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

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Introduction 20 (NXF1=TAP; NXF2, NXF3, NXF5) (Figure 5; Herold et al., 2001; Herold et al., 2000; Tan et al., 2000). In addition, the human genome also encodes two NXF-related sequences, which are likely to represent pseudogenes (NXF4, NXF6). Phylogenetic analysis indicates that the gene duplication events leading to multiple nxf genes have occurred independently in several eukaryotic lineages, so that there is no clear one-to-one relationship of any isoform (Figure 5; Herold et al., 2000). The overall domain organization of NXF proteins is conserved, although not all domains are present in all members (Figure 5; Herold et al., 2001; Herold et al., 2000). The essential roles of S. cerevisiae Mex67p and human TAP in mRNA export have been described above. Interestingly, although there is only one NXF protein in S. pombe (Mex67p), it not essential for mRNA export in this organism. Nevertheless, it has been implicated in this process, as it interacts genetically and physically with Rae1p, an essential mRNA export factor in this organism (Yoon et al., 2000). When this study was initiated, it was unclear whether apart from TAP and Mex67p, other members of this family would be involved in mRNA export. Therefore, it has been a central objective of this thesis to investigate the role of different (human and Drosophila) NXF proteins in mRNA export (see 1.3). A B Figure 5: The NXF family (A) Phylogenetic tree of NXF family sequences as available in June 2002. The tree was drawn by the neighbor-joining method. Only the most conserved region of NXF proteins (corresponding to amino acids 212-539 of human TAP) was considered in the analysis. DmNXF4 and HsNXF6 were excluded from the analysis as they contain few residues in this region. It is evident that in insects the gene duplication resulting in nxf1 and nxf2 occurred before the splitting into Drosophila and Anopheles species. No sequences related to Dm nxf3 and Dm nxf4 could be found in Anopheles, indicating that Dm nxf3 and Dm nxf4 might be evolved by gene duplication after speciation, or the corresponding genes were lost in Anopheles (courtesy of Mikita Suyama). (B) Domain organization of NXF proteins. The Nterminal domains of Mex67p and DmNXF2 do not show any similarity to TAP as indicated by the different colors. The relative sizes of the individual domains are not drawn to scale. Abbreviations: Ag: Anopheles gambiae. Cc: Coturnix coturnis (quail). Ce: Caenorhabditis elegans. Dm: Drosophila melanogaster. Hs: Homo sapiens. Mm: Mus musculus. Rn: Rattus norvegicus. Sc: Saccharomyces cerevisiae. Sp: Schizosaccharomyces pombe.
Introduction 21 Association of the heterodimeric mRNA export receptor with cellular mRNAs: the role of REF proteins and UAP56/Sub2p As noted above, the efficient recruitment of NXF:p15 or Mex67p:Mtr2p heterodimers to cellular mRNAs is believed to depend on adapter proteins. Different RNAbinding proteins that could bridge the interaction between mRNAs and the actual export receptor have been identified. Among these are members of the conserved RNA and export factor-binding (REF) family. The protein Yra1p, one of the two REF members in S. cerevisiae, was originally isolated as a protein with strong RNA-RNA annealing activity, but the relevance of this activity for Yra1p function is still obscure (Portman et al., 1997). A few years later, Yra1p has been implicated in mRNA export as it interacts genetically and physically with Mex67p, and yra1 mutants display strong defects in mRNA nuclear export. Yra1p also displays RNA-binding activity in vitro (Strasser and Hurt, 2000; Stutz et al., 2000; Zenklusen et al., 2001). Similarly, mammalian REF proteins have been shown to bind RNA and TAP directly in vitro (Stutz et al., 2000). Thus, REF proteins could fulfill their role as adapters by directly interacting with RNA and NXF/Mex67p, forming a bridge between the RNA substrate and its export receptor. Furthermore, REFs are shuttling proteins (Rodrigues et al., 2001; Stutz et al., 2000; Zhou et al., 2000). The role of mammalian REF proteins in export was demonstrated in microinjection experiments: recombinant murine REF2 can stimulate the export of mRNAs when injected into Xenopus oocytes. Anti-REF antibodies preventing the interaction of REFs with RNA inhibit the nuclear export of spliced and unspliced mRNAs in this system. Aly (also called REF1-I), another member of the REF family in mouse, is able to interact with Mex67p and partially complements the otherwise lethal yra1 null mutation, showing that the function of REF proteins has been conserved throughout evolution (Strasser and Hurt, 2000). Moreover, mammalian REF is a component of the EJC whose presence on spliced mRNAs also seems to facilitate the recruitment of the nuclear export machinery (see below). As REF proteins are largely dispensable for mRNA export in Drosophila, other adapter proteins which are able to mediate the interaction of NXFs with mRNAs must exist in this organism (Figure 3; see also below). Another protein implicated in the efficient recruitment of Mex67p:Mtr2p or NXF:p15 to mRNAs, is the putative RNA helicase UAP56 (called Sub2p in yeast) which had previously been implicated in spliceosome assembly (Fleckner et al., 1997; Libri et al., 2001). More recently, Sub2p/UAP56 was shown to be essential for bulk mRNA export in S. cerevisiae and D. melanogaster. The export defect caused by the inactivation of Sub2p/UAP56 affects intron-containing and intronless mRNAs (Gatfield et al., 2001; Jensen et al., 2001a; Strasser and Hurt, 2001). This is in agreement with the
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: Introduction 19 The NPC-binding dom
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
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
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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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