scholarly journals Dissecting the import and export pathways of the human RNA helicase UPF1

2021 ◽  
Author(s):  
Andrea B. Eberle ◽  
Karin Schranz ◽  
Sofia Nasif ◽  
Lena Grollmus ◽  
Oliver Muehlemann
Keyword(s):  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Rna Helicase ◽  
Helicase Domain ◽  
Cellular Processes ◽  
Mrna Surveillance ◽  
Localization Signal ◽  
Import And Export ◽  
Export Signal

The RNA helicase UPF1 is best known for its key role in mRNA surveillance but has been implicated in additional cellular processes both in the nucleus and in the cytoplasm. In human cells, the vast majority of UPF1 resides in the cytoplasm and only small amounts can be detected in the nucleus at steady state. It was previously shown that its export from the nucleus to the cytoplasm is Crm1-dependent, yet neither the nuclear export signal (NES) nor the nuclear localization signal (NLS) has been identified. Here, we provide evidence for a noncanonical NLS in UPF1, map the NES to amino acids 89-105 and show that L103 and F105 are essential for UPF1's export to the cytoplasm. Examination of additional UPF1 mutants revealed that a functional helicase domain but not the association with RNA is crucial for the shuttling capacity of UPF1.

p65 controls NF-κB activity by regulating cellular localization of IκBβ

2011 ◽  
Vol 434 (2) ◽  
pp. 253-263 ◽  
Author(s):  
Taras Valovka ◽  
Michael O. Hottiger
Keyword(s):  
Dna Binding ◽  
Nuclear Export ◽  
Cellular Localization ◽  
Nuclear Export Signal ◽  
Nuclear Factor Κb ◽  
Binding Activity ◽  
Cellular Processes ◽  
Dna Binding Activity ◽  
Localization Signal ◽  
Export Signal

NF-κB (nuclear factor κB) controls diverse cellular processes and is frequently misregulated in chronic immune diseases or cancer. The activity of NF-κB is regulated by IκB (inhibitory κB) proteins which control nuclear–cytoplasmic shuttling and DNA binding of NF-κB. In the present paper, we describe a novel role for p65 as a critical regulator of the cellular localization and functions of NF-κB and its inhibitor IκBβ. In genetically modified p65−/− cells, the localization of ectopic p65 is not solely regulated by IκBα, but is largely dependent on the NLS (nuclear localization signal) and the NES (nuclear export signal) of p65. Furthermore, unlike IκBα, IκBβ does not contribute to the nuclear export of p65. In fact, the cellular localization and degradation of IκBβ is controlled by the p65-specific NLS and NES. The results of our present study also reveal that, in addition to stimulus-induced redistribution of NF-κB, changes in the constitutive localization of p65 and IκBβ specifically modulate activation of inflammatory genes. This is a consequence of differences in the DNA-binding activity and signal responsiveness between the nuclear and cytoplasmic NF-κB–IκBβ complexes. Taken together, the findings of the present study indicate that the p65 subunit controls transcriptional competence of NF-κB by regulating the NF-κB/IκBβ pathway.

scholarly journals Control of NF–κB transcriptional activation by signal induced proteolysis of IκBα

1999 ◽  
Vol 354 (1389) ◽  
pp. 1601-1609 ◽  
Author(s):  
R. T. Hay ◽  
L. Vuillard ◽  
J. M. P. Desterro ◽  
M. S. Rodriguez
Keyword(s):  
Nuclear Localization ◽  
Transcriptional Activation ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Cytoplasmic Process ◽  
Leptomycin B ◽  
Rapid Degradation ◽  
Localization Signal ◽  
Export Signal

In unstimulated cells the transcription factor NF–κB is held in the cytoplasm in an inactive state by IκB inhibitor proteins. Ultimately activation of NF–κB is achieved by ubiquitination and proteasome–mediated degradation of IκBα and we have therefore investigated factors which control this proteolysis. Signal–induced degradation of IκBα exposes the nuclear localization signal of NF–κB, thus allowing it to translocate into the nucleus and activate transcription from responsive genes. An autoregulatory loop is established when NF–κB induces expression of the IκBα gene and newly synthesized IκBα accumulates in the nucleus where it negatively regulates NF–κB–dependent transcription. As part of this post–induction repression, the nuclear export signal on IκBα mediates transport of NF–κB–IκBα complexes from the nucleus to the cytoplasm. As nuclear export of IκBα is blocked by leptomycin B this drug was used to examine the effect of cellular location on susceptibility of IκBα to signal–induced degradation. In the presence of leptomycin B, IκBα is accumulated in the nucleus and in this compartment is resistant to signal–induced degradation. Thus signal–induced degradation of IκBα is mainly, if not exclusively a cytoplasmic process. An efficient nuclear export of IκBα is therefore essential for maintaining a low level of IκBα in the nucleus and allowing NF–κB to be transcriptionally active upon cell stimulation. We have detected a modified form of IκBα, conjugated to the small ubiquitin–like protein SUMO–1, which is resistant to signal–induced degradation. SUMO–1 modified IκBα remains associated with NF–κB and thus overexpression of SUMO–1 inhibits the signal–induced activation of NF–κB–dependent transcription. Reconstitution of the conjugation reaction with highly purified proteins demonstrated that in the presence of a novel E1 SUMO–1 activating enzyme, Ubch9 directly conjugated SUMO–1 to IκBα on residues K21 and K22, which are also used for ubiquitin modification. Thus, while ubiquitination targets proteins for rapid degradation, SUMO–1 modification acts antagonistically to generate proteins resistant to degradation.

Nuclear-localization-signal-dependent and nuclear-export-signal-dependent mechanisms determine the localization of 5-lipoxygenase

2002 ◽  
Vol 361 (3) ◽  
pp. 505-514 ◽  
Author(s):  
Hiromi HANAKA ◽  
Takao SHIMIZU ◽  
Takashi IZUMI
Keyword(s):  
Fusion Protein ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Molecular Mechanisms ◽  
Nuclear Export Signal ◽  
Specific Localization ◽  
Localization Signal ◽  
Leukotriene A4 ◽  
Export Signal

5-Lipoxygenase (5-LO) metabolizes arachidonic acid to leukotriene A4, a key intermediate in leukotriene biosynthesis. To explore the molecular mechanisms of its cell-specific localization, a fusion protein between green fluorescent protein (GFP) and human 5-LO (GFP—5LO) was expressed in various cells. GFP—5LO was localized in the cytosol in HL-60 cells and in both the nucleus and the cytosol in RBL (rat basophilic leukaemia) cells, similarly to the native enzyme in these cells. The localization of GFP fusion proteins for mutant 5-LOs in a putative bipartite nuclear localization signal (NLS), amino acids 638–655, in Chinese hamster ovary (CHO)-K1 and Swiss3T3 cells revealed that this motif is important for the nuclear localization of 5-LO. A GFP fusion protein of this short peptide localized consistently in the nucleus. Leptomycin B, a specific inhibitor of nuclear export signal (NES)-dependent transport, diminished the cytosolic localization of 5-LO in HL-60 cells and that of GFP—5LO in CHO-K1 cells, suggesting that an NES-system might also function in determining 5-LO localization. Analysis of the localization of 5-LO during the cell cycle points to a controlled movement of this enzyme. Thus we conclude that a balance of NLS- and NES-dependent mechanisms determines the cell-type-specific localization of 5-LO, suggesting a nuclear function for this enzyme.

scholarly journals Identification of a nuclear localization signal and nuclear export signal of the umbraviral long-distance RNA movement protein

2004 ◽  
Vol 85 (5) ◽  
pp. 1329-1333 ◽  
Author(s):  
Eugene V. Ryabov ◽  
Sang Hyon Kim ◽  
Michael Taliansky
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Viral Rna ◽  
Protein Accumulation ◽  
Long Distance ◽  
Orf3 Protein ◽  
Localization Signal ◽  
Export Signal

The 27 kDa protein encoded by ORF3 of Groundnut rosette virus (GRV) is required for viral RNA protection and movement of viral RNA through the phloem. Localization studies have revealed that this protein is located in nuclei, preferentially targeting nucleoli. We have demonstrated that amino acids (aa) 108–122 of the GRV ORF3 protein contain an arginine-rich nuclear localization signal. Arginine-to-asparagine substitutions in this region decreased the level of the ORF3 protein accumulation in nuclei. A leucine-rich nuclear export signal (NES) was located at aa 148–156 of the GRV ORF3 protein. Leucine-to-alanine substitutions in this region resulted in a dramatic increase in GRV ORF3 protein accumulation in both nuclei and nucleoli. Consistent with this, we also showed that the previously identified NES of BR1 protein of Squash leaf curl virus can functionally replace the leucine-rich region of GRV ORF3 in nuclear export.

scholarly journals Nucleocytoplasmic Recycling of the Nuclear Localization Signal Receptor α Subunit In Vivo Is Dependent on a Nuclear Export Signal, Energy, and RCC1

1997 ◽  
Vol 139 (2) ◽  
pp. 313-325 ◽  
Author(s):  
Irene Boche ◽  
Ellen Fanning
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Nonpermissive Temperature ◽  
Α Subunit ◽  
Localization Signal ◽  
Tsbn2 Cells ◽  
Export Signal

Nuclear protein import requires a nuclear localization signal (NLS) receptor and at least three other cytoplasmic factors. The α subunit of the NLS receptor, Rag cohort 1 (Rch1), enters the nucleus, probably in a complex with the β subunit of the receptor, as well as other import factors and the import substrate. To learn more about which factors and/or events end the import reaction and how the import factors return to the cytoplasm, we have studied nucleocytoplasmic shuttling of Rch1 in vivo. Recombinant Rch1 microinjected into Vero or tsBN2 cells was found primarily in the cytoplasm. Rch1 injected into the nucleus was rapidly exported in a temperature-dependent manner. In contrast, a mutant of Rch1 lacking the first 243 residues accumulated in the nuclei of Vero cells after cytoplasmic injection. After nuclear injection, the truncated Rch1 was retained in the nucleus, but either Rch1 residues 207–217 or a heterologous nuclear export signal, but not a mutant form of residues 207–217, restored nuclear export. Loss of the nuclear transport factor RCC1 (regulator of chromosome condensation) at the nonpermissive temperature in the thermosensitive mutant cell line tsBN2 caused nuclear accumulation of wild-type Rch1 injected into the cytoplasm. However, free Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported. These results suggested that RCC1 acts at an earlier step in Rch1 recycling, possibly the disassembly of an import complex that contains Rch1 and the import substrate. Consistent with this possibility, incubation of purified RanGTP and RCC1 with NLS receptor and import substrate prevented assembly of receptor/substrate complexes or stimulated their disassembly.

Sign in / Sign up

Export Citation Format

Share Document