scholarly journals zGrad is a nanobody-based degron system that inactivates proteins in zebrafish

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Naoya Yamaguchi ◽  
Tugba Colak-Champollion ◽  
Holger Knaut
Keyword(s):  
Rna Interference ◽  
Protein Function ◽  
Fusion Proteins ◽  
Gene Activity ◽  
Modern Biology ◽  
Tissue Specific ◽  
Inducible Promoters ◽  
Protein Depletion ◽  
Protein Inactivation ◽  
Versatile Tool

The analysis of protein function is essential to modern biology. While protein function has mostly been studied through gene or RNA interference, more recent approaches to degrade proteins directly have been developed. Here, we adapted the anti-GFP nanobody-based system deGradFP from flies to zebrafish. We named this system zGrad and show that zGrad efficiently degrades transmembrane, cytosolic and nuclear GFP-tagged proteins in zebrafish in an inducible and reversible manner. Using tissue-specific and inducible promoters in combination with functional GFP-fusion proteins, we demonstrate that zGrad can inactivate transmembrane and cytosolic proteins globally, locally and temporally with different consequences. Global protein depletion results in phenotypes similar to loss of gene activity, while local and temporal protein inactivation yields more restricted and novel phenotypes. Thus, zGrad is a versatile tool to study the spatial and temporal requirement of proteins in zebrafish.

scholarly journals zGrad: A nanobody-based degron system to inactivate proteins in zebrafish

2019 ◽  
Author(s):  
Naoya Yamaguchi ◽  
Tugba Colak-Champollion ◽  
Holger Knaut
Keyword(s):  
Rna Interference ◽  
Protein Function ◽  
Fusion Proteins ◽  
Gene Activity ◽  
Nuclear Proteins ◽  
Modern Biology ◽  
Tissue Specific ◽  
Inducible Promoters ◽  
Protein Inactivation ◽  
Versatile Tool

AbstractThe analysis of protein function is essential to modern biology. While protein function has mostly been studied through gene or RNA interference, more recent approaches to degrade proteins directly have been developed. Here, we adapted the anti-GFP nanobody-based system deGradFP from flies to zebrafish. We named this system zGrad and show that zGrad efficiently degrades transmembrane, cytosolic and nuclear GFP-tagged proteins in zebrafish in an inducible and reversible manner. Using tissue-specific and inducible promoters in combination with functional GFP-fusion proteins, we demonstrate that zGrad can inactivate transmembrane, cytosolic and nuclear proteins globally, locally and temporally with different consequences. Global protein depletion results in phenotypes similar to loss of gene activity while local and temporal protein inactivation yields more restricted and novel phenotypes. Thus, zGrad is a versatile tool to study the spatial and temporal requirement of proteins in zebrafish.

scholarly journals Targeted Protein Degradation Tools: Overview and Future Perspectives

Biology ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 421
Author(s):  
Yuri Prozzillo ◽  
Gaia Fattorini ◽  
Maria Virginia Santopietro ◽  
Luigi Suglia ◽  
Alessandra Ruggiero ◽  
...  
Keyword(s):  
Rna Interference ◽  
Protein Degradation ◽  
Protein Function ◽  
Signal Sequence ◽  
The Other ◽  
Future Perspectives ◽  
Wild Type ◽  
Design And Optimization ◽  
Protein Inactivation ◽  
Target Effects

Targeted protein inactivation (TPI) is an elegant approach to investigate protein function and its role in the cellular landscape, overcoming limitations of genetic perturbation strategies. These systems act in a reversible manner and reduce off-target effects exceeding the limitations of CRISPR/Cas9 and RNA interference, respectively. Several TPI have been developed and wisely improved, including compartment delocalization tools and protein degradation systems. However, unlike chemical tools such as PROTACs (PROteolysis TArgeting Chimeras), which work in a wild-type genomic background, TPI technologies require adding an aminoacidic signal sequence (tag) to the protein of interest (POI). On the other hand, the design and optimization of PROTACs are very laborious and time-consuming. In this review, we focus on anchor-away, deGradFP, auxin-inducible degron (AID) and dTAG technologies and discuss their recent applications and advances. Finally, we propose nano-grad, a novel nanobody-based protein degradation tool, which specifically proteolyzes endogenous tag-free target protein.

scholarly journals Impact of fluorescent protein fusions on the bacterial flagellar motor

2017 ◽  
Author(s):  
M Heo ◽  
AL Nord ◽  
D Chamousset ◽  
E van Rijn ◽  
HJE Beaumont ◽  
...  
Keyword(s):  
Side Effects ◽  
Protein Function ◽  
Fusion Proteins ◽  
Fluorescent Proteins ◽  
Protein Complexes ◽  
Switching Frequency ◽  
Flagellar Motor ◽  
Internal Dynamics ◽  
Biologically Relevant ◽  
Bacterial Flagellar Motor

AbstractFluorescent fusion proteins open a direct and unique window onto protein function. However, they also introduce the risk of perturbation of the function of the native protein. Successful applications of fluorescent fusions therefore rely on a careful assessment and minimization of the side effects. Such insight, however, is still lacking for many applications of fluorescent fusions. This is particularly relevant in the study of the internal dynamics of motor protein complexes, where both the chemical and mechanical reaction coordinates can be affected. Fluorescent proteins fused to thestatorof the bacterial flagellar motor (BFM) complex have previously been used to successfully unveil the internal subunit dynamics of the motor. Here we report the effects of three different fluorescent proteins fused to the stator, all of which altered BFM behavior. The torque generated by individual stators was reduced while their stoichiometry in the complex remained unaffected. MotB fusions decreased the rotation-direction switching frequency of single motors and induced a novel BFM behavior: a bias-dependent asymmetry in the speed attained in the two rotation directions. All these effects could be mitigated by the insertion of a linker at the fusion point. These findings provide a quantitative account of the effects of fluorescent fusions on BFM dynamics and their alleviation—new insights that advance the use of fluorescent fusions to probe the dynamics of protein complexes.Author summaryMuch of what is known about the biology of proteins was discovered by fusing them to fluorescent proteins that allow detection of their location. But the label comes at a cost: the presence of the tag can alter the behavior of the protein of interest in unforeseen, yet biologically relevant ways. These side effects limit the depth to which fluorescent proteins can be used to probe protein function. One of the systems that has been successfully studied with fluorescent fusions for which these effects have not been addressed are dynamic protein complexes that carry out mechanical work. We examined how fluorescent proteins fused to a component of the bacterial flagellar motor complex impacts its function. Our findings show that the fusion proteins altered biologically relevant dynamical properties of the motor, including induction of a novel mechanical behavior, and demonstrate an approach to alleviate this. These results advance our ability to dissect the bacterial flagellar motor, and the internal dynamics of protein complexes in general, with fluorescent fusion proteins while causing minimal perturbation.

scholarly journals Inducible Systemic RNA Silencing in Caenorhabditis elegans

2003 ◽  
Vol 14 (7) ◽  
pp. 2972-2983 ◽  
Author(s):  
Lisa Timmons ◽  
Hiroaki Tabara ◽  
Craig C. Mello ◽  
Andrew Z. Fire
Keyword(s):  
Rna Interference ◽  
Caenorhabditis Elegans ◽  
Rna Silencing ◽  
Molecular Mechanisms ◽  
Normal Animal ◽  
Cell Types ◽  
Animal Cells ◽  
Tissue Specific ◽  
C Elegans ◽  
Systemic Rna

Introduction of double-stranded RNA (dsRNA) can elicit a gene-specific RNA interference response in a variety of organisms and cell types. In many cases, this response has a systemic character in that silencing of gene expression is observed in cells distal from the site of dsRNA delivery. The molecular mechanisms underlying the mobile nature of RNA silencing are unknown. For example, although cellular entry of dsRNA is possible, cellular exit of dsRNA from normal animal cells has not been directly observed. We provide evidence that transgenic strains of Caenorhabditis elegans transcribing dsRNA from a tissue-specific promoter do not exhibit comprehensive systemic RNA interference phenotypes. In these same animals, modifications of environmental conditions can result in more robust systemic RNA silencing. Additionally, we find that genetic mutations can influence the systemic character of RNA silencing in C. elegans and can separate mechanisms underlying systemic RNA silencing into tissue-specific components. These data suggest that trafficking of RNA silencing signals in C. elegans is regulated by specific physiological and genetic factors.

scholarly journals Alternative promoters and repetitive DNA elements define the species-dependent tissue-specific expression of the FMO1 genes of human and mouse

2007 ◽  
Vol 406 (3) ◽  
pp. 491-499 ◽  
Author(s):  
Elizabeth A. Shephard ◽  
Pritpal Chandan ◽  
Milena Stevanovic-Walker ◽  
Mina Edwards ◽  
Ian R. Phillips
Keyword(s):  
Reporter Gene ◽  
Adult Mouse ◽  
Direct Repeat ◽  
Mouse Gene ◽  
Gene Activity ◽  
Upstream Sequence ◽  
Alternative Promoters ◽  
Specific Expression ◽  
Tissue Specific ◽  
Reporter Gene Activity

In humans, expression of the FMO1 (flavin-containing mono-oxygenase 1) gene is silenced postnatally in liver, but not kidney. In adult mouse, however, the gene is active in both tissues. We investigated the basis of this species-dependent tissue-specific transcription of FMO1. Our results indicate the use of three alternative promoters. Transcription of the gene in fetal human and adult mouse liver is exclusively from the P0 promoter, whereas in extra-hepatic tissues of both species, P1 and P2 are active. Reporter gene assays showed that the proximal P0 promoters of human (hFMO1) and mouse (mFmo1) genes are equally effective. However, sequences upstream (−2955 to −506) of the proximal P0 of mFmo1 increased reporter gene activity 3-fold, whereas hFMO1 upstream sequences (−3027 to −541) decreased reporter gene activity by 75%. Replacement of the upstream sequence of human P0 with the upstream sequence of mouse P0 increased activity of the human proximal P0 8-fold. Species-specific repetitive elements are present immediately upstream of the proximal P0 promoters. The human gene contains five LINE (long-interspersed nuclear element)-1-like elements, whereas the mouse gene contains a poly A region, an 80-bp direct repeat, an LTR (long terminal repeat), a SINE (short-interspersed nuclear element) and a poly T tract. The rat and rabbit FMO1 genes, which are expressed in adult liver, lack some (rat) or all (rabbit) of the elements upstream of mouse P0. Thus silencing of FMO1 in adult human liver is due apparently to the presence upstream of the proximal P0 of L1 (LINE-1) elements rather than the absence of retrotransposons similar to those found in the mouse gene.

Tissue-specific RNA Interference

2006 ◽  
Vol 22 (1) ◽  
pp. 63-76 ◽  
Author(s):  
Melanie Philipp ◽  
Esther T. Stoeckli
Keyword(s):  
Rna Interference ◽  
Tissue Specific

scholarly journals Real Time de novo Deposition of Centromeric Histone-associated Proteins Using the Auxin Inducible Degradation system

2018 ◽  
Author(s):  
Sebastian Hoffmann ◽  
Daniele Fachinetti
Keyword(s):  
Real Time ◽  
Protein Dynamics ◽  
Protein Function ◽  
Genome Engineering ◽  
De Novo ◽  
Protein Complexes ◽  
Versatile Tool ◽  
Associated Proteins ◽  
Endogenous Proteins ◽  
Set Up

i.Summary/AbstractMeasuring protein dynamics is essential to uncover protein function and to understand the formation of large protein complexes such as centromeres. Recently, genome engineering in human cells has improved our ability to study the function of endogenous proteins. By combining genome editing techniques with the Auxin Inducible Degradation (AID) system, we created a versatile tool to study protein dynamics. This system allows us to analyze both protein function and dynamics by enabling rapid protein depletion and re-expression in the same experimental set-up. Here, we focus on the dynamics of the centromeric histone-associated protein CENP-C, responsible for the formation of the kinetochore complex. Following rapid removal and re-activation of a fluorescent version of CENP-C by auxin treatment and removal, we could follow CENP-C de novo deposition at centromeric regions during different stages of the cell cycle. In conclusion, the auxin degradation system is a powerful tool to assess and quantify protein dynamics in real time.

scholarly journals Mapping protein function with CRISPR/Cas9-mediated mutagenesis

2016 ◽  
Author(s):  
Katherine F Donovan ◽  
Mudra Hegde ◽  
Meagan Sullender ◽  
Emma W Vaimberg ◽  
Cory M Johannessen ◽  
...  
Keyword(s):  
Protein Function ◽  
Allelic Diversity ◽  
Mapk Signaling ◽  
Cell Populations ◽  
End Joining ◽  
Allelic Variants ◽  
Genomics Research ◽  
Versatile Tool ◽  
Non Homologous End Joining ◽  
Or Genes

CRISPR/Cas9 screening has proven to be a versatile tool for genomics research. We describe a CRISPR/Cas9-mediated approach to mutagenesis, exploiting the allelic diversity generated by error-prone non-homologous end-joining (NHEJ) to identify gain-of-function alleles of the MAPK signaling pathway genes MEK1 and BRAF. These results illustrate a scalable technique to easily generate cell populations containing thousands of endogenous allelic variants of any gene or genes to map variant functions.

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