Optogenetic control of nuclear protein export

Niopek, Dominik; Wehler, Pierre; Roensch, Julia; Eils, Roland; Ventura, Barbara Di

doi:10.1038/ncomms10624

Cited by 204 publications

(250 citation statements)

References 57 publications

(89 reference statements)

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“…1A). To test this hypothesis, we exploited a cutting-edge optogenetic approach recently developed by Niopek et al wherein a C-terminal masked NES was designed to be conditionally unmasked under the control of blue light (75). In this system, blue light (480 to 500 nm excitation) destabilizes an Avena sativa phototropin-1 LOV2 core domain (AsLOV2), thus unmasking a rationally designed NES peptide.…”

Section: Resultsmentioning

confidence: 99%

“…A subset of Rev-mChe variants were modified to encode the mApple fluorophore instead of mCherry to avoid spectral overlap with the Quasar 670 dye used for RNA FISH (described below). The LEXY domain was derived from plasmid pLexATF-T2A-NLS-LexA-KRABmCherry-LEXY (kindly provided by Barbara Di Ventura and Roland Eils; Addgene plasmid 72662) (75) and added to the C terminus of Rev-and RevM10-mChe constructs using BsrGI and XhoI cut sites. Plasmids encoding visible intron-retaining gag-pol mRNAs modified to produce Gag fused to cyan fluorescent protein (Gag-CFP) and bearing 24 copies of the MS2 bacteriophage stem-loop (24XMSL) are described elsewhere (76).…”

Section: Methodsmentioning

confidence: 99%

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Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export

Behrens

Aligeti

Pocock

et al. 2017

J Virol

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HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding.IMPORTANCE HIV-1 infects more than 34 million people worldwide causing Ͼ1 million deaths per year. Infectious virion production is activated by the essential viral Rev protein that mediates nuclear export of intron-bearing late-stage viral mRNAs. Rev's shuttling into and out of the nucleus is regulated by the antagonistic activities of both a peptide-encoded N-terminal nuclear localization signal and C-terminal nuclear export signal (NES). How Rev and related viral proteins balance strong import and export activities in order to achieve optimal levels of viral gene expression is incompletely understood. We provide evidence that multimerization provides a mechanism by which Rev transiently masks its NES peptide, thereby biasing its trafficking to and retention within the nucleus. Targeted pharmacological disruption of Rev-Rev interactions should perturb multiple Rev activities, both Rev-RNA binding and Rev's trafficking to the nucleus in the first place.KEYWORDS CRM1, Gag, RNA trafficking, Rev, exportin-1, human immunodeficiency virus, nuclear export signal, nuclear pore complex, nucleolus, retroviruses A core challenge to eukaryotic gene expression is ensuring strong but transient interactions between newly transcribed messenger RNAs (mRNAs) in the nucleus and export receptors at nuclear pore complexes (NPCs) (1-3). For spliced mRNAs, posttranscriptional regulatory factors program export receptor recruitment, the formation of export complexes, and subsequent transit through the hydrophobic core of the NPC (4, 5). mRNA dissociation from the NPC is also crucial and is regulated by RNA

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export

Behrens

Aligeti

Pocock

et al. 2017

J Virol

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“…However, currently all these tools are only bidirectional, either recruiting the protein of interest from the cytoplasm to a specific compartment 4–7,10,15 or translocating it between two different compartments 40 (one in darkness and another in lit conditions). This limitation results from the monochromatic nature of the available protein-targeting tools as they sense light in the same spectral region.…”

Section: Discussionmentioning

confidence: 99%

“…A LOV2 domain of phototropin 1 from Avena sativa ( As LOV2) and a modified PDZ domain (ePDZ) are combined into an optogenetic system based on heterodimerization 4 . As LOV2-based optogenetic tools enable light control of nuclear–cytoplasmic protein shuttling 5–7 . Cryptochrome 2 (CRY2) from Arabidopsis thaliana is another photoreceptor, which initially was applied to PPI heterodimerization approaches 8 .…”

mentioning

confidence: 99%

Near-infrared optogenetic pair for protein regulation and spectral multiplexing

et al. 2017

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Multifunctional optogenetic systems are in high demand for use in basic and biomedical research. Near-infrared-light-inducible binding of bacterial phytochrome BphP1 to its natural PpsR2 partner is beneficial for simultaneous use with blue-light-activatable tools. However, applications of the BphP1–PpsR2 pair are limited by the large size, multidomain structure and oligomeric behavior of PpsR2. Here, we engineered a single-domain BphP1 binding partner, Q-PAS1, which is three-fold smaller and lacks oligomerization. We exploited a helix–PAS fold of Q-PAS1 to develop several near-infrared-light-controllable transcription regulation systems, enabling either 40-fold activation or inhibition. The light-induced BphP1–Q-PAS1 interaction allowed modification of the chromatin epigenetic state. Multiplexing the BphP1–Q-PAS1 pair with a blue-light-activatable LOV-domain-based system demonstrated their negligible spectral crosstalk. By integrating the Q-PAS1 and LOV domains in a single optogenetic tool, we achieved tridirectional protein targeting, independently controlled by near-infrared and blue light, thus demonstrating the superiority of Q-PAS1 for spectral multiplexing and engineering of multicomponent systems.

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“…Indeed, there is a growing need to dissect the circuits that regulate cell and tissue morphology, and uncover the contribution of each node in the development of a new shape. Several optogenetic approaches to modulate gene expression in cell culture and model organisms, including Drosophila , zebrafish, and mouse have been recently developed 45, 67, 68, 69, 70, 71, 72, 73. In combination with gene editing, these tools could be used to control gene expression endogenously.…”

Section: Discussionmentioning

confidence: 99%

Optogenetic Control of Protein Function: From Intracellular Processes to Tissue Morphogenesis

Guglielmi

Falk

Renzis

2016

Trends in Cell Biology

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Optogenetics is an emerging and powerful technique that allows the control of protein activity with light. The possibility of inhibiting or stimulating protein activity with the spatial and temporal precision of a pulse of laser light is opening new frontiers for the investigation of developmental pathways and cell biological bases underlying organismal development. With this powerful technique in hand, it will be possible to address old and novel questions about how cells, tissues, and organisms form. In this review, we focus on the applications of existing optogenetic tools for addressing issues in animal morphogenesis.

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Optogenetic control of nuclear protein export

Cited by 204 publications

References 57 publications

Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export

Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export

Near-infrared optogenetic pair for protein regulation and spectral multiplexing

Optogenetic Control of Protein Function: From Intracellular Processes to Tissue Morphogenesis

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