Characterization of a cis-acting regulatory element in the protein-coding region of human dihydrofolate reductase mRNA

Ningwen Tai, John C. Schmitz, Tian Min Chen, Edward Chu

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

Abstract

Previous studies have shown that human DHFR (dihydrofolate reductase), in addition to its critical role in DNA biosynthesis, functions as an RNA-binding protein. The interaction between DHFR and its own mRNA results in translational repression. In this study, we characterized the cis-acting elements on human DHFR mRNA that are required for the DHFR mRNA-DHFR protein interaction. Using a series of gel-shift and nitrocellulose filter-binding assays, a 164 nt RNA sequence, corresponding to nt 401-564, was identified within the coding region that binds to DHFR protein with an affinity similar to that of full-length DHFR mRNA. To document in vivo biological activity, various DHFR sequences contained within the coding region were cloned on to the 5' end of a luciferase reporter plasmid, and transient transfection experiments were performed using human colon cancer RKO cells. In cells transfected with p644/DHFR:401-564, luciferase activity was decreased by 50 % when compared with cells transfected with the p644 plasmid alone. Luciferase mRNA levels were identical under each of these conditions, as determined by Northern-blot analysis. In cells transfected with p644/ DHFR:401-564, luciferase activity was restored to almost 100 % of control when cells were treated with the antifolate analogue methotrexate or with a short-interfering RNA targeting DHFR mRNA. These findings provide evidence that the DHFR 401-564 sequence is a DHFR-response element. In vitro and in vivo studies further localized this cis-element to an 82 nt sequence corresponding to nt 401-482. This work provides new insights into critical elements that mediate RNA-protein interactions.

Original languageEnglish (US)
Pages (from-to)999-1006
Number of pages8
JournalBiochemical Journal
Volume378
Issue number3
DOIs
StatePublished - Mar 15 2004
Externally publishedYes

Keywords

  • Dihydrofolate reductase
  • RNA-binding protein
  • Rna-protein interaction
  • Translational regulation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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