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

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Table of Contents II 2.1.2 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 ................ 49 2.2 Role of Drosophila NXFs, p15 and UAP56 in nuclear mRNA export.................... 62 2.2.1 Paper 3: NXF1/p15 heterodimers are essential for mRNA nuclear export in Drosophila.................................................................................................. 62 2.2.2 Paper 4: Genome-wide analysis of mRNA nuclear export pathways in Drosophila.................................................................................................. 78 2.3 Functional analysis of TAP/NXF1 domains .......................................................... 117 2.3.1 Paper 5: Overexpression of TAP/p15 heterodimers bypasses nuclear retention and stimulates nuclear mRNA export ............................................ 117 2.3.2 Paper 6: Nuclear export of mRNA by TAP/NXF1 requires two nucleoporin binding sites but not p15............................................................................... 127 2.4 Role of Cdc37 in chromosome segregation and cytokinesis................................. 143 2.4.1 Paper 7: Cdc37 is essential for chromosome segregation and cytokinesis in higher eukaryotes ..................................................................................... 143 3 Discussion and Perspectives ........................................................ 156 3.1 The NXF:p15 heterodimer acts as a functional unit in mRNA export................ 156 3.2 NXF1:p15 and UAP56 define a single mRNA export pathway in Drosophila................................................................................................................ 159 3.3 Cotranscriptional surveillance might account for decreased mRNA levels after export inhibition .................................................................................. 161 3.4 A role for Crm1 and NXF proteins in mediating alternative mRNA export pathways? ....................................................................................... 163 3.5 RNAi and microarrays: novel approaches to study mRNA export in vivo ........ 169 References .......................................................................................... 171 Abbreviations..................................................................................... 181 Curriculum vitae................................................................................ 183 Publications........................................................................................ 184
Summary Summary 1 A distinguishing feature of eukaryotic cells is the spatial separation of the site of mRNA synthesis (nucleus) from the site of mRNA function (cytoplasm) by the nuclear envelope. As a consequence, mRNAs need to be actively exported from the nucleus to the cytoplasm. At the time when this study was initiated, both human TAP and yeast Mex67p had been proposed to play a role in this process. Work presented in this thesis (section 2.1) revealed that TAP and Mex67p belong to an evolutionarily conserved family of proteins which are characterized by a conserved modular domain organization. This family was termed nuclear export factor (NXF) family. While the yeast genome encodes only one NXF protein (Mex67p), the genomes of higher eukaryotes encode several NXF proteins. There are two nxf genes in C. elegans and A. gambiae, four in D. melanogaster, and at least four in H. sapiens and M. musculus. It was unclear whether, apart from TAP and Mex67p, other members of this family would also be involved in mRNA export. In the first part of this thesis (2.1), several human NXF members were tested for a possible function in nuclear mRNA export. They were analyzed for their interaction with RNA, nucleoporins and other known TAP partners in vitro, and tested for their ability to promote nuclear export of a reporter mRNA in vivo. Using these assays, human NXF2, NXF3 and NXF5 were all shown to interact with the known NXF partner p15. NXF2 and NXF5 were also found to bind directly to RNA, but only NXF2 was able to bind directly to nucleoporins and to promote the nuclear export of an (untethered) reporter mRNA. Thus NXF2 possesses many and NXF3 and NXF5 possess some of the features required to serve as an export receptor for cellular mRNAs. As NXF2 and NXF3 transcripts were mainly found in testis, and the closest orthologue of NXF5 in mouse has the highest levels of expression in brain, these NXF members could potentially serve as tissue-specific mRNA export receptors. In the second part of this work (2.2), the role of different Drosophila NXF proteins and other export factors in mRNA export was investigated using double-stranded RNA interference (RNAi) in Drosophila Schneider cells. Three of the four predicted Drosophila NXF members (NXF1-3) were found to be expressed in this cell line and could be targeted by RNAi. Depletion of endogenous NXF1 inhibited growth and resulted in the nuclear accumulation of polyadenylated RNA. Fluorescence in situ hybridization revealed that export of both heat shock and non-heat shock mRNAs, including intron-containing and intronless mRNAs, was inhibited. Depleting endogenous NXF2 or NXF3 had no apparent phenotype. These results suggested that NXF1 (but not NXF2-NXF4) mediates the export of bulk mRNA in Drosophila cells.
Page 1 and 2: The role of human and Drosophila NX
Page 3 and 4: This thesis describes work carried
Page 5: Table of Contents Table of Contents
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 and 38: Introduction 31 efficiently blocks
Page 39 and 40: 2 Results/Publications Results/Publ
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Page 43 and 44: VOL. 20, 2000 NXF MULTIGENE FAMILY
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Page 55 and 56: Results/Publications 49 2.1.2 Paper
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Research Paper 1381 NXF5, a novel m
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Research Paper Loss of the NXF5 gen
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Figure 4 Research Paper Loss of the
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Results/Publications 63 with the nu
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RNA (2001), 7:1768-1780+ Cambridge
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1770 A. Herold et al. FIGURE 1. Com
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1772 A. Herold et al. FIGURE 3. Dep
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1774 A. Herold et al. FIGURE 5. Dep
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1776 A. Herold et al. FIGURE 7. hsp
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1778 A. Herold et al. 2001)+ Simila
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1780 A. Herold et al. Black BE, Hol
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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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