Abstract
Heterogeneous ribonucleoprotein K (hnRNP K) binds to the 5' untranslated region of the hepatitis C virus (HCV) and is required for HCV RNA replication. The hnRNP K binding site on HCV RNA overlaps with the sequence recognized by the liver-specific microRNA, miR-122. A proteome chip containing ∼17,000 unique human proteins probed with miR-122 identified hnRNP K as one of the strong binding proteins. In vitro kinetic study showed hnRNP K binds miR-122 with a nanomolar dissociation constant, in which the short pyrimidine-rich residues in the central and 3' portion of the miR-122 were required for hnRNP K binding. In liver hepatocytes, miR-122 formed a coprecipitable complex with hnRNP K. High throughput Illumina DNA sequencing of the RNAs precipitated with hnRNP K was enriched for mature miR-122. SiRNA knockdown of hnRNP K in human hepatocytes reduced the levels of miR-122. These results show that hnRNP K is a cellular protein that binds and affects the accumulation of miR-122. Its ability to also bind HCV RNA near the miR-122 binding site suggests a role for miR-122 recognition of HCV RNA.
Highlights
MicroRNAs are a class of noncoding RNA of ϳ22-nucleotides in length that can regulate gene expression by either targeting RNA for degradation or suppressing their translation through base pairing to the RNAs [1]
We previously reported that the hepatitis C virus (HCV) RNA sequence that anneals to miR-122 is recognized by the heterogeneous ribonucleoprotein K, a multifunctional RNA-binding protein known to be involved in RNA processing, translation, and the replication of several RNA viruses [13,14,15]
A 22-nt miR-122 modified with a Cyanine 5 (Cy5) dye at its 5Ј terminus was used as a probe (Fig. 1A)
Summary
HnRNP K Binds miR-122 scribed [15] and stored in aliquots at Ϫ80 °C until use. MiR-122 and variants, including those modified with fluorophores, were chemically synthesized (Bioneer, Alameda CA). The coverslip was removed and the chip was washed with 500 ml of phosphate buffered saline (PBS) amended with diethylpyrocarbonate and 0.05% Triton-X100 for 10 min. To quantify the relative amount of each protein spot on the human proteome chip, the chip was further probed with 80 l of 1 g/ml diluted DyLightTM 549-conjugated anti-GST monoclonal antibody (Rockland) in diethylpyrocarbonate-PBS and incubated for 45 min at 37 °C, 8 rpm. The lysate was centrifuged at 16,000 g for 30 min, and the supernatant was collected and incubated at 4 °C for 3 h with protein A/G beads conjugated with mouse anti-human hnRNP K antibody (Abcam, Thermo Fisher Scientific, catalogue number Ab39975) equilibrated with Nonidet P-40 lysis buffer or a goat anti-mouse IgG control antibody treated in the same way (Santa Cruz). MiR-122 levels were quantified using the protocol of Livak and Schmittgen [19]
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