microrna-122 stimulates translation of Hepatitis C Virus RNA

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1 Supplementary information for: microrna-122 stimulates translation of Hepatitis C Virus RNA Jura Inga Henke 1,5, Dagmar Goergen 1,5, Junfeng Zheng 3, Yutong Song 4, Christian G. Schüttler 2, Carmen Fehr 1, Christiane Jünemann 1, Michael Niepmann 1,6 (1) Institute of Biochemistry, (2) Institute of Medical Virology, Justus-Liebig-University, Friedrichstrasse 24, Giessen, Germany; (3) present address: Dept. of Neurobiology, Univ. of Muenster, Muenster, Germany, (4) present address: Dept. of Molecular Genetics and Microbiology, Stony Brook University, NY 11794, USA (5) these authors contributed equally to this study (6) To whom correspondence should be addressed. michael.niepmann@biochemie.med.uni-giessen.de

2 Figure S1. Time course of HCV translation after sequestration of mir-122. Huh-7 cells were transfected with the HCV reporter RNA shown in Figure 1A in the absence or presence of anti-mir-122 as indicated. After the indicated times, cells were harvested, lysed in Passive Lysis Buffer (Promega), and luciferase activity expressed in the cytosol was determined. Values were normalized to the highest expression value set as 100 %. The result shows that HCV IRES directed luciferase translation increases with time, and the difference between the expression in the absence versus the presence of anti-mir-122 is highest after 4 hrs. Consequently, in all other transient transfection assays the cells were harvested and luciferase values measured after 4 hrs, unless otherwise indicated.

3 Figure S2. Transfection of the HCV reporter RNAs together with micrornas by electroporation. The HCV-Fluc-3 -UTR reporter RNA (see Figure 1A) was transfected into Huh-7 cells together with the mir duplex or the 2 -O-methylated antisense oligonucleotides as indicated. In this case, the transfection was performed by electroporation instead of liposome-mediated transfection. 4 hrs after transfection, the cells were harvested and cell lysates used for measuring Fluc activity. The results show that the differences in translation efficiency in this experiment are the same after electroporation transfection compared with liposome-mediated transfection. This confirms the results obtained with the routine liposome-mediated transfection.

4 Figure S3. 3 -UTR substitution mutants. This illustration details the 3 -region of the HCV-IRES-Fluc-3 -UTR reporter RNA shown in Figure 2A. At the top, the variable region of the 3 -UTR is shown with the potential mir-122 target site in red (boxed). At the bottom, the two mutations in the variable region are shown. In mutant "mut bulge" (left), the sequence of the potential mir-122 target site (ACACUCC) was mutated to change its primary sequence but not the secondary structure of the variable region. In mutant "closed stem" (right), the potential mir-122 target site was mutated to change both its primary sequence and its secondary structure, allowing it to largely base-pair with the sequence located opposite to it.

5 Figure S4. The 3 -UTR is not involved in translation stimulation by mir-122. (A) The structure of the delta-vr and delta 3 -UTR mutants. In this mutant, most of the variable region and the mir-122 complementary sequence had been deleted from the variable region (indicated by the dotted line). In contrast, mutant delta-3 -UTR lacks the entire 3 -UTR. (B) Sequestration of mir-122 in Huh-7 cells decreases the translation efficiency of delta- VR RNA like that of wild-type RNA. (C) In Huh-7 cells, duplex mir-122 but not mir-124 stimulates HCV IRES-directed translation in the absence of the HCV 3 -UTR. (D) In reticulocyte lysate, mature mir-122 but not mir-124 stimulates HCV IRES-directed translation in the absence of the HCV 3 -UTR. Taken together, these results confirm the conclusion drawn from Figure 2 that the mir-122 complementary sequence in the 3 -UTR is not significantly involved in translation stimulation.

6 Figure S5. mir-122 does not stimulate formation of ribosomal 48S complexes with a Fluc control RNA. Initiation complexes were formed with a radiolabeled capped and polyadenylated Fluc RNA (without any HCV sequences) in the absence (solid line) or presence (broken line) of non-labeled mir-122 and analyzed by sucrose gradient analysis. The elongation inhibitor anisomycin was present in both samples to enrich 80S initiation complexes.

7 Figure S6. mir-124 is not incorporated into 48S initiation complexes with the HCV Fluc-3 -UTR reporter RNA. Incorporation of radiolabeled mir-124 into ribosomal initiation complexes in the presence (solid line) or absence (broken line) of non-labeled short HCV RNA (see Figure 5A). The elongation inhibitor anisomycin was present in both samples to enrich 80S initiation complexes.

8 Figure S7. Comparison of ribosomal initiation complexes with HCV (reporter) RNAs of different length. Formation of ribosomal 48S and 80S complexes in RRL with the radiolabeled short HCV RNA (see Figure 5A, 658 nucleotides) (black solid line), the HCV-Fluc reporter RNA (see Figure 6C, nucleotides) (blue broken line) and the fulllength HCV RNA genome (see Figure 5H, nucleotides) (red dotted line). The sum of all fractions of a respective gradient is set 100 %, and the reading of each fraction of this gradient is then shown in % of the complete gradient. The result shows that the "background" curve of a given RNA is "lifted" with increasing RNA length. This effect is most likely due to the fact that more unspecific RNA-binding proteins from the reticulocyte lysate can bind to a longer RNA molecule than to a shorter RNA molecule. Consequently, a longer RNA species sediments in the gradient as a population of RNA-protein complexes in which individual RNA molecules are associated with different amounts of RNA-binding proteins, causing different sizes of these RNAprotein complexes. This in turn results in the distribution of these RNA-protein complexes over the entire gradient. Therefore, the "background" curve of the gradient profile with a longer RNA is "lifted", while the peak size caused by the single ribosomes decreases relative to the total amount of protein associated with the RNA.

9 Figure S8. mir-122 accelerates the formation of ribosomal 48S initiation complexes with the HCV RNA. (A-F) Kinetics of formation of ribosomal 48S and 80S complexes in RRL with the radiolabeled short HCV RNA (see Figure 5A) in RRL in the absence of microrna (solid black line), in the presence of mir-122 (broken red line) or in the presence of mir-124 (dotted blue line). The initiation complexes formed were "frozen" by adding MgCl 2 to 30 mm final concentration after the reactions. The values of fractions 5 to 7 and 11 to 13, respectively, of each of these gradients, are summed up and shown in Figure 6B to illustrate the increase in 48S and 80S initiation complex formation.