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

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9004 HEROLD ET AL. MOL. CELL. BIOL. FIG. 6. A fraction of NXF2 localizes to the nuclear rim. (A) GST pull-down assays were performed with [ 35 S]methionine-labeled TAP, NXF2, or NXF3, and the recombinant proteins indicated above the lanes. One-tenth of the inputs (lanes 1, 4, and 7) and one-third of the bound fractions (lanes 2, 3, 5, 6, 8, and 9) were analyzed by SDS-PAGE followed by fluorography. (B) NXF2 interacts with multiple components of the NPC. GST pull-down assays were performed with the [ 35 S]methioninelabeled nucleoporins indicated on the left of the panels and recombinant GST or TAP, TAP W594A, TAP D595R, or NXF2 fused to GST, as indicated above the lanes. One-tenth of the input (lane 1) and one-third of the bound fractions (lanes 2 to 6) were analyzed on SDS-PAGE followed by fluorography. (C) GST pull-down assays were performed with the [ 35 S]methionine-labeled nucleoporins indicated on the left of the panels and recombinant GST, GST-NXF2, or various NXF2 mutants as indicated above the lanes. Samples were analyzed as indicated for panel A. (D and E) HeLa cells were transfected with pEGFP-C1 plasmid derivatives expressing GFP fusions of TAP, NXF2, and NFX3 or various TAP or NXF2 mutants as indicated on the left. Approximately 20 h after transfection, the cells were fixed in formaldehyde, permeabilized with Triton X-100, and directly observed with a fluorescence microscope. For all proteins, the GFP signal was detected throughout the nucleoplasm (Tx). Cytoplasmic staining was also detected for NXF3. On the right (Tx), HeLa cells were extracted with Triton X-100 prior to fixation. A punctate labeling pattern was visible at the nuclear periphery for TAP and NXF2, while in cells transfected with NXF3, TAP D595R, or the NXF2 mutants, no GFP signal was detected at the nuclear rim. binding (40). We tested the effect of replacing the three loop residues of NXF2 with alanines. This substitution (3xAla598) disrupts nucleoporin binding in vitro as efficiently as the deletion of the entire NXF2 C-terminal domain (598–626) (Fig. 6C, lanes 7 and 8). As is the case for TAP, a single substitution of alanine for W599 was sufficient to disrupt nucleoporin binding (Fig. 6C, lane 5), while substitution of alanine for N600 or of arginine for E598 had no significant effect (Fig. 6C, lanes 4 and 6).
VOL. 20, 2000 NXF MULTIGENE FAMILY OF TAP-LIKE PROTEINS 9005 FIG. 7. NXF2 exhibits general RNA nuclear export activity. (A to D) Human 293 cells were transfected with a mixture of plasmids encoding -Gal, CAT, and either GFP alone () or fused to the N termini of TAP, NXF2, NXF3, and various TAP and NXF2 mutants as indicated on the left. pEGFP-N3 derivatives encoding zz-tagged versions of p15-1 and p15-2a were cotransfected as indicated. The cells were collected 40 h after transfection, and -Gal and CAT activities were determined. Data from three separate experiments were expressed relative to the activities measured when GFP alone was coexpressed with pDM138. The data are means standard deviations. (B and C) Protein expression levels were analyzed by Western blotting with anti-GFP antibodies. (B) Arrowheads indicate the positions of NXF proteins fused to GFP or of GFP itself, while the asterisks show the positions of p15 proteins. To analyze nucleoporin binding in vivo, the subcellular localization of NXF2 and NXF3 fused to GFP was investigated in transfected HeLa cells and compared to that of GFP-tagged TAP. GFP-tagged NXF2 and NXF3 were evenly detected in the nucleoplasm and were excluded from the nucleolus. Moreover, a fraction of NXF3 could be detected in the cytoplasm (Fig. 6D, Tx). To visualize a potential nuclear envelope association, transfected cells were extracted with Triton X-100 prior to fixation (Fig. 6D, Tx). As previously observed for TAP, a fraction of NXF2 was resistant to detergent extraction and remained associated with the nuclear envelope while NXF3 was not detected at the nuclear rim following Triton extraction. Furthermore, NXF2 and TAP mutants that do not interact with nucleoporins in vitro were not detected at the nuclear envelope upon Triton extraction (Fig. 6E). NXF2, but not NXF3, can stimulate RNA export. We have developed an assay that tests mRNA export stimulation of TAP and its homologues. In this assay, TAP and TAP-like proteins are cotransfected with the reporter plasmid pDM138 (14) in human 293 cells. As mentioned above, transfection with this plasmid yields only trace levels of CAT enzyme activity (13, 14). However, cotransfection with vectors expressing TAP and p15 nuclear retention to be bypassed and export of the unspliced transcripts to be promoted, increasing CAT activity (Fig. 7A). To test the effect of TAP-like proteins on cat gene expression, human 293 cells were cotransfected with a constant amount of pDM138 reporter plasmid, plasmids encoding TAP, NXF2, or NXF3 together with plasmids encoding p15-1 or p15-2a (Fig. 7A and D). Protein expression levels were analyzed by Western blotting (Fig. 7B and C). Overexpression of the TAP protein together with p15-1 increased cat expression by 15- to 17-fold compared to the expression levels obtained when only pDM138 vector was transfected, whereas coexpression of TAP with p15-2a resulted in 13-fold activation of cat expression (Fig. 7A). Coexpression of NXF2 with p15-1 or p15-2a activated cat expression up to 10- or 6-fold, respectively.
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 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 49: VOL. 20, 2000 NXF MULTIGENE FAMILY
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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