Table 1. Contributions of impaired translation and diminished mRNA concentration to repression by perfectly or imperfectly complementary siRNA and miRNA.

The effect of siEGFP or miR-125b on reporter gene expression was determined at both the protein and mRNA level by measuring firefly luciferase activity (normalized to Renilla luciferase activity) and by Northern blot analysis of the firefly luciferase reporter transcript (normalized to Renilla luciferase mRNA) (Supplementary Figure 1) 36 hr after transfection of 293T cells. Repression ratios for protein production and cytoplasmic mRNA concentration were calculated from normalized levels of firefly luciferase protein and uncleaved mRNA in the absence versus the presence of siEGFP or miR-125b. By dividing these two repression ratios, the repression ratio for translation efficiency (protein yield per mRNA molecule) was determined. A repression ratio of 1 indicates no repression. Calculations of repression ratio per element assume that the fold effects of all copies of an element are equal and multiplicative, as observed for E1 and miR-125b [10].

			Repression ratio
3′ UTR element	Copies	mi/siRNA	Protein production	mRNA concentration		Translation efficiency
				Total	Per element	Total	Per element
None	0	siEGFP	00.98 ± 0.05	1.00 ± 0.06		0.98 ± 0.08
GP	1	siEGFP	11.43 ± 0.96	3.37 ± 0.26	3.37 ± 0.26	3.39 ± 0.38	3.39 ± 0.38
GM	1	siEGFP	02.79 ± 0.17	1.60 ± 0.08	1.60 ± 0.08	1.74 ± 0.14	1.74 ± 0.14
GM	4	siEGFP	10.83 ± 0.71	2.27 ± 0.20	1.23 ± 0.03	4.76 ± 0.52	1.48 ± 0.04
None	0	miR-125b	01.02 ± 0.05	1.00 ± 0.05		1.02 ± 0.07
P	2	miR-125b	08.07 ± 0.39	3.10 ± 0.22	1.76 ± 0.06	2.60 ± 0.22	1.61 ± 0.07
E1	2	miR-125b	02.02 ± 0.19	1.39 ± 0.06	1.18 ± 0.03	1.45 ± 0.15	1.20 ± 0.06
E1	6	miR-125b	09.09 ± 0.44	2.81 ± 0.14	1.19 ± 0.01	3.23 ± 0.22	1.22 ± 0.02