The role of human and Drosophila NXF proteins in nuclear mRNA ...

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1.3.2 Goals and techniques Introduction 32 The central objective of my Ph.D. project was to investigate the role of different human and Drosophila NXF members in nuclear mRNA export (sections 2.1, 2.2 and 2.3). In the first part of this project (section 2.1), I cloned and characterized two human NXF proteins, NXF2 and NXF3, mainly using biochemical approaches. I analyzed their interaction with RNA, nucleoporins and other known TAP partners in vitro, and used reporter-gene assays to test these human TAP homologues for CTE-dependent and general RNA export activity in cultured cells (2.1.1). I have also contributed to the biochemical characterization of human NXF5 (2.1.2). In the second part of this work (section 2.2), I investigated the function of the different NXF members and other mRNA export factors in D. melanogaster. We have used Drosophila Schneider cells in which the expression of endogenous proteins can be efficiently silenced by means of double-stranded RNA interference (RNAi), providing an excellent tool to analyze the function of the individual (potential) export factors in vivo. Three (NXF1-3) of the four NXF members were found to be expressed in this cell line and could be studied. First, a combination of "classical" biochemical and cell biological techniques, such as RNA localization by in situ hybridization and metabolic labeling was applied to analyze the effects caused by the depletion of different NXF members and their heterodimeric interaction partner p15 (2.2.1). Subsequently, a genome-wide analysis of different mRNA export pathways using a combination of RNAi and microarray technology was performed (2.2.2). Apart from screening for potential mRNA substrates of NXF2, NXF3 and Crm1, I also analyzed the effects caused by depleting the essential export factors NXF1, p15 or UAP56. The following questions were addressed in this large-scale analysis: what fraction of mRNAs is affected when NXF1:p15 or UAP56 function is abolished? Are there mRNAs which are still exported despite the general mRNA export block? Can we identify mRNAs requiring NXF1:p15, but not UAP56 function, or vice versa? I also participated in the analysis of the role of the different TAP/NXF1 domains in nuclear mRNA export (section 2.3). To study the human TAP protein, two different mRNA export assays in mammalian cells (both based on CAT reporter mRNAs) were used (2.3.1 and 2.3.2). RNAi in Schneider cells was used to investigate the role of the NTF2-and the UBA-like domain of Drosophila NXF1 in mRNA export in vivo (2.3.2). The last part of this thesis (section 2.4) describes results that were obtained as part of a collaboration using the technique of RNAi in Schneider cells to study the function of Cdc37 (2.4.1).
2 Results/Publications Results/Publications 33 In the following sections, the results obtained during my Ph.D. are presented in the form of journal articles. Each paper is preceded by a short introduction and a summary of results and conclusions. My contribution to each publication is also indicated. The papers are organized into four sections: 2.1 The nuclear export factor family: characterization of human NXF proteins Paper 1: TAP (NXF1) belongs to a multigene family of putative RNA export factors with a conserved modular architecture Herold, A., Suyama, M., Rodrigues, J. P., Braun, I., Kutay, U., Carmo-Fonseca, M., Bork, P., and Izaurralde, E. (2000) Mol Cell Biol 20, 8996-9008. Paper 2: NXF5, a novel member of the nuclear RNA export factor family, is lost in a male patient with a syndromic form of mental retardation Jun, L., Frints, S., Duhamel, H., Herold, A., Abad-Rodrigues, J., Dotti, C., Izaurralde, E., Marynen, P., Froyen, G. (2001) Curr Biol 11, 1381-1391. 2.2 Role of Drosophila NXFs, p15 and UAP56 in nuclear mRNA export Paper 3: NXF1/p15 heterodimers are essential for mRNA nuclear export in Drosophila Herold, A., Klymenko, T., Izaurralde, E. (2001) RNA 7, 1768-1780. Paper 4: Genome-wide analysis of mRNA nuclear export pathways in Drosophila Herold, A., Teixeira L., and Izaurralde, E. (2003) Submitted. 2.3 Functional analysis of TAP/NXF1 domains Paper 5: Overexpression of TAP/p15 heterodimers bypasses nuclear retention and stimulates nuclear mRNA export Braun, I.C., Herold, A., Rode, M., Conti, E., and Izaurralde, E. (2001) J Biol Chem, 276, 20536- 20543. Paper 6: Nuclear export of mRNA by TAP/NXF1 requires two nucleoporin binding sites but not p15 Braun, I. C., Herold, A., Rode, M., and Izaurralde, E. (2002) Mol Cell Biol 22, 5405-5418. 2.4 Role of Cdc37 in chromosome segregation and cytokinesis Paper 7: Cdc37 is essential for chromosome segregation and cytokinesis in higher eukaryotes Lange, B.M.H., Rebollo, E., Herold, A., and Gonzalez, C. (2002) EMBO J 21, 5364-5374. Wherever one of these publications is cited in the thesis, the citation is underlined to indicate that the citation refers to results obtained and presented in this thesis.
Page 1 and 2: The role of human and Drosophila NX
Page 3 and 4: This thesis describes work carried
Page 5 and 6: Table of Contents Table of Contents
Page 7 and 8: Summary Summary 1 A distinguishing
Page 9 and 10: Summary 3 Crm1 resulted in decrease
Page 11 and 12: Zusammenfassung 5 NXF1 durch RNAi v
Page 13 and 14: 1 Introduction 1.1 Nucleocytoplasmi
Page 15 and 16: 1.1.4 The importin ββ-like family
Page 17 and 18: 1.2 The nuclear export of RNAs Intr
Page 19 and 20: Introduction 13 with the export of
Page 21 and 22: Introduction 15 What are the critic
Page 23 and 24: Introduction 17 mRNA export are ind
Page 25 and 26: Introduction 19 The NPC-binding dom
Page 27 and 28: Introduction 21 Association of the
Page 29 and 30: Introduction 23 could trigger ATP-d
Page 31 and 32: Introduction 25 Sub2p and Yra1p and
Page 33 and 34: Introduction 27 In S. cerevisiae, s
Page 35 and 36: Introduction 29 export block of bul
Page 37: Introduction 31 efficiently blocks
Page 41 and 42: Results/Publications 35 shown to in
Page 43 and 44: VOL. 20, 2000 NXF MULTIGENE FAMILY
Page 47 and 48: 9001
Page 55 and 56: Results/Publications 49 2.1.2 Paper
Page 57 and 58: Research Paper 1381 NXF5, a novel m
Page 59 and 60: Research Paper Loss of the NXF5 gen
Page 63 and 64: Figure 4 Research Paper Loss of the
Page 69 and 70: Results/Publications 63 with the nu
Page 71 and 72: RNA (2001), 7:1768-1780+ Cambridge
Page 73 and 74: 1770 A. Herold et al. FIGURE 1. Com
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Page 85 and 86: Results/Publications 79 mRNAs had a
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Herold et al. Results/Publications
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Herold et al. (Supplemental Materia
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Herold et al. (Supplemental Materia
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A 10000 reference 1000 100 10 10000
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A 10 1 -10 3.07 (11) 2.37 4.74 (20)
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A B Northern blot dsRNA sd06908: cl
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GFP day4 NXF1 day2 NXF1 day4 p15 da
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Supplemental Table I. mRNAs not aff
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GH06306 1.86 1.49 2.60 1.65 2.95 2.
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Supplemental Table III: mRNAs that
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Supplemental Table IV: mRNAs that a
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2.3 Functional analysis of TAP/NXF1
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THE JOURNAL OF BIOLOGICAL CHEMISTRY
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20538 FIG. 1. Domain organization o
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20540 FIG. 4. TAP stimulates export
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20542 CRM1-mediated export of U snR
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Results/Publications 127 2.3.2 Pape
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MOLECULAR AND CELLULAR BIOLOGY, Aug
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VOL. 22, 2002 p15-INDEPENDENT NUCLE
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Results/Publications 143 2.4 Role o
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The EMBO Journal Vol. 21 No. 20 pp.
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B.M.H.Lange et al. somes at metapha
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B.M.H.Lange et al. Fig. 5. Aurora B
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B.M.H.Lange et al. Fig. 6. Aurora B
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B.M.H.Lange et al. released along t
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B.M.H.Lange et al. protein kinase i
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Discussion 157 strain, already poin
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Discussion 159 by a (second) UBA-li
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Discussion 161 gradual process requ
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Discussion 163 Although there is no
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Discussion 165 identify mRNAs which
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Human NXFs Discussion 167 While the
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Discussion 169 Therefore, DmNXF1 sh
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References References 171 Aitchison
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References 173 Gadal, O., Strauss,
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References 175 Katahira, J., Strass
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References 177 Murphy, R., Watkins,
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References 179 Strasser, K., Masuda
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Abbreviations Abbreviations 181 ARE
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Curriculum vitae Personal Details N
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Erklärung Hiermit erkläre ich ehr
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