Structure-function analysis of heterogeneous nuclear ribonucleoproteins L and LL [Elektronische Ressource] / vorgelegt von Inna Grishina

Inna, Girshina

Sujets

• Biologie

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2010
• Nombre de lectures: 28
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Structure - function analysis of heterogeneous
nuclear ribonucleoproteins L and LL





Dissertation zur Erlangung des akademischen Grades des
Doktors der Naturwissenschaften (Dr. rer. nat.)


eingereicht im Fachbereich Biologie und Chemie
der Justus-Liebig-Universität Gießen






vorgelegt von
Inna Grishina
aus Tula, Russland






Gießen, Juli 2010

Die vorliegende Arbeit wurde am Institut für Biochemie des Fachbereichs 08
der Justus-Liebig-Universität Gießen in der Zeit von Juli 2007 bis Juli 2010
unter der Leitung von Prof. Dr. Albrecht Bindereif angefertigt.



















Dekan: Prof. Dr. Volkmar Wolters
Institut für Tierökologie
Justus-Liebig-Universität Gießen

1. Gutachter: Prof. Dr. Albrecht Bindereif
Institut für Biochemie
Justus-Liebig-Universität Gießen

2. Gutachter: Prof. Dr. Michael Niepmann
Biochemisches Institut,
Fachbereich Medizin
Justus-Liebig-Universität Gießen Contents
Contents

Contents................................................................................................................... 1
Zusammenfassung................................................................................................... 4
Summary.................................................................................................................. 6
1. Introduction........................................................................................................... 7
1.1 Splicing of pre-mRNA ................................................................................................................ 7
1.2 Spliceosome assembly .............................................................................................................. 8
1.3 Alternative splicing ................................................................................................................... 11
1.4 Mechanism of splicing regulation............................................................................................. 13
1.4.1 Splicing enhancers and silencers..................................................................................... 13
1.4.2 SR and SR-related proteins.............................................................................................. 15
1.4.3 HnRNP proteins................................................................................................................ 18
1.5 RNA-binding motifs .................................................................................................................. 19
1.5.1 RNA recognition motif....................................................................................................... 20
1.6 HnRNP L and LL...................................................................................................................... 21
1.7 Alternative splicing and disease............................................................................................... 24
1.8 Specific aims............................................................................................................................ 25
2. Materials and Methods ....................................................................................... 26
2.1 Materials................................................................................................................................... 26
2.1.1 Chemicals and reagents................................................................................................... 26
2.1.2 Nucleotides ....................................................................................................................... 28
2.1.3 Enzymes and enzyme inhibitors....................................................................................... 28
2.1.4 Reaction buffers................................................................................................................ 28
2.1.5 Molecular weight markers................................................................................................. 29
2.1.6 Kits.................................................................................................................................... 29
2.1.7 Plasmids ........................................................................................................................... 29
2.1.8 E.coli strains and cell lines ............................................................................................... 29
2.1.9 Antibodies ......................................................................................................................... 30
2.1.10 DNA oligonucleotides ..................................................................................................... 30
2.1.11 RNA oligonucleotides ..................................................................................................... 34
2.1.12 Other materials ............................................................................................................... 34
2.2 Methods ................................................................................................................................... 34
2.2.1 DNA cloning...................................................................................................................... 34
2.2.1.1 Preparation of plasmid DNA...................................................................................... 34
2.2.1.2 Agarose gel electrophoresis...................................................................................... 35
2.2.1.3 DNA extraction from agarose gels ............................................................................ 35
2.2.1.4 DNA cleavage with restriction enzymes.................................................................... 35
2.2.1.5 Dephosphorylation of DNA........................................................................................ 35
2.2.1.6 Ligation...................................................................................................................... 36
2.2.1.7 Transformation of E.coli cells .................................................................................... 36
2.2.1.8 PCR amplification of DNA ......................................................................................... 36
2.2.2 Generation of hnRNP L and hnRNP LL mutants.............................................................. 36
2.2.3 Expression and purification of proteins in E.coli............................................................... 37
1 Contents
2.2.3.1 GST-tagged hnRNP L, hnRNP LL and their derivatives........................................... 37
2.2.3.2 His-tagged hnRNP L ................................................................................................. 38
2.2.4 Generation and purification of recombinant baculovirus-expressed His-tagged hnRNP L38
2.2.4.1 Infection of insect cells .............................................................................................. 39
2.2.4.2 Purification of His-tagged hnRNP L from baculovirus-infected insect cells .............. 39
2.2.5 In vitro transcription .......................................................................................................... 40
2.2.5.1 Annealing of DNA oligos ........................................................................................... 40
32
2.2.5.2 Transcription of P-labeled RNA .............................................................................. 41
32
2.2.5.3 Gel purification of P-labeled RNA........................................................................... 41
32
2.2.5.4 Transcription without P-label .................................................................................. 42
2.2.5.5 Biotin attachment....................................................................................................... 42
2.2.6 In vitro splicing of pre-mRNAs.......................................................................................... 42
2.2.6.1 Splicing reaction........................................................................................................ 42
2.2.6.2 Proteinase K treatment ............................................................................................. 42
2.2.6.3 Analysis of in vitro splicing by RT-PCR..................................................................... 42
2.2.7 Depletion of hnRNP L from HeLa nuclear extract ............................................................ 43
2.2.8 Electrophoresis of proteins ............................................................................................... 43
2.2.9 Coomassie staining .......................................................................................................... 44
2.2.10 Western blotting.............................................................................................................. 44
2.2.11 Electromobility shift assay (band shift) ..............................................................