Additional file 1 of miRA: adaptable novel miRNA identification in plants using small RNA sequencing data

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microRNA-122 Dependent Binding of Ago2 Protein to Hepatitis C Virus RNA Is Associated with Enhanced RNA Stability and Translation Stimulation

<div><p>Translation of Hepatitis C Virus (HCV) RNA is directed by an internal ribosome entry site (IRES) in the 5′-untranslated region (5′-UTR). HCV translation is stimulated by the liver-specific microRNA-122 (miR-122) that binds to two binding sites between the stem-loops I and II near the 5′-end of the 5′-UTR. Here, we show that Argonaute (Ago) 2 protein binds to the HCV 5′-UTR in a miR-122-dependent manner, whereas the HCV 3′-UTR does not bind Ago2. miR-122 also recruits Ago1 to the HCV 5’-UTR. Only miRNA duplex precursors of the correct length stimulate HCV translation, indicating that the duplex miR-122 precursors are unwound by a complex that measures their length. Insertions in the 5′-UTR between the miR-122 binding sites and the IRES only slightly decrease translation stimulation by miR-122. In contrast, partially masking the miR-122 binding sites in a stem-loop structure impairs Ago2 binding and translation stimulation by miR-122. In an RNA decay assay, also miR-122-mediated RNA stability contributes to HCV translation stimulation. These results suggest that Ago2 protein is directly involved in loading miR-122 to the HCV RNA and mediating RNA stability and translation stimulation.</p> </div

Argonaute gene expression in different melanoma and non-melanoma cell lines.

<p>Relative (A) AGO1, (B) AGO2, (C) AGO3 and (D) AGO4 mRNA expression in melanoma cell lines derived from primary tumors (Mel Ei, Mel Juso, Mel Ho and Mel Wei) and metastases (Mel Ju, Mel Im, SkMel28 and Hmb2) and other non-melanoma cell lines (HeLa, CaCo2, PLC, Jurkat, Hep3b, SW1353 and MCF7). Each point shows the measurement of one independently derived cDNA sample. Bars show mean and S.D. (E) The compilation of percentage distribution of each AGO to the aggregate AGO amount. (F) Entire mRNA expression of all four AGOs compared to actin in melanoma cell lines derived from primary tumors or metastases and in other non-melanoma cell lines.</p

Argonaute gene expression in different human healthy tissues compared to NHEMs.

<p>Relative (A) AGO1, (B) AGO2, (C) AGO3 and (D) AGO4 mRNA expression in NHEMs (derived from different cultivation passages (P4-P6) from three different donors respectively), skin (derived from three different tissue samples) and heart, kidney, bone marrow, fetal brain, bowel, thymus, uterus, trachea, brain, muscle, bone marrow, liver, fetal liver, brain and fetal brain (derived from a total RNA bank and shown as technical replicates). (E) The compilation of percentage distribution of each AGO to the total AGO amount in the respective cell line or tissue.</p

Ago2 protein interacts miR-122-dependently with the HCV 5′-UTR.

<p>(A) Immunoblot of HeLa cell lysate with the anti-Ago2 antibody monoclonal 11A9 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056272#pone.0056272-Rdel1" target="_blank">[32]</a>. (B–D) Analysis of ³²P-labelled HCV 5′-UTR RNA after transfection into HeLa cells in the presence or absence of miR-122 or control miR-124 duplexes as indicated. The cells were lysed 6 h after transfection. (B) Aliquots of the cell lysate were used for RNA re-extraction to check the integrity of the input RNA prior to immunoprecipitation. The re-extracted radioactive HCV RNA was visualized after gel electrophoresis and autoradiography. A quantification of the RNA amounts in lanes 2–5 is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056272#pone.0056272.s001" target="_blank">Fig. S1A</a>. (C) Immunoprecipitation (IP) of Ago2-HCV 5′-UTR RNA complexes. RNA-Ago2 protein complexes were immunoprecipitated from the cell lysates with anti-Ago2 antibodies. From the precipitate, RNA was re-extracted, and the radiolabelled HCV RNA was visualized after gel electrophoresis and autoradiography. In lane 1, the input RNA is shown (1∶100 dilution). In lane 2, an anti-eIF3 antibody was used as a positive control. In lane 3, an anti-Flag antibody was used as a negative control. (D) Aliquots of the immunoprecipitates used in lanes 2–5 in (C) were checked for Ago2 in an immunoblot.</p

Oligonucleotide sequences and qRT-PCR conditions.

<p>Oligonucleotide sequences and qRT-PCR conditions.</p

Argonaute protein distribution in melanoma and non-melanoma cell lines.

<p>(A) AGO protein percentage distribution of each AGO to total AGO protein amount in non-melanoma cell lines CaCo2, HepG2, SW1353, MCF7, HeLa and melanoma cell lines Mel Ju, Mel Im, Mel Wei, Mel Ei and Mel Ho derived from AGO-APP. (B) Total protein amount determination of each AGO enriched by AGO-APP in CaCo2, HepG2, SW1353, MCF7, HeLa, Mel Ju, Mel Im, Mel Wei, Mel Ei and Mel Ho. (C) Average AGO protein amount of each AGO in non-melanoma compared to melanoma cell lines. The reduced AGO concentration in melanoma cell lines compared to non-melanoma cell lines is only significant for AGO2. ** = p<0.01 (D) AGO1 western blot analysis and corresponding (E) Western blot quantification of melanoma cell lines (Mel Ju, Mel Im, Mel Wei, Mel Ei, Mel Ho) and non-melanoma cell lines (CaCo2, SW1353, MCF7, HeLa, HepG2). The two additional values in the AGO1 western blot quantification illustrate the average AGO1 concentration in melanoma and non-melanoma cell lines. Quantification was done relative to Actin in the respective blot.</p

Hypothetical mechanism of the effect of miR-122 on HCV RNA stability and translation stimulation in cells.

<p>The mature miR-122 guide strand is processed from its duplex precursor and incorporated into functional microRNA-protein (miRNP) complexes. These complexes target one or two miR-122 target sites (grey boxes) in the HCV 5′-UTR and confer HCV RNA stability and translation stimulation (indicated by the (+)).</p

Influence of insertions between miR-122 target sites and IRES on miR-122-mediated stimulation of HCV translation.

<p>(A) In the HCV reporter RNA, 5 or 10 nucleotides (nt) were inserted between the second miR-122 binding site and the base of stem-loop II. (B) An insert between the second miR-122 target site and the stem-loop II was designed to mask the two miR-122 target sites in an additional stem (st). (C) Relative translation activities of the wild-type (wt) and mutant HCV reporter RNAs in HuH-7 cells. Additional ectopic miR-122 (or miR-124 as a control) was added as indicated. A capped and polyadenylated Renilla-Luciferase (Rluc) was co-transfected. The Rluc readouts were used for normalization of the Fluc readouts from the HCV reporter RNA. The expression of the HCV reporter RNA in the presence of the control miR-124 was set 100%. The p-value of the statistical difference is indicated for the stem insertion construct compared with the wt HCV reporter RNA (* = p<0.05). (D–F) Immunoprecipitation of Ago2-HCV 5′-UTR RNA complexes with the constructs shown in A and B. The experiments were performed essentially as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056272#pone-0056272-g002" target="_blank">Fig. 2 B–D</a>. (D) Examination of the radioactive HCV RNA re-extracted from the cell lysate prior to immunoprecipitation (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056272#pone-0056272-g002" target="_blank">Fig. 2B</a>). A quantification of the RNA amounts in lanes 2–7 is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056272#pone.0056272.s001" target="_blank">Fig. S1D</a>. (E) Radiolabelled HCV RNA recovered by anti-Ago2 immunoprecipitation. In lanes 2–5, the HCV RNA with the additional stem (st) was used, in lanes 6–7, the wt HCV 5′-UTR RNA was used. In lane 1, the input RNA with the stem insert, in lane 8 the input wt HCV 5′-UTR RNA is shown (each in 1∶100 dilution). In lane 2, an anti-eIF3 antibody was used as a positive control. In lane 3, an anti-Flag antibody was used as a negative control. (F) Anti-Ago2 immunoblot control of the samples analyzed in (E).</p

Length variation of the miR-122 duplex precursor impairs stimulation of HCV translation.

<p>(A) Structure of the miR-122 duplex (22 nucleotides) and its length variants. The base-pairing conditions at the thermodynamically less stable end (left side) and the 2 nucleotide 3′-overhangs at both ends were retained in all cases. (B) Stimulation of HCV reporter RNA translation in HeLa cells after co-transfection of the RNA duplexes shown in (A) as indicated. miR-124 was used as negative control, and its readout was set to 100%.</p