Genetic, biochemical, and electron microscopic analysis of components involved in transcription coupled mRNA export [Elektronische Ressource] / presented by Maria Luisa Oppizzi

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2004
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 10 Mo

Extrait

Dissertation
Submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the Degree of
Doctor of Natural Sciences
Presented by
Diplom-Biologin: Maria Luisa Oppizzi
Born in : Milan, Italy
Oral examination :Genetic, Biochemical and Electron Microscopic Analysis
of Components Involved in Transcription-Coupled mRNA
Export
Referees: Prof. Dr. Ed Hurt
Dr. Anne EphrussiAcknowledgements
I would like to thank Ed Hurt for giving me the possibility to work on this project and for
being always available to discuss it. I would also like to thank all the members of the lab for
creating a nice and interactive environment.
Grazie alla mia numerosa famiglia, che mi sostiene sempre con forza e amore, anche da
lontano. E alle mie angeli custodi, Alessia, Alessandra e Simona. Grazie per aver creduto in
me in quest’avventura. Sarebbe stata molto piu’ dura senza sapervi e sentirvi vicine. Grazie
anche a tutti i fantastici amici che sono rimasti a Milano, ma hanno la pazienza di seguirmi
sempre per e-mail.
Of course, a very big thank to the great friends I met in the lab and in Heidelberg. Silvi,
Vikram, Susana, Kiki, Tracy, Alex, Oliver and Maribel, thank you for the friendly atmosphere
in the lab and for sharing with me so many moments during these years. It was great to work
with you and to have fun together. A special thanks to Vikram, Susana, Kiki, Tracy, Silvi,
Jochen and Thomas for critically reading the thesis and providing helpful comments on it. I
would like also to thank Anne Ephrussi for support and constructive discussions.
Thomas, grazie per la pazienza, l’incitamento e l’amore che mi dai ogni giorno.Table of Contents
Summary ....................................................................................................................................................... 1
Abbreviations................................................................................................................................................ 3
Introduction .................................................................................................................................................. 5
The nuclear pore complex......................................................................................................................... 5
Nucleocytoplasmic transport .................................................................................................................... 7
The Ran-GTPase system............................................................................................................................ 8
Nuclear export of RNAs............................................................................................................................. 9
Yeast tRNA export.............................................................................................................................. 10
Export of rRNAs ................................................................................................................................. 13
Export of snRNA ................................................................................................................................ 14
Nuclear export of mRNA.................................................................................................................... 15
Cis and trans acting signals that stimulate mRNA export ............................................................ 15
The role of the Mex67/Mtr2 complex in mRNA export............................................................... 16
The role of Yra1 and Sub2 in mRNA export ................................................................................ 17
Nucleoporins, Gle1 and Dbp5 in mRNA export ........................................................................... 19
The role of Gle2 in mRNA export................................................................................................. 20
Aim of the work.......................................................................................................................................... 22
Results.......................................................................................................................................................... 23
Part I: Analysis of the genetic network of GLE2 reveals extended interactions with the NPC....... 23
The gle2Δ sl screen reveals a pleiotropic network of Gle2 interactions at the NPC............................ 23
GLE2 genetically interacts with nucleoporins ....................................................................................... 25
GLE2 is genetically connected to components of the protein import machinery.................................. 27
Components of the mRNA export machinery are genetically linked to GLE2 ...................................... 28
+GLE2 is not required for nuclear poly(A) RNA export. ....................................................................... 29
Part II: TREX is a conserved complex coupling transcription with mRNA export ......................... 31
SUB2 is genetically linked to the THO complex, which acts in transcription elongation.................... 31Table of Contents
Sub2, Yra1, Tho2, Hpr1, Mft1, Thp2 and Tex1 form the transcription-export (TREX) complex......... 32
The core of the TREX complex adopts a symmetric butterfly-like structure......................................... 34
Alternative purifications of the TREX complex...................................................................................... 39
Components of the TREX complex genetically interact with RRP6 and with MTR10, a member of
the importin β-family that might regulate their import.......................................................................... 41
Discussion.................................................................................................................................................... 44
Is Gle2 a nucleoporin or an mRNA export factor? ................................................................................ 44
Coupling transcription elongation to mRNA export via the TREX complex......................................... 49
The TREX complex at the electron microscope...................................................................................... 52
Publications................................................................................................................................................. 55
Materials and Methods.............................................................................................................................. 56
Molecular biological methods................................................................................................................. 56
DNA manipulation.............................................................................................................................. 56
Cloning of plasmids ............................................................................................................................ 56
Genetic methods ...................................................................................................................................... 60
Media for yeast growth and microbiological techniques................................................................... 60
Yeast transformation, plasmid selection, complementation test ....................................................... 61
Yeast plasmids and genomic preparation........................................................................................... 61
Yeast strains ........................................................................................................................................ 61
Isolation of synthetic lethal mutants starting with the gle2Δ, sub2-85, tho2Δ and thp2Δ allele...... 63
Biochemical methods............................................................................................................................... 65
Whole yeast protein extract ................................................................................................................ 65
TAP purification ................................................................................................................................. 66
GST-fusion protein purification ......................................................................................................... 67
Gel filtration chromatography ............................................................................................................ 67
Immunological methods .......................................................................................................................... 67
Western blot ........................................................................................................................................ 67
Transfer of proteins from SDS-PAGE to nitrocellulose.................................................................... 67
Immunological detection of proteins immobilized on nitrocellulose filters..................................... 68
Other methods.......................................................................................................................................... 69Table of Contents
Fluorescence microscopy.................................................................................................................... 69
Electron Microscopy (EM)..................................................