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

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Introduction 28 antibodies (directed against human Dbp5), or of recombinantly expressed mutant (human) Dbp5 protein inhibits mRNA export in Xenopus oocytes, indicating that the role of Dbp5 in mRNA export is conserved (Schmitt et al., 1999). Both yeast and human Dbp5 have been shown to have ATP-dependent RNA unwinding activity in vitro, although this activity is dependent on the presence of other proteins ("cofactors") (Schmitt et al., 1999; Tseng et al., 1998). The observation that Dbp5 is located at the cytoplasmic side of the NPC has led to the proposal that it might function in the remodeling of mRNPs just after their translocation through the NPC (Schmitt et al., 1999; Tseng et al., 1998). Such remodeling events, which would be dependent on ATP hydrolysis by Dbp5, could confer directionality to mRNA export, and could also contribute to the energy requirements of this process. However, the essential function of Dbp5 in mRNA export does not seem to be conserved in all eukaryotic species, as Dbp5 has recently been shown to be dispensable for mRNA export in Drosophila (Gatfield et al., 2001). Regulated mRNA export Nearly all steps of gene expression can be regulated in eukaryotic cells. Wellknown examples for posttranscriptional regulatory events include alternative splicing, regulated polyadenylation or differential translation. Recently, evidence has been collected that nuclear mRNA export might also be subjected to regulation under certain conditions. Examples of regulated export include the preferential nuclear export of mRNAs encoding heat shock proteins induced by stress, and regulated mRNA export during development. Cells suffering from stress quickly respond by synthesizing heat shock proteins which serve as chaperones to maintain protein stability and folding. Apart from a massive transcriptional induction of heat shock genes, paralleled by the transcriptional downregulation of non-heat shock genes, several posttranscriptional mechanisms ensure the preferential expression of heat shock factors in eukaryotes under stress. In yeast, it has been reported that heat shock mRNAs are selectively and efficiently exported to the cytoplasm under stress, while non-heat shock mRNAs are blocked in the nuclear compartment (Saavedra et al., 1996). A possible mechanism for the export inhibition of non-heat shock mRNAs involves the hnRNP-like protein Nlp3p, which is normally bound to poly(A) + RNA, but dissociates from mRNAs under stress. As Npl3p is essential for general mRNA export, this uncoupling of Nlp3p (and possibly of other RNAbinding proteins) from non-heat shock mRNAs could trigger their retention in the nucleus, rendering the RNPs export-incompetent (Krebber et al., 1999). Furthermore, heat shock mRNA export was initially suggested to occur via a distinct pathway defined by the nucleoporin Rip1p, which is essential for heat shock mRNA export, but was proposed to be dispensable for bulk mRNA export (Saavedra et al., 1997b; Stutz et al., 1997). However, both the selective involvement of Rip1p in heat shock mRNA export and the general
Introduction 29 export block of bulk poly(A) + RNA under stress are discussed controversially in the field, and could not be reproduced in an independent study (Vainberg et al., 2000). In mammalian cells, heat shock induces transcription and enhances stability of mRNAs encoding heat shock proteins. Non-heat shock mRNAs are transcriptionally downregulated and not exported to the cytoplasm, resulting in their nuclear accumulation (Gallouzi et al., 2000; Sadis et al., 1988). Despite this export inhibition affecting bulk mRNA, heat shock mRNAs are exported to the cytoplasm, suggesting that heat shock mRNAs might have access to an export pathway which is not available to non-heat shock mRNAs (Gallouzi et al., 2001). The following model has been proposed to explain these observations: Most stress-related mRNAs contain AU-rich elements (AREs), which are recognized by the HuR protein. HuR binding affects the stability of these mRNAs, and also influences their nuclear export. Under normal growth conditions HuR shuttling can occur through two separate pathways. One pathway depends on the importin β-like protein transportin-2. The other pathway is thought to be mediated by two adapter proteins (pp32 and APRIL) which contain leucine-rich export signals recognized by Crm1, another importin β-like export factor (see 1.1.4). Upon heat shock, the interaction of HuR with transportin-2 is lost, and HuR export depends solely on Crm1. As it is believed that Crm1 is not involved in the export of bulk mRNA (see below), the specific interaction of HuR with ARE-containing mRNAs might allow these mRNAs to be exported (via Crm1), while bulk mRNA export is inhibited (Gallouzi et al., 2001; Gallouzi and Steitz, 2001). This model is in agreement with the observation that the nuclear export of transcriptionally induced hsp70 mRNA is blocked by leptomycin B (LMB), a drug specifically inhibiting Crm1 function. In contrast, general mRNA export is not affected by LMB (Brennan et al., 2000; Gallouzi et al., 2001). One example for a developmentally regulated mRNA export event involves C. elegans tra-1 and tra-2, both being implicated in the terminal steps of sex differentiation. In the absence of TRA-1 protein, tra-2 mRNA is retained in the nucleus. However, binding of TRA-1 to the 3' UTR of tra-2 mRNA overcomes this retention and results in the export of the TRA-1 protein:tra-2 mRNA complex to the cytoplasm. The export of this complex is dependent on Crm1 (see also Discussion; Graves et al., 1999; Segal et al., 2001). Another case of regulated mRNA export occurs in Drosophila tendon differentiation. The transcription factor Stripe plays a key role in this process. Its expression is regulated by two distinct versions of the How protein. In tendon precursor cells, the long version [How(l)] is present, which binds stripe mRNA and inhibits its nuclear export, thus keeping Stripe protein levels low. In later stages of differentiation, a short form of How, How(s), is thought to compete with How(l) for binding stripe mRNA thus releasing the nuclear export block of stripe mRNA. The regulated nuclear export of stripe mRNA is only one of several ways to modulate Stripe activity, as the two How
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: Introduction 27 In S. cerevisiae, s
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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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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