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Proteomic and Functional Characterization of Human Argonaute Complexes

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Proteomic and Functional Characterization of Human Argonaute Complexes Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Proteomic and functional characterization of human Argonaute complexes Julia Regina Stöhr, geb. Höck aus Augsburg 2011 Erklärung Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar 1998 (in der Fassung der vierten Änderungssatzung vom 26. November 2004) von Herrn Prof. Gunter Meister betreut und von Herrn Prof. Patrick Cramer von der Fakultät für Chemie und Pharmazie vertreten. Ehrenwörtliche Versicherung Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet. München, den 22.02.2011 _______________________________________ Dissertation eingereicht am 22.02.2011 1. Gutachter Prof. Patrick Cramer 2. Gutachter Prof. Gunter Meister Mündliche Prüfung am 06.04.2011 SUMMARY Proteins from the Argonaute (Ago) family act as key factors of small RNA function. In mammalian somatic cells, the predominant class of small inhibitory RNAs is constituted by microRNAs (miRNAs) with a size of 21-24 nucleotides. They are bound by Ago proteins and guide them to their target mRNAs, thereby facilitating regulation of transcription, mRNA stability and translational repression. The complexity of miRNA-guided cellular events implies that a considerable number of additional factors is involved in controlling and fine-tuning these processes. Details on the underlying regulatory mechanisms, however, remain largely unknown. Therefore, protein complexes containing Ago1 or Ago2 were analyzed for their RNA content as well as for associated protein factors and enzymatic activities. Gradient centrifugation of lysates from human cells revealed three distinct Ago-containing complexes, termed complex I-III, which differed in catalytic activities. While only the smallest complex (complex I) was cleavage competent, both complex I and the largest complex III were able to process a miRNA precursor into mature miRNA. Complex I consists of multiple sub- complexes with distinct enzymatic activities. While all three complexes contain miRNAs, only complex III associates with a translationally repressed mRNA target. For a comprehensive proteomic analysis, proteins that co-purified with ectopically expressed FLAG/HA-tagged Ago1 and Ago2 were identified by mass spectrometry. Besides factors with reported functions in the miRNA pathway, the majority of Ago-associated proteins were implicated in mRNA binding or RNA metabolism. DEAD/DEAH-box containing proteins and heterogeneous nuclear ribonucleoprotein particles were represented in large numbers as well as ribosomal proteins. Ago interaction was verified for a subset of the identified proteins using immunoprecipitation and in vitro pull-down approaches. Luciferase reporter experiments supported a functional relevance for the RNA binding protein RBM4 in miRNA-mediated repression. Moreover, in vitro pull-down approaches confirmed the interaction of RBM4 with all four human Ago proteins. Furthermore, the interaction interface could be narrowed down to the PIWI domain of Ago2, presumably with minor contributions of the N-terminal domain. RBM4 binding appears to be mediated by the second of the two RNA recognition motifs of RBM4 in concert with the Zinc finger domain and the intermediate linker region. Attempting to finally clarify the molecular mechanism of Ago binding to RBM4, first approaches were made towards the identification of common in vivo mRNA targets as well as mRNA binding requirements that allow for efficient interaction of RBM4 and Ago proteins. While a relevance in small RNA biogenesis or RISC activity was not observed, RBM4 might cooperate with Ago proteins in target binding and stabilize the Ago- target interaction, thereby increasing the effectiveness of miRNA-mediated gene regulation. 1 TABLE OF CONTENTS SUMMARY ....................................................................................................................................................... 1 TABLE OF CONTENTS ........................................................................................................................................ 2 1. INTRODUCTION ....................................................................................................................................... 5 1.1. CLASSES OF SMALL RNAS ..................................................................................................................... 6 1.1.1. Small inhibitory RNAs (siRNAs) ........................................................................................................ 6 1.1.2. MicroRNAs (miRNAs) ....................................................................................................................... 8 1.1.2.1. MiRNA biogenesis .................................................................................................................................. 8 1.1.2.2. MiRNA binding to target mRNAs ......................................................................................................... 10 1.1.2.3. Mechanisms of miRNA function .......................................................................................................... 11 1.1.2.3.1. Translational repression ................................................................................................................. 11 1.1.2.3.2. mRNA deadenylation and decay .................................................................................................... 13 1.1.2.3.3. Translational activation .................................................................................................................. 14 1.1.3. Piwi-interacting RNAs (piRNAs) ..................................................................................................... 14 1.2. PROTEINS INVOLVED IN SMALL RNA FUNCTION ............................................................................... 16 1.2.1. The RNase III ribonucleases Drosha and Dicer .............................................................................. 16 1.2.2. Argonaute proteins ....................................................................................................................... 18 1.2.2.1. Structure of Argonaute proteins ......................................................................................................... 18 1.2.2.2. Argonaute loading ............................................................................................................................... 20 1.2.2.3. Argonaute localization to processing bodies ....................................................................................... 21 1.3. THE RNA BINDING PROTEIN RBM4 .................................................................................................... 23 1.4. AIM OF THE THESIS ............................................................................................................................ 25 2. RESULTS ................................................................................................................................................ 26 2.1. ANALYSIS OF ARGONAUTE CONTAINING MRNA-PROTEIN COMPLEXES ............................................ 26 2.1.1. Human Ago1 and Ago2 form distinct protein complexes .............................................................. 26 2.1.2. Ago distribution in nuclear and cytoplasmic extracts ................................................................... 28 2.1.3. Ago complexes I-III associate with miRNAs ................................................................................... 30 2.1.4. Ago complex III co-sediments with the KRAS mRNA ..................................................................... 31 2.1.5. Analysis of Ago-associated RISC and Dicer activity ....................................................................... 32 2.2. ARGONAUTE PROTEINS AND THEIR INTERACTION PARTNERS .......................................................... 35 2.2.1. Proteomic analysis of Ago complexes I-III ..................................................................................... 35 2.2.2. Ago complex I distribution into distinct subcomplexes ................................................................. 40 2.2.3. Sedimentation of co-purified proteins with Ago complexes .......................................................... 42 2.2.4. Verification of Ago-protein-interactions by co-immunoprecipitation ........................................... 43 2.2.5. Analysis of Ago-interactions by in vitro pull-down experiments ................................................... 45 2.2.6. Characterization of the Ago interaction factor PTCD3 .................................................................. 47 2.3. RBM4 AND ITS FUNCTION AS ARGONAUTE INTERACTION PARTNER ................................................ 49 2.3.1. RBM4 is required for miRNA-guided gene silencing ...................................................................... 49 2.3.2. RBM4 characterization and Ago interaction ................................................................................. 53 2.3.3. Identification of the RBM4 domains involved in Ago2 binding ..................................................... 55 2 2.3.4. Effects of RBM4 on RISC activity, Dicer activity and binding ......................................................... 60 2.3.5. RNA recognition motifs as a potential binding platform for Ago proteins .................................... 61 2.3.6. Sequence and structure analysis of the RBM4 RNA recognition
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