The reaction mechanism of cellular U snRNP assembly [Elektronische Ressource] / vorgelegt von Ashwin Chari

Ashwin Chari

Sujets

• Biologie

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2009
• Nombre de lectures: 28
• Langue: Deutsch
• Poids de l'ouvrage: 11 Mo

Extrait

The Reaction Mechanism of Cellular U
snRNP Assembly






Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Bayerischen Julius-Maximilians-Universität Würzburg



vorgelegt von


Ashwin Chari


Aus Bangalore (Indien)


Würzburg 2009















Eingereicht am:

Mitglieder der Promotionskommission:
Vorsitzender: Prof. Dr. M. Müller
1. Gutachter: Prof. Dr. U. Fischer
2. Gutachter: Prof. Dr. U. Scheer


Tag des Promotionskolloquiums:
Doktorurkunde ausgehändigt am:
Erklärung

Erklärung gemäss §4 Absatz 3 der Promotionsordnung der Fakultät für Biologie der
Bayerischen Julius-Maximilians-Universität Würzburg vom 15. März 1999

1. Hiermit erkläre ich ehrenwörtlich, dass ich die vorliegende Dissertation selbstständig
angefertigt und keine anderen als die angegebenen Quellen und Hilfsmittel benutzt
habe.

2. Ich erkläre, dass die vorliegende Dissertation weder in gleicher noch in ähnlicher Form
bereits in einem Prüfungsverfahren vorgelegen hat.

3. Ich erkläre, dass ich ausser den mit dem Zulassungsantrag urkundlich vorgelegten
Graden keine weiteren akademischen Grade erworben oder zu erwerben versucht habe.




Würzburg, 2009



Ashwin Chari Table of Contents

1. Summary 1

2. Zusammenfassung 5

3. Introduction 9
3.1 Principles Governing Macromolecular Complex Assembly in Vivo 9
3.2 Pre-mRNA Splicing 12
3.3 Architecture of Spliceosomal U snRNPs 14
3.4 The Cell Biology of U snRNP Biogenesis 16
3.5 U snRNP Assembly in Vivo is an Active, Factor-Mediated Process 19
3.6 References 22

4. Goals of this Thesis 29

5. Results 31
5.1 Taking an Inventory of the Subunits of the Human SMN-Complex 31
5.2 Definition of the Basic Architecture of the Human SMN-49
5.3 Mechanistic Aspects of Cellular U snRNP Assembly 65
5.4 Evolution of the SMN-Complex 115

6. Discussion 129
6.1 The Etiology of Spinal Muscular Atrophy 131
6.2 The Mechanistic Basis of Cellular U snRNP Formation 147

7. Conclusions 169

8. Appendix 171
8.1 The Role of the LSm 1-7 Complex in the Translation an Replication
of Positive-Strand RNA Virus Genomes 173
8.2 Arginine Methylation of Mammalian Pre-mRNA Cleavage Factor I 235
8.3 IGHMBP2 is a Ribosome-Associated Helicase Inactive in the
Neuromuscular Disorder Distal SMA Type 1 291
8.4 A 5´-Fluorobenzoyladenosine-Based Method to Identify
Physiological Substrates of a Drosophila p21-Activated Kinase 325 1. Summary

Macromolecular complexes, also termed molecular machines, facilitate a large spectrum of
biological reactions and tasks crucial to the survival of cells. These complexes are composed
of either protein only, or proteins bound to nucleic acids (DNA or RNA). Prominent examples
for each class are the proteosome, the nucleosome and the ribosome. How such units are
assembled within the context of a living cell is a central question in molecular biology. Earlier
studies had indicated that even very large complexes such as ribosomes could be reconstituted
from purified constituents in vitro. The structural information required for the formation of
macromolecular complexes, hence, lies within the subunits itself and, thus, allow for self-
assembly. However, increasing evidence suggests that in vivo many macromolecular
complexes do not form spontaneously but require assisting factors (“assembly chaperones”)
for their maturation.

In this thesis the assembly of RNA-protein (RNP) complexes has been studied by a
combination of biochemical and structural approaches. A resourceful model system to study
this process is the biogenesis pathway of the uridine-rich small nuclear ribonucleoproteins (U
snRNPs) of the spliceosome. This molecular machine catalyzes pre-mRNA splicing, i.e. the
removal of non-coding introns and the joining of coding exons to functional mRNA. The
composition and architecture of U snRNPs is well defined, also, the nucleo-cytoplasmic
transport events enabling the formation of these particles in vivo have been analyzed in some
detail. Furthermore, recent studies suggest that the formation of U snRNPs in vivo is mediated
by an elaborate assembly machinery consisting of protein arginine methyltransferase
(PRMT5)- and survival motor neuron (SMN)-complexes. The elucidation of the reaction
mechanism of cellular U snRNP assembly would serve as a paradigm for our understanding
of how RNA-protein complexes are formed in the cellular environment.

The following key findings were obtained as part of this study:

1) Efforts were made to establish a full inventory of the subunits of the SMN-complex.
This was achieved by the biochemical definition and characterization of an atypical
component of this complex, the unrip protein. This protein is associated with the SMN-
complex exclusively in the cytoplasm and influences its subcellular localization.

12) With a full inventory of the components in hand, the architecture of the SMN-complex
was defined on the basis of an interaction map of all subunits. This study elucidated that
the proteins SMN, Gemin7 and Gemin8 form a backbone, onto which the remaining
subunits adhere in a modular manner.

3) The two studies mentioned above formed the basis to elucidate the reaction mechanism
of cellular U snRNP assembly. Initially, an early phase in the SMN-assisted formation
of U snRNPs was analyzed. Two subunits of the U7 snRNP (LSm10 and 11) were
found to interact with the PRMT5-complex, without being methylated. This report
suggests that the stimulatory role of the PRMT5-complex is independent of its
methylation activity.

4) Key reaction intermediates in U snRNP assembly were found and characterized by a
combination of biochemistry and structural studies. Initially, a precursor to U snRNPs
with a sedimentation coefficient of 6S is formed by the pICln subunit of the PRMT5-
complex and Sm proteins. This intermediate was shown to constitute a kinetic trap in
the U snRNP assembly reaction. Progression towards the assembled U snRNP depends
on the activity of the SMN-complex, which acts as a catalyst. The formation of U
snRNPs is shown to be structurally similar to the way clamps are deposited onto DNA
to tether poorly processive polymerases.

5) The human SMN-complex is composed of several subunits. However, it is unknown
whether all subunits of this entity are essential for U snRNP assembly. A combination
of bioinformatics and biochemistry was applied to tackle this question. By mining
databases containing whole-genome assemblies, the SMN-Gemin2 heterodimer is
recognized as the most ancestral form of the SMN-complex. Biochemical purification
of the Drosophila melanogaster SMN-complex reveals that this complex is composed of
the same two subunits. Furthermore, evidence is provided that the SMN-Gemin2
heterodimer is necessary and sufficient to promote faithful U snRNP assembly.

Future studies will adress further details in the reaction mechanism of cellular U snRNP
assembly. The results obtained in this thesis suggest that the SMN and Gemin2 subunits are
sufficient to promote U snRNP formation. What then is the function of the remaining subunits
of the SMN-complex? The reconstitution schemes established in this thesis will be
2instrumental to address this question. Furthermore, additional mechanistic insights into the U
snRNP assembly reaction will require the elucidation of structures of the assembly machinery
trapped at various states. The prerequisite for these structural studies, the capability to
generate homogenous complexes in sufficient amounts, has been accomplished in this thesis.
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