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

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9002 HEROLD ET AL. MOL. CELL. BIOL. FIG. 4. Characterization of the N-terminal domains of NXF2 and NXF3. (A) GST pull-down assays were performed with [ 35 S]methionine-labeled TAP, NXF2, NXF3, and the recombinant proteins indicated above the lanes. Lanes 2, background obtained with glutathione agarose beads coated with GST; lanes 3, proteins selected on immobilized GST-E1B-AP5 (fragment 101–453); lanes 4 to 6, binding to GST-REF1-II or fragments of this protein as indicated. In all panels, 1/10 of the inputs (lanes 1) and 1/3 of the bound fractions (lanes 2 to 6) were analyzed on SDS-PAGE followed by fluorography. Supernatant fractions were analyzed in parallel in order to confirm that the absence of binding was not due to protein degradation (data not shown). (B and C) Gel mobility retardation assays were performed using a radiolabeled RNA probe derived from pBS polylinker and purified recombinant proteins fused to GST. (B) TAP or NXF2 N-terminal fragments (50 ng) were used; (C), 1.5 g of the corresponding RNA binding domains were used. Unlabeled competitor tRNA was added as indicated above the lanes. The position of the free RNA probe is indicated. The asterisk indicates the positions of the RNA-protein complexes. (D) A gel mobility retardation assay was performed with reticulocyte lysates unprogrammed (R) or programmed with cDNAs encoding TAP, NXF2, or NXF3. In lane 4, unlabeled M36 competitor RNA was added, while in lanes 5 and 6, CTE competitor RNA was included in the reaction mixtures. The amounts of the competitor RNAs are indicated above the lanes. The position of the free RNA probe is indicated on the left. (E) Schematic representation of pDM138 and pDM138-CTE vectors (14). (F) Quail cells were transfected with plasmids pDM138 or pDM138-CTE along with various plasmids encoding either GFP alone or fused to the N termini of TAP, NXF2, NXF3, or the TAP mutants indicated on the left. TAPNPC has a deletion of residues 541 to 613. The cells were collected 40 h after transfection, and CAT activity was determined. Data from three separate experiments were expressed relative to the activities measured when GFP alone was coexpressed with pDM138 with or without CTE. The data are means standard deviations.
VOL. 20, 2000 NXF MULTIGENE FAMILY OF TAP-LIKE PROTEINS 9003 FIG. 5. p15-2a, a human p15-1 homologue, interacts with TAP and localizes to the nuclear rim. (A) Intron-exon structures of p15 family sequences. Protein coding regions and untranslated regions are colored red and white, respectively. The positions of initiation and termination codons are indicated by green and red triangles, respectively. For human p15-2a and -b, the alternative splicing pathway is shown by lines above the gene. The 5 exon of the p15-2a gene contains an open reading frame of five amino acids, while p15-2b gene 5 exon sequences contain an in-frame stop codon but no in-frame ATG. Therefore, translation of this mRNA may generate a truncated protein starting at methionine 29 in the p15-2a sequence. Alternatively, translation may start at the GTG codon (green open triangle), resulting in an open reading frame (gray), since this region does not show any similarity to the other p15 sequences. (B) Multiple sequence alignment of p15 family sequences. The symbols are as in Fig. 3. (C) Phylogenetic tree of the NTF2 family sequences. The tree was drawn by the neighbor-joining method (34). Abbreviations (other than those in Fig. 1 and 2): At, Arabidopsis thaliana; Nt, Nicotiana tabacum; G3BP, Ras-GAP SH3 domain binding protein; MKK3, MAP kinase kinase 3; NPK2, a tobacco protein kinase. (D) Subcellular localization of p15-2a. HeLa cells were transfected with a pEGFP-N3 plasmid derivative expressing a zz fusion of p15-2a. The fusion protein was detected throughout the nucleoplasm and cytoplasm and was excluded from the nucleolus (left). On the right, HeLa cells were extracted with () Triton X-100 prior to fixation. A punctate labeling pattern was visible at the nuclear periphery for the p15-2a protein. (E) Lysates from E. coli expressing the Ran binding domain of Importin (fragment 1–452) or supplemented with equimolar amounts of NTF2, p15-1, or p15-2a were incubated with IgG-Sepharose beads coated with purified zzRanGDP, zzRanQ69L-GTP, or zzTAP (fragment 61–619). After extensive washes, the bound proteins were eluted. One-hundredth of the inputs (lanes 1 to 4)and 1/10 of the bound fractions (lanes 6 to 16) were analyzed by SDS-PAGE followed by Coomassie blue staining. (F) Lysates from E. coli expressing GST fusions of TAP, NXF2, or NXF3 together with untagged versions of p15-1 or p15-2a were incubated with glutathione agarose beads. After extensive washes, the bound proteins were eluted with SDS sample buffer and analyzed by SDS-PAGE followed by Coomassie blue staining. Lanes 1 to 9, input lysates; lanes 11 to 19, bound fractions; lane 10, molecular mass markers (116, 97, 84, 66, 55, 45, 36, 29, 24, 20, and 14.2 kDa).
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: 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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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. 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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