Supplementary Table 1: Quantification of transcript abundance of luciferase-UTR
fusions. For qPCR, RNA was extracted from S2 cells (5x105) 20 h after transfection
using RNeasy (Quiagen); to avoid contamination with genomic DNA, samples were
vortexed (1’) prior to loading onto the RNeasy columns. 1µg of total RNA was reverse
transcribed using an oligo dT primer and Superscript III (Invitrogen; 20µl reaction
volume; 30’ at 42°C; termination by heat inactivation 10’ at 99°C). qPCR was carried out
following the Biorad iQ SYBR Green Supermix protocol using 100 ng cDNA template
and 800mM primers designed with VectorNTI software (20-22 bp; Tm 60-62°C; length of
amplification product 100-200 bp). We compared the transcript levels of Renilla (FW:
TGAGGAG TTCGCTGCCTACC; REV: TGCGGACAATCTGGACGACG) with Firefly
(FW: ATCGTGGACCGCCTGAAGTC; REV: ACGACGGCGGCAGGCAGC) luciferase
and RP49 (FW:GATGCCCAACATCGGTTACG; REV: TTGTGCACCAGG AACTTCTT)
as internal control. Each data point is the result of two independent experiments done in
triplicate. Relative changes in gene expression were calculated using the 2-Ct method,
see Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-8 (2001).
Backbone
Psicheck empty
RPR_N
RPR_C
HID_N
HID_C
GRIM_N
GRIM_C
HID_N>RPR
HID_N>GRIM
HID_∆5
Mean Fold changes
1.00 ( 1.02 – 0.98 )
0.98 ( 1.02 – 0.93)
1.02 ( 1.04 – 1.00)
1.01 ( 1.03 - 1.00)
1.01 ( 1.04 – 1.00)
1.02 ( 1.04 – 1.00)
1.01 ( 1.04 – 0.98)
0.98 ( 1.04 – 0.93)
0.97 ( 0.99 – 0.94)
1.11 ( 1.17 – 1.05)