State-of-the-art and preparatory work

Translation initiation on eukaryotic mRNAs is mediated by the 5´‐cap, guiding the ribosomal 40S subunit to the mRNA´s 5´‐end. In contrast, translation initiation of Hepatitis C Virus (HCV) RNA is directed by an internal ribosome entry site (IRES) (1), stimulated by the viral 3´‐UTR (2, 3) to ensure that only intact viral RNAs are efficiently translated. The HCV IRES can initiate translation with a minimal set of initiation factors (4). In addition, other RNA‐binding proteins modulate HCV IRES activity (1). However, a tissue specific stimulation of HCV translation could only be attributed to the liver‐specific microRNA‐122 (5). In contrast to the inhibitory interaction of miRNAs with 3´‐UTRs of cellular mRNAs (6), the stimulation of HCV translation by miR‐122 occurs in the 5´‐UTR of the viral RNA (5).

In translation‐competent rabbit reticulocyte lysate (RRL) miR‐122 stimulates HCV translation by interfering with an inhibitory long‐range HCV RNA‐RNA interaction (D. Goergen, MN, unpublished results). In RRL miR‐122 acts only when supplied single‐stranded but not as duplex (5). Thus, RRL does not support unwinding of duplex miRNA precursors and loading of single‐stranded miRNA guide strand into microRNA‐protein (miRNP) complexes, consistent with the finding that miRNA‐mediated inhibition works in RRL only after artificial pre‐hybridization of single‐stranded miRNA to its target mRNA prior to translation (7).

In contrast, in living cells the displacement of the inhibitory long‐range HCV RNA‐RNA interaction plays a minor role, but other mechanisms mediate miR‐122 action (DG, MN, unpublished). This likely involves miRNP complexes (6) forming from unwound miR‐122 duplexes, HCV RNA and proteins. In this project, such miRNP complexes forming with the HCV RNA in living cells will be isolated and their protein components will be analyzed.

Aims

In this project the action of miR‐122 in HCV translation in living cells will be investigated by

I. isolation of miRNP complexes with the HCV RNA from living cells.

II. identification of the miRNP protein components by mass spectrometry.

III. characterization of the function of the identified proteins.

Work programme and methods

I. α‐32P‐UTP‐labeled HCV RNA hybridized to a biotinylated LNA‐oligo will be transfected together with miR‐122 into Huh‐7 hepatoma cells. RNA will be cross‐linked to miRNP proteins in the living cells by UV 254 nm. Alternatively, the use of 4‐thio‐UTP (4‐tU) (8) and cross‐link with UV 366 nm can enhance cross‐link efficiency (D. Dittrich, MN, unpublished). Also miR‐122 can be labeled with α‐32P‐UTP and/or 4‐tU by in vitro‐transcription of an RNA with a hammerhead ribozyme to process the miRNA´s 5´‐end, the miR‐122 sequence and a BsmAI site to process the miRNA´s 3´‐end (J. Henke, MN, unpublished). After UV cross‐link and cell lysis, miRNP complexes can be enriched by sucrose gradient sedimentation (5) and purified with streptavidin beads or antibodies (9) or by gel electrophoresis (10).

II. miRNP complexes will be released from the beads with S7 nuclease, ensuring a low background. Proteins can be separated on an Acclaim C18 RP‐HPLC column (G. Lochnit, Biochemisches Institut), further separated on Laemmli gels, excised and digested. Proteins will be identified on a Bruker Ultraflex I mass spectrometer (GL). This approach has already been successfully used to identify proteins binding to HCV RNA (JH, GL, MN, unpublished).

III. When miRNP complex proteins have been identified, their identity can be confirmed by cross‐link immunoprecitation (CLIP) (9), and their function can be analyzed using RNAi and overexpression approaches. In vitro reconstitution of ribosomal initiation complexes in a system using purified initiation factors, HeLa extract and excess miRNP proteins can result in toeprint analysis of initiation steps (4) in the lab of Ivan Shatsky (Moscow).

References

1. Niepmann, M. (2009) Internal translation initiation of picornaviruses and hepatitis C virus. BBA 1789, 529‐541.

2. Song, Y., Friebe, P., Tzima, E., Junemann, C., Bartenschlager, R. & Niepmann, M. (2006) The hepatitis C virus RNA 3'‐ untranslated region strongly enhances translation directed by the internal ribosome entry site. J Virol 80, 11579‐11588.

3. Bung, C., Bochkaeva, Z., Terenin, I., Zinovkin, R., Shatsky, I.N. & Niepmann, M. (2010) Influence of the hepatitis C virus 3'‐untranslated region on IRES‐dependent and cap‐dependent translation initiation. FEBS Lett 584, 837‐842.

4. Terenin, I. M., Dmitriev, S. E., Andreev, D. E., Shatsky, I. N. (2008) Eukaryotic translation initiation machinery can operate in a bacterial‐like mode without eIF2. Nat Struct Mol Biol 15, 836‐841.

5. Henke, J.I., Goergen, D., Zheng, J., Song, Y., Schuttler, C.G., Fehr, C., Junemann, C. & Niepmann, M. (2008) microRNA‐ 122 stimulates translation of hepatitis C virus RNA. EMBO J 27, 3300‐3310.

6. Chekulaeva, M. & Filipowicz, W. (2009) Mechanisms of miRNA‐mediated post‐transcriptional regulation in animal cells. Curr Opin Cell Biol 21, 452‐460.

7. Wang, B., Love, T.M., Call, M.E., Doench, J.G. & Novina, C.D. (2006) Recapitulation of Short RNA‐Directed Translational Gene Silencing In Vitro. Mol Cell 22, 553‐560.

8. Bartholomew, B., Meares, C.F. & Dahmus, M.E. (1990) Photoaffinity labeling of RNA polymerase III transcription complexes by nascent RNA. J Biol Chem 265, 3731‐3737.

9. Ule, J., Jensen, K., Mele, A. & Darnell,R. B. (2005) CLIP: a method for identifying protein‐RNA interaction sites in living cells. Methods 37, 376‐386.

10. Kawamata, T., Seitz, H. & Tomari, Y. (2009) Structural determinants of miRNAs for RISC loading and slicer‐independent unwinding. Nat Struct Mol Biol 16, 953‐960.

Title for dissertation (prospective)

• Identification of microRNA‐protein complex components acting on the HCV RNA

Relationships/connections within the research training group

Shatsky: Trans‐acting factors in the translation initiation on viral RNAs

Bindereif, Hartmann, Klug: Detailed methodological expertise in studying RNA‐protein interactions by different approaches in vivo and in vitro

Ziebuhr: Analysis of specific protein‐RNA interactions of viral proteins with 5´‐ and 3´‐UTRs

Benefits of the scientific exchange

Cooperation with the Shatsky group has resulted in the successful analysis of HCV 3´‐5´‐end communication (3) and of initiation complexes with the poliovirus IRES (J. Hirnet, IS, MN, unpublished). The future cooperation will allow analysis of initiation complexes together with miRNP proteins, helping to understand their function. With the Bindereif, Hartmann and Klug groups methodological expertise in RNA‐protein interactions is continuously exchanged; this will in particular apply also to the future cooperation with the Ziebuhr group.