193b-3p acts as a tumor suppressor by targeting the MYB oncogene in T-cell acute lymphoblastic leukemia Mets et al. (2014) Leukemia
Human microRNA-24 modulates highly pathogenic avian-origin H5N1 influenza A virus infection in A549 cells by targeting secretory pathway furin Loveday et al. (2014) Journal of General Virology
Renilla Luciferase Reporter Assay to Study 3´UTR-Driven Posttranscriptional Regulations of OPRM1 Vincelli et al. (2014) Methods in Molecular Biology
microRNA100 inhibits self-renewal of breast cancer stem-like cells and breast tumor development Deng et al. (2014) Cancer Research
Selective repression of the oncogene cyclin D1 by the tumor suppressor miR-206 in cancers Elliman et al. (2014) Oncogenesis
Stromal fibroblast-derived miR-409 promotes epithelial-to-mesenchymal transition and prostate tumorigenesis Josson et al. (2014) Oncogene
MicroRNA-138 is a potential regulator of memory performance in humans Schröder et al. (2014) Frontiers in Human Neuroscience
miR-150 influences B-cell receptor signaling in chronic lymphocytic leukemia by regulating expression of GAB1 and FOXP1 Mraz et al. (2014) Blood
MicroRNA-34a Modulates Neural Stem Cell Differentiation by Regulating Expression of Synaptic and Autophagic Proteins Morgado et al. (2014) Molecular Neurobiology
Circulating microRNA signature in non-alcoholic fatty liver disease: from serum non-coding RNAs to liver histology and disease pathogenesis Pirola et al. (2014) Gut
A Novel Function for Lysyl Oxidase in Pluripotent Mesenchymal Cell Proliferation and Relevance to Inflammation-Associated Osteopenia Khosravi et al. (2014) PLOS One
Regulation of COX-2 expression by miR-146a in lung cancer cells Cornett et al. (2014) RNA
MicroRNA-205 signaling regulates mammary stem cell fate and tumorigenesis Chao et al. (2014) Journal of Clinical Investigation
CFIm25 links alternative polyadenylation to glioblastoma tumour suppression Masamha et al. (2014) Nature
Therapeutic Delivery of miR-200c Enhances Radiosensitivity in Lung Cancer Cortez et al. (2014) Molecular Therapy
miR-145 suppresses thyroid cancer growth and metastasis and targets AKT3 Boufraqech et al. (2014) Endocrine-Related Cancer
Mapping posttranscriptional regulation of the human glycome uncovers microRNA defining the glycocode Agrawal et al. (2014) PNAS
Assessment of microRNA-related SNP effects in the 3´ untranslated region of the IL22RA2 risk locus in multiple sclerosis Lill et al. (2014) Neurogenetics
Long Non-coding RNA HOTAIR Is Targeted and Regulated by miR-141 in Human Cancer Cells Chiyomaru et al. (2014) Journal of Biological Chemistry
Systematic design and functional analysis of artificial microRNAs Arroyo et al. (2014) Nucleic Acids Research
Identifying microRNAs regulating B7-H3 in breast cancer: the clinical impact of microRNA-29c Nygren et al. (2014) British Journal of Cancer