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RNAi Screen Reveals Candidate miRNAs Associated with the NF‐κB Pathway
Author(s) -
Redan Benjamin W.,
Motobar Omid,
Tuzmen Pinar,
Guha Rajarshi,
Caplen Natasha,
Martin Scott E.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.780.1
Subject(s) - microrna , biology , gene silencing , rna interference , downregulation and upregulation , gene , genetics , untranslated region , three prime untranslated region , computational biology , microbiology and biotechnology , messenger rna , rna
Gene silencing through RNAi has emerged as a powerful tool for genetic loss‐of‐function studies. This study screened the druggable genome (≈7,000 genes) to illuminate novel components of NF‐kB signaling. A cell line with a β‐lactamase reporter gene was used, which was assayed after stimulation with TNF‐α. Downregulation of IKKα and proteasome constituents were identified as top NF‐κB antagonists. Follow‐up of hits helped us identify novel components of NF‐κB signaling, namely microRNAs (miRNA). miRNAs are about 22 nucleotides in length and function as endogenous post‐transcriptional regulators. Of primary importance is the seed sequence—positions 2–8 of miRNA—because it can bind to the 3’ UTR of a mRNA transcript in order to repress translation and/or facilitate sequestration and degradation of targets. Approximately 900 human miRNA mimics were screened and candidate target genes were identified. One such target is RELA. TargetScan ( targetscan.org ) predicts that RELA is targeted by the miR‐302 family and miR‐372, supporting our screening data. There is both a conserved and a poorly conserved possible heptamer binding site on the RELA 3′ UTR (5’ AGCACUU 3’). Thus far, the putative properties of two of the miRNAs were confirmed by showing downregulation of RELA by RT‐PCR. Also, an immunofluorescence method has been developed that will investigate downregulation of RELA protein.