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

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Introduction 22 observation that the association of UAP56 with the mRNP occurs cotranscriptionally and independently of splicing, as shown by immunoelectron microscopy using the C. tetans Balbiani ring pre-RNP as a model system (Kiesler et al., 2002). The cotranscriptional association of Sub2p/UAP56 might be related to its ability to interact with a set of factors involved in transcription (see below; Strasser et al., 2002). Mammalian UAP56 interacts directly with Aly and is present together with Aly in the spliced mRNP. Furthermore, microinjection experiments in Xenopus oocytes showed that an excess of human UAP56 inhibits mRNA export, with a concomitant failure to recruit Aly to the spliced mRNP. This export block can be released by coinjecting high amounts of REF (Luo et al., 2001). These data suggest that UAP56 interacts with REF in vivo and facilitates its recruitment to the mRNA. Similarly, yeast Sub2p and Yra1p interact directly in vivo and in vitro. The function of Sub2p in mRNA export has been shown to be related to its ability to bind to Yra1p (Strasser and Hurt, 2001). This and the discovery that the interaction of Sub2p and Mex67p with Yra1p are mutually exclusive lead to the following model: Sub2p associates with the nascent mRNP during transcription and recruits Yra1p to the transcript. The interaction of Mex67p:Mtr2p with Yra1p then displaces Sub2p, rendering the mRNP export-competent (Strasser and Hurt, 2001). By analogy to the yeast model, and as the mammalian proteins UAP56 and Aly were also shown to associate with spliced mRNPs in sequential order, it has been proposed that the function of UAP56 is to recruit Aly, which in turn recruits the heterodimeric export receptor NXF:p15. While the above model of how the heterodimeric mRNA export receptor is recruited to the mRNP (UAP56/Sub2p recruits REF/Yra1p, REF/Yra1p recruits TAP/Mex67p) might be true in yeast, it is probably an oversimplification for higher eukaryotic systems. A study using an RNA interference (RNAi) approach in Drosophila cells demonstrated that the depletion of REF (and of other components of the EJC) does not lead to a complete block of nuclear mRNA export, whereas the depletion of UAP56, NXF1 or p15 does (Gatfield and Izaurralde, 2002; and this study (2.2)). These results show that the essential role of UAP56 in mRNA nuclear export is not restricted to the recruitment of REF, and that there are additional (unidentified) adapter proteins in higher eukaryotes mediating the association of NXF1:p15 heterodimers with cellular mRNAs. These experiments are supported by electron microscopic studies in C. tetans which demonstrate that UAP56 is present on the mRNP in exonic and intronic regions, while REF1 is restricted to regions of the transcripts in which introns have been removed (Kiesler et al., 2002). This shows that the presence of UAP56 on the mRNA does not necessarily result in the recruitment of REF. In this context, it has been suggested that the role of UAP56 in mRNA could be more general. As a putative RNA-helicase, UAP56
Introduction 23 could trigger ATP-dependent rearrangements of the mRNP. This may then facilitate the binding of different proteins which can serve as adapters for NXF:p15 heterodimers (Izaurralde, 2002). Nuclear mRNA export is coupled to other processes: efficient recruitment and quality control Studies over the last few years have shown that virtually all steps in gene expression are closely connected to one another (reviewed by: Maniatis and Reed, 2002; Proudfoot et al., 2002). Likewise, nuclear mRNA export has been linked to transcription, splicing and 3' end formation (Figure 6). Even though the functional relevance of each of these connections is not completely clear yet, the coupling of mRNA export with splicing and 3' end formation would be one way to ensure that incompletely or improperly processed mRNAs do not reach the cytoplasm. mRNA export and splicing In higher eukaryotes, the majority of transcripts contain introns which have to be removed by splicing before nuclear export. Microinjection experiments in Xenopus oocytes demonstrated that, at least in some cases, splicing can enhance subsequent nuclear export, since spliced mRNAs are exported more efficiently than identical mRNAs that have been produced by in vitro transcription using the corresponding cDNA as a template (Le Hir et al., 2001b; Luo and Reed, 1999). This effect was subsequently shown to depend on the deposition of a stable multiprotein-complex, the so-called exon-exon junction complex (EJC). The EJC is deposited 20-24 nucleotides upstream of exon-exon junctions, independent of the sequence there. It is approximately 335 kDa in size and minimally consists of the proteins SRm160, RNPS1, Y14, Magoh, DEK and REF (Kataoka et al., 2001; Le Hir et al., 2001a; Le Hir et al., 2001b; Le Hir et al., 2000a; Le Hir et al., 2000b). Most of the EJC-components had already been directly or indirectly associated with splicing before (e.g. SRm160, DEK, RNPS1), and/or are localized in splicing speckles (e.g. REF, Y14, Magoh) (Blencowe et al., 1998; Kataoka et al., 2000; Le Hir et al., 2001a; Mayeda et al., 1999; McGarvey et al., 2000; Zhou et al., 2000). The EJC is thought to influence different aspects in post-splicing mRNA metabolism, such as mRNA export, translation and stability. Its function in nuclear mRNA export is supported by the fact that some of its constituents (REF, Y14 and Magoh) interact with TAP, possibly facilitating the binding of TAP:p15 heterodimers to spliced mRNPs (Kataoka et al., 2001; Kim et al., 2001; Le Hir et al., 2001b; Stutz et al., 2000). While the EJC is clearly able to promote the association with the export machinery, it is certainly not the only mechanism to recruit TAP:p15 to the mRNP. As mentioned above, the components of the EJC are dispensable for nuclear export in Drosophila (Gatfield and Izaurralde, 2002). Furthermore, both
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: Introduction 21 Association of the
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
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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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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