Characterization of protein and small RNA components of Argonaute complexes [Elektronische Ressource] / Christine Ender

Ender Christine

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 28 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilian-Universität München







Characterization of protein and small RNA
components of Argonaute complexes





Christine Ender

aus

Neuchâtel, Schweiz




2009 Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 der Promotionsordnung vom 29. Januar
1998 von Herrn Prof. Klaus Förstemann betreut.


Ehrenwörtliche Versicherung
Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.


München, am 9.11.2009






_______________________________________






Dissertation eingereicht am 9.11.2009

1. Gutachter Prof. Klaus Förstemann

2. Gutachter Prof. Gunter Meister

Mündliche Prüfung am 17.12.2009
Summary
Members of the Argonaute (Ago) protein family associate with small RNAs and regulate gene
expression at the level of transcription, mRNA stability or translation. Although a number of small
RNAs and proteins involved in gene silencing have been identified so far, the spectrum of Ago-
mediated silencing implies an interplay of various additional factors. We therefore purified Ago
complexes from human cells in order to analyze their protein as well as their RNA content. We
found that Ago1 and Ago2 reside in three complexes of different sizes, showing distinct Dicer
and RNA-induced silencing complex (RISC) activities. The smallest complex is formed by
multiple subcomplexes with distinct enzymatic activities. The two larger complexes form large
ribonucleoprotein particles (RNPs) carrying a variety of different RNA binding or regulatory
proteins, providing new insights into the function of Ago proteins.
The second part of this work focuses on the identification of small RNAs associated with human
Ago1 and Ago2. Endogenous Ago proteins were purified, followed by small RNA cloning and
large-scale sequencing. The majority of the cloned RNAs belong to the class of miRNAs.
Interestingly, individual read numbers suggest preferential Ago binding for at least some
miRNAs. Besides miRNAs, we found RNA fragments mapping to mRNAs, transposons and
other non-coding RNAs like tRNAs. Moreover, small RNAs with a length of about 20-22
nucleotides originating from snoRNAs have been found. The most prominent sequence read
mapping to snoRNA H/ACA45 is capable of guiding Ago2-mediated cleavage of complementary
target RNAs. Furthermore, processing of H/ACA45 is Drosha-independent but depending on
Dicer. Remarkably, the seed sequence of the H/ACA45 processing product is perfectly
complementary in the 3' UTR of several mRNAs. We could demonstrate that H/ACA45-derived
small RNAs possess an endogenous mRNA target that is translationally repressed. Together
with the finding that many snoRNA-derived reads from various deep sequencing libraries are
conserved in mammals, the data suggest that snoRNA processing is a more general mechanism
and snoRNA-derived small RNAs can act as miRNAs.

SUMMARY.................................................................................................................................. 1
1 INTRODUCTION................................................................................................................... 1
1.1 RNA silencing, a historical overview....................................1
1.2 Classes of small RNAs in gene silencing ............................................................................................2
1.3 Biogenesis of miRNAs and siRNAs......................................4
1.3.1 The RNAse III enzymes Drosha and Dicer .......................................................................................7
1.3.2 Argonaute proteins..............................................................9
1.4 MiRNA and siRNA effector complexes ...............................................................................................11
1.5 Regulation of gene expression by miRNAs and siRNAs................................13
1.5.1 Translational repression ...................................................................................................................13
1.5.1.1 Inhibition of translation at initiation steps.................13
1.5.1.2 Inhibition of translation at post-initiation steps ........................................................................15
1.5.1.3 Inhibition of translation: initiation versus post-initiation steps................15
1.5.1.4 MiRNA-mediated deadenylation and destabilization of target mRNAs.15
1.5.2 Other miRNA functions (miRNAs activating gene expression)......................................................17
1.5.3 Transcriptional gene silencing..........................................................................18
1.6 MiRNAs and target mRNAs...................................................19
1.7 PiRNAs......................................................................................................................21
1.7.1 PiRNA function and transposon silencing.......................................................21
1.7.2 PiRNA biogenesis.............................22
1.8 Aim of the thesis.....................................................................................................................................24
2 RESULTS ........................................................... 25
2.1 Analysis of Argonaute containing mRNA protein complexes in human cells ............................25
2.1.1 Ago1 and Ago2 are incorporated into three distinct protein complexes........................................25
2.1.2 RISC and Dicer activity associate with distinct Ago complexes....................27
2.1.3 Ago complex I consists of distinct subcomplexes...........................................29
2.2 Argonaute proteins and their small RNA binding partners ............................................................34
2.2.1 Small RNAs associated with human Ago1 and Ago2.....................................35
2.2.2 SnoRNAs: structure, assembly and function...................................................38
2.2.3 ACA45 processing products are conserved across species..........................................................42
2.2.4 SnoRNA ACA45 is processed to small RNAs.................................................44
2.2.5 ACA45 small RNAs can function like miRNAs................................................46
2.2.6 ACA45 processing is independent of the Drosha/DGCR8 complex..............47
2.2.7 ACA45 processing requires Dicer....................................................................................................48
2.2.8 Validation of an endogenous ACA45-derived small RNA target ...................................................52
2.2.9 Small RNA processing signatures are not restricted to snoACA45...............54
2.2.10 Small RNA reads mapping to tRNAs immunoprecipitate with Ago.............58
2.2.11 tRNA fragments mediate RNA cleavage in vitro...........................................................................58
2.2.12 tRNA processing is Dicer independent..........................................................................................60
3 DISCUSSION...................................................... 62
3.1 Characterization of Ago-containing protein complexes..................................................................62
3.2 Small RNA binding partners of Argonaute proteins........64
3.2.1 A human snoRNA acting as a miRNA .............................................................................................64
3.2.2 Processing of ACA45........................................................65
3.2.3 SnoRNA-derived miRNAs are a general mechanism of small RNA generation...........................68
3.2.4 Orphan snoRNAs..............................................................................................69
3.2.5 tRNA processing ...............................................................70
4 MATERIAL & METHODS.................................................................... 72
4.1 Material .....................................................................................72
4.1.1 Chemicals and enzymes..................................................72
4.1.2 Plasmids ............................................................................................................72
4.1.3 Antibodies..........72
4.1.4 Bacterial strains and cell lines..........................................73
4.1.5 Cell culture media..............................................................................................................................73
4.1.6 Buffers and solutions........................73
4.2 Methods....................................................................................................................................................79
4.2.1 Molecular biological methods...........79
4.2.1.1 General methods.......................79
4.2.1.2 Cloning of 3ʼUTRs from genomic DNA into pMIR-RNL ..........................................................79
4.2.1.3 RNA Extraction from cultured cells ................