Nuclear Import Mechanism of the EJC Component Mago-Y14 Revealed by Structural Studies of Importin 13

Bono, Fulvia; Cook, Atlanta G.; Grünwald, Marlene; Ebert, J.; Conti, Elena

doi:10.1016/j.molcel.2010.01.007

Cited by 60 publications

(107 citation statements)

References 46 publications

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“…The closest paralog of Tnpo3 is Importin 13 (Imp13), with which it shares 21.6% amino acid sequence identity. The overall structures of isolated Tnpo3 and of its complex with RanGTP are predictably similar to the corresponding Imp13 structures reported previously (37)(38)(39). Akin to Imp13 and some other β-karyopherins, Tnpo3 is composed of 20 HEAT repeats and adopts an open circular/toroidal shape ( Fig.…”

Section: Resultssupporting

confidence: 82%

“…1A). As in Imp13 and some other β-karyopherins (37,42), the C-terminal HEAT repeat of Tnpo3 is expanded by an additional α-helix. Unliganded Tnpo3 crystallized in the triclinic space group P1 with four molecules per unit cell, forming a pair of homodimers composed of interlocking protein chains (Movie S1 and SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 91%

“…Although Tnpo3 and Imp13 are close paralogs, their modes of cargo engagement are very different. Imp13 has been cocrystallized with two of its nuclear import substrates: Ubc9 and the Mago-Y14 complex (37,38). Unlike Tnpo3, which primarily relies on recognition of the flexible RS domains, Imp13 adapts to engage folded regions of its cargoes.…”

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

Structural basis for nuclear import of splicing factors by human Transportin 3

Maertens

Cook

Wang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

128

167

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Transportin 3 (Tnpo3, Transportin-SR2) is implicated in nuclear import of splicing factors and HIV-1 replication. Herein, we show that the majority of cellular Tnpo3 binding partners contain arginineserine (RS) repeat domains and present crystal structures of human Tnpo3 in its free as well as GTPase Ran-and alternative splicing factor/splicing factor 2 (ASF/SF2)-bound forms. The flexible β-karyopherin fold of Tnpo3 embraces the RNA recognition motif and RS domains of the cargo. A constellation of charged residues on and around the arginine-rich helix of Tnpo3 HEAT repeat 15 engage the phosphorylated RS domain and are critical for the recognition and nuclear import of ASF/SF2. Mutations in the same region of Tnpo3 impair its interaction with the cleavage and polyadenylation specificity factor 6 (CPSF6) and its ability to support HIV-1 replication. Steric incompatibility of the RS domain and RanGTP engagement by Tnpo3 provides the mechanism for cargo release in the nucleus. Our results elucidate the structural bases for nuclear import of splicing factors and the Tnpo3-CPSF6 nexus in HIV-1 biology.T he transport of macromolecules between cytoplasm and nucleus is orchestrated by a family of nuclear import and export receptors (1). Referred to as importins and exportins, these proteins bind their specific cargoes and translocate them across the nuclear pore complex. The process is regulated by the small GTPase Ran that partitions between cytoplasm and nucleus in the predominantly GDP-and GTP-bound form, respectively. Importins associate with their cargoes in the cytoplasm, and the competitive binding of RanGTP induces them to release their cargoes in the nucleus (2). Most nuclear import/export receptors belong to the β-karyopherin family of proteins, with 22 members encoded in the human genome (3). The majority of β-karyopherins bind their cargoes directly, recognizing a linear nuclear localization or export signal and/or a specific tertiary/quaternary structural feature (4, 5).A fundamentally important type of a nuclear localization signal (NLS), comprising sequences rich in Arg-Ser and/or ArgAsp/Glu/Gly dipeptides (referred to as RS or RS-like domains), belongs to the family of Ser/Arg-rich (SR) proteins. These nuclear proteins also contain RNA recognition motif (RRM) domains and play essential roles in pre-mRNA splicing and 3′ processing and participate in transcription regulation, mRNA transport, translation, and nonsense-mediated mRNA decay (6). The splicing factors alternative splicing factor/splicing factor 2 (ASF/SF2) and SC35 along with the cleavage and polyadenylation specificity factor 6 (CPSF6, also known as CF-Im-68) are among the best-characterized metazoan SR proteins (7-10). The SR protein family can be further extended by inclusion of a structurally and functionally diverse group of nuclear proteins that possess RS repeats but lack RRM domains (11). The RS domains are processively phosphorylated on their Ser residues by a set of dedicated kinases (12-16). Phosphorylation of SR proteins is thought to...

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Section: Resultssupporting

confidence: 82%

Section: Resultsmentioning

confidence: 91%

See 1 more Smart Citation

Structural basis for nuclear import of splicing factors by human Transportin 3

Maertens

Cook

Wang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

128

167

View full text Add to dashboard Cite

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“…Similarities between these two proteins are further evident from their primary structure comparisons indicating 25% sequence identity and 44% similarity. Therefore, it was sensible to use the available x-ray crystal structures of Importin-13 (51,66) for the molecular modeling of TNPO3. Although the free full-length Importin-13 structure is not available, the structures of Importin-13 in its complexes with different cargoes revealed a remarkable conformational flexibility of the protein (51,66).…”

Section: Tnpo3 Does Not Interact Directly With Ca Tubes In Vitro-mentioning

confidence: 99%

“…Therefore, it was sensible to use the available x-ray crystal structures of Importin-13 (51,66) for the molecular modeling of TNPO3. Although the free full-length Importin-13 structure is not available, the structures of Importin-13 in its complexes with different cargoes revealed a remarkable conformational flexibility of the protein (51,66). For example, Importin-13 in the complex with Mago-Y14 adopts an open super-helical conformation (51), whereas the Importin 13-Ubc9 complex formed a closed ring conformation (66).…”

Section: Tnpo3 Does Not Interact Directly With Ca Tubes In Vitro-mentioning

confidence: 99%

Interaction of the HIV-1 Intasome with Transportin 3 Protein (TNPO3 or TRN-SR2)

Larue

Gupta

Wuensch

et al. 2012

Journal of Biological Chemistry

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Background: TNPO3 is a key cellular factor involved in early steps of HIV-1 replication. Results: TNPO3 is highly structured, interacts with the HIV-1 intasome by engaging the C-terminal domain of integrase, and does not directly bind capsid tubes. Conclusion: TNPO3 interacts with HIV-1 intasomes and not capsid cores. Significance: Our findings aid future genetic analysis to elucidate the role of TNPO3 in HIV-1 replication.

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Immuno‐detection of mRNA‐binding protein complex in human cells under transmission electron microscopy

Tatsuno

Nakamura

et al. 2019

Microscopy Res & Technique

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Transit from the nuclear complex to the cytoplasm through the nuclear pore complex permits modification of mRNA, including processing such as splicing, capping, and polyadenylation, etc. At each of these events, mRNA interacts with various proteins to form mRNA‐protein complex. Visualizing the mRNA is crucial for understanding the mechanisms underlying mRNA processing and elucidating its structure and recent advances in mRNA imaging allow detection of real‐time mRNA localization in living cells. However, these techniques revealed only the location of mRNA but cannot visualize the conformation of mRNA‐protein complex in cells. On the other hand, transmission electron microscopy has been used to visualize the structure of the Balbiani ring‐derived large mRNA, but their observations were limited to the insect cells. In this study, we visualized the structure of mRNA‐protein complex in human culture cells by using immuno‐electron microscopy. Through immuno‐detection, an mRNA exon junction binding complex Y14, and its binding protien Upf2, different gold particle patterns were imaged with transmission electron microscopy and analyzed. Characteristic linear and stacked particle orientation were observed. Across the nuclear membrane, only linear aggregation pattern was observed, whereas the stacked aggregation pattern was detected in the cytoplasm. Our method is able to visualize mRNA‐conformation and applicable to many cell types, including mammalian cells, where genes can easily be manipulated.

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Nuclear Import Mechanism of the EJC Component Mago-Y14 Revealed by Structural Studies of Importin 13

Cited by 60 publications

References 46 publications

Structural basis for nuclear import of splicing factors by human Transportin 3

Structural basis for nuclear import of splicing factors by human Transportin 3

Interaction of the HIV-1 Intasome with Transportin 3 Protein (TNPO3 or TRN-SR2)

Immuno‐detection of mRNA‐binding protein complex in human cells under transmission electron microscopy

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