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Target prediction and assembly of 3′-UTR reporter gene constructs

CD Caroline Diener
MH Martin Hart
DA Dalia Alansary
VP Vanessa Poth
BW Barbara Walch-Rückheim
JM Jennifer Menegatti
FG Friedrich Grässer
TF Tobias Fehlmann
SR Stefanie Rheinheimer
BN Barbara A. Niemeyer
HL Hans-Peter Lenhof
AK Andreas Keller
EM Eckart Meese
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We used miRWalk 2.0 (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/index.html) for an in silico prediction of miR-34a-5p-binding sites within the 3′-UTR of possible target genes78. For that purpose, all through miRWalk 2.0 available databases were independently selected to find putative 3′-UTR target sides for seed binding, with a minimum of a 6 nt seed sequence. Results were filtered for a prediction by more than four databases. Subsequently, we chose predicted target genes related to SOCE or calcineurin/NFAT signaling. The respective 3′-UTR sequences of predicted miR-34a-5p target genes were amplified by PCR using specific primer pairs (Supplementary Table 1) and cloned into the multiple cloning site of pMIR-RNL-TK plasmid79, using SpeI and SacI restriction sites. Jurkat cDNA was used for template. The denoted parts of human GRCh38/hg38 genome have been cloned utilizing the declared NCBI sequences for reference: ATP2A2 3ʹ-UTR (chr12:110,347,119–110,348,110; NM_170665.3), ATP2A3 3ʹ-UTR (chr17:3,923,898–3,924,783; NM_005173.3), CALM3 3ʹ-UTR (chr19:46,609,301–46,610,572; NM_005184.3), CAMLG 3ʹ-UTR (chr5:134,750,951–134,752,087; NM_001745.3), ITPR1 3ʹ-UTR (chr3:4,846,620–4,847,798; NM_001168272.1), ITPR2 3ʹ-UTR (chr12:26,337,690–26,338,767; NM_002223.3), ITPR3 3ʹ-UTR (chr6:33,695,799–33,696,154; NM_002224.3), NFATC4 3ʹ-UTR (chr14:24,378,485–24,379,520; NM_001136022.2), ORAI3 3ʹ-UTR (chr16:30,953,784–30,954,826; NM_152288.2), PPP3R1 3ʹ-UTR (chr2:68,179,165–68,180,278; NM_000945.3), RCAN1 3ʹ-UTR (chr21:34,516,824–34,517,736; NM_004414.6), STIM1 3ʹ-UTR (chr11:4,092,219–4,093,198; NM_001277961.1). MiR-34a-5p-binding sites were mutated by technique of overlap extension PCR using specific primers80 (Supplementary Table 2).

Copyright and License information: The Author(s) ©2018
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