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How to Measure UTR Regulation with Optimized Reporter Assays

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Abstract

Post-transcriptional regulatory mechanisms are important in the control of gene expression affecting localization, translation efficiency and stability of messenger RNA (mRNA). Regulation generally occurs through interactions of a regulatory protein or small noncoding RNA with specific sequences located in untranslated regions (UTRs) of the target mRNA sequence. Luciferase reporter assays are well suited to the study of mRNA regulatory mechanisms. Specific regulatory elements can be inserted downstream of the reporter gene sequence, and the impact on reporter gene expression can be assayed easily by examining reporter protein activity or by quantifying reporter mRNA levels. In addition, the reporter gene can be assayed after the regulatory sequence is manipulated or mutated for further identification and characterization of the sequence required for regulation.

A paper published in Methods outlines the steps that are needed to develop and optimize a luciferase-based reporter model for studying mammalian mRNA regulation. A detailed workflow is presented, including steps for reporter gene construction, transfection and measurement of reporter protein activity and RNA levels. For each step a discussion of the important considerations and controls and suggestions for optimization and troubleshooting are included. In addition, detailed protocols and example results from the authors’ studies of mRNA regulation by the human PUF proteins PUM1 and PUM2 are included. In their study, the authors introduce wildtype and mutated PUM response elements (PREs) into the 3´ UTR of the Renilla luciferase reporter gene using the psiCHECK™-1 Vector and demonstrate that both PUM1 and PUM2 repress reporter protein activity and decrease reporter mRNA levels when the native, but not mutant, PRE is present in the 3´ UTR sequence.

Jamie Van Etten1, Trista L. Schagat1,2 and Aaron C. Goldstrohm1

1University of Michigan Medical School 2Promega Corporation

Publication Date: 2013

How to Cite This Article

Van Etten, J., Schagat, T.L. and Goldstrohm, A.C. Abstract: How to Measure UTR Regulation with Optimized Reporter Assays. [Internet] 2013. [cited: year, month, date]. Available from: http://www.promega.com/resources/pubhub/how-to-measure-utr-regulation-with-optimized-reporter-assays/

Van Etten, J., Schagat, T.L. and Goldstrohm, A.C. Abstract: How to Measure UTR Regulation with Optimized Reporter Assays. Promega Corporation Web site. http://www.promega.com/resources/pubhub/how-to-measure-utr-regulation-with-optimized-reporter-assays/ Updated 2013. Accessed Month Day, Year.

psiCHECK is a trademark of Promega Corporation.