Browse
Submit Data
Databases
API
Help

Dataset Information

8 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Nucleolus localization of SpyCas9 affects its stability and interferes with host protein translation in mammalian cells.

ABSTRACT: The CRISPR/Cas9 system, originally derived from the prokaryotic adaptive immune system, has been developed as efficient genome editing tools. It enables precise gene manipulation on chromosomal DNA through the specific binding of programmable sgRNA to target DNA, and the Cas9 protein, which has endonuclease activity, will cut a double strand break at specific locus. However, Cas9 is a foreign protein in mammalian cells, and the potential risks associated with its introduction into mammalian cells are not fully understood. In this study, we performed pull-down and mass spectrometry (MS) analysis of Streptococcus pyogenes Cas9 (SpyCas9) interacting proteins in HEK293T cells and showed that the majority of Cas9-associated proteins identified by MS were localized in the nucleolus. Interestingly, we further discovered that the Cas9 protein contains a sequence encoding a nucleolus detention signal (NoDS). Compared with wild-type (WT) Cas9, NoDS-mutated variants of Cas9 (mCas9) are less stable, although their gene editing activity is minimally affected. Overexpression of WT Cas9, but not mCas9, causes general effects on transcription and protein translation in the host cell. Overall, identification of NoDS in Cas9 will improve the understanding of Cas9's biological function in vivo, and the removal of NoDS in Cas9 may enhance its safety for future clinical use.

SUBMITTER: Tan R

PROVIDER: S-EPMC9243344 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Mammalian TRIM71 as repressor of mRNAs that inhibits translation and affects mRNA stability

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2013-01-22 | E-GEOD-37714 | biostudies-arrayexpress

Mammalian TRIM71 as repressor of mRNAs that inhibits translation and affects mRNA stability

Project description:This SuperSeries is composed of the SubSeries listed below.

2013-01-22 | GSE37714 | GEO

The DFNA15 deafness mutation affects POU4F3 protein stability, localization, and transcriptional activity.

Project description:A mutation in the POU4F3 gene (BRN-3.1, BRN3C) is responsible for DFNA15 (MIM 602459), autosomal-dominant nonsyndromic hearing loss. POU4F3 is a member of the POU family of transcription factors and is essential for inner-ear hair cell maintenance. To test the potential effects of the human POU4F3 mutation, we performed a series of experiments in cell culture to mimic the human mutation. Mutant POU4F3 loses most of its transcriptional activity and most of its ability to bind to DNA and does not function in a dominant-negative manner. Moreover, whereas wild-type POU4F3 is found exclusively in the nucleus, our studies demonstrate that the mutant protein is localized both to the nucleus and the cytoplasm. Two nuclear localization signals were identified; both are essential for proper nuclear entry of POU4F3 protein. We found that the mutant protein half-life is longer than that of the wild type. We propose that the combination of defects caused by the mutation on the function of the POU4F3 transcription factor eventually leads to hair cell morbidity in affected family H members.

| S-EPMC262385 | biostudies-literature

NOA36 protein contains a highly conserved nucleolar localization signal capable of directing functional proteins to the nucleolus, in mammalian cells.

Project description:NOA36/ZNF330 is an evolutionarily well-preserved protein present in the nucleolus and mitochondria of mammalian cells. We have previously reported that the pro-apoptotic activity of this protein is mediated by a characteristic cysteine-rich domain. We now demonstrate that the nucleolar localization of NOA36 is due to a highly-conserved nucleolar localization signal (NoLS) present in residues 1-33. This NoLS is a sequence containing three clusters of two or three basic amino acids. We fused the amino terminal of NOA36 to eGFP in order to characterize this putative NoLS. We show that a cluster of three lysine residues at positions 3 to 5 within this sequence is critical for the nucleolar localization. We also demonstrate that the sequence as found in human is capable of directing eGFP to the nucleolus in several mammal, fish and insect cells. Moreover, this NoLS is capable of specifically directing the cytosolic yeast enzyme polyphosphatase to the target of the nucleolus of HeLa cells, wherein its enzymatic activity was detected. This NoLS could therefore serve as a very useful tool as a nucleolar marker and for directing particular proteins to the nucleolus in distant animal species.

| S-EPMC3596294 | biostudies-literature

RNA-GPS Predicts SARS-CoV-2 RNA Localization to Host Mitochondria and Nucleolus.

Project description:The SARS-CoV-2 coronavirus is driving a global pandemic, but its biological mechanisms are less well understood. SARS-CoV-2 is an RNA virus whose multiple genomic and subgenomic RNA (sgRNA) transcripts hijack the host cell's machinery, located across distinct cytotopic locations. Subcellular localization of its viral RNA could play important roles in viral replication and host antiviral immune response. Here we perform computational modeling of SARS-CoV-2 viral RNA localization across eight subcellular neighborhoods. We compare hundreds of SARS-CoV-2 genomes to the human transcriptome and other coronaviruses and perform systematic sub-sequence analyses to identify the responsible signals. Using state-of-the-art machine learning models, we predict that the SARS-CoV-2 RNA genome and all sgRNAs are enriched in the host mitochondrial matrix and nucleolus. The 5' and 3' viral untranslated regions possess the strongest and most distinct localization signals. We discuss the mitochondrial localization signal in relation to the formation of double-membrane vesicles, a critical stage in the coronavirus life cycle. Our computational analysis serves as a hypothesis generation tool to suggest models for SARS-CoV-2 biology and inform experimental efforts to combat the virus.

| S-EPMC7263502 | biostudies-literature

The mammalian host protein DAP5 facilitates the initial round of translation of Coxsackievirus B3 RNA.

Project description:During enteroviral infections, the canonical translation factor eukaryotic translation initiation factor 4 γ I (eIF4GI) is cleaved by viral protease 2A. The resulting C-terminal fragment is recruited by the viral internal ribosome entry site (IRES) for efficient translation of the viral RNA. However, the 2A protease is not present in the viral capsid and is synthesized only after the initial round of translation. This presents the conundrum of how the initial round of translation occurs in the absence of the C-terminal eIF4GI fragment. Interestingly, the host protein DAP5 (also known as p97, eIF4GIII, and eIF4G2), an isoform of eIF4GI, closely resembles the eIF4GI C-terminal fragment produced after 2A protease-mediated cleavage. Using the Coxsackievirus B3 (CVB3) IRES as a model system, here we demonstrate that DAP5, but not the full-length eIF4GI, is required for CVB3 IRES activity for translation of input viral RNA. Additionally, we show that DAP5 is specifically required by type I IRES but not by type II or type III IRES, in which cleavage of eIF4GI has not been observed. We observed that both DAP5 and C-terminal eIF4GI interact with CVB3 IRES in the same region, but DAP5 exhibits a lower affinity for CVB3 IRES compared with the C-terminal eIF4GI fragment. It appears that DAP5 is required for the initial round of viral RNA translation by sustaining a basal level of CVB3 IRES activity. This activity leads to expression of 2A protease and consequent robust CVB3 IRES-mediated translation by the C-terminal eIF4GI fragment.

| S-EPMC6802510 | biostudies-literature

Localization of RNA and translation in the mammalian oocyte and embryo.

Project description:The tight correlation between mRNA distribution and subsequent protein localization and function indicate a major role for mRNA localization within the cell. RNA localization, followed by local translation, presents a mechanism for spatial and temporal gene expression regulation utilized by various cell types. However, little is known about mRNA localization and translation in the mammalian oocyte and early embryo. Importantly, fully-grown oocyte becomes transcriptionally inactive and only utilizes transcripts previously synthesized and stored during earlier development. We discovered an abundant RNA population in the oocyte and early embryo nucleus together with RNA binding proteins. We also characterized specific ribosomal proteins, which contribute to translation in the oocyte and embryo. By applying selected markers to mouse and human oocytes, we found that there might be a similar mechanism of RNA metabolism in both species. In conclusion, we visualized the localization of RNAs and translation machinery in the oocyte, that could shed light on this terra incognita of these unique cell types in mouse and human.

| S-EPMC5846722 | biostudies-literature

High affinity RNA for mammalian initiation factor 4E interferes with mRNA-cap binding and inhibits translation.

Project description:The eukaryotic translation initiation factor 4F (eIF4F) consists of three polypeptides (eIF4A, eIF4G, and eIF4E) and is responsible for recruiting ribosomes to mRNA. eIF4E recognizes the mRNA 5'-cap structure (m7GpppN) and plays a pivotal role in control of translation initiation, which is the rate-limiting step in translation. Overexpression of eIF4E has a dramatic effect on cell growth and leads to oncogenic transformation. Therefore, an inhibitory agent to eIF4E, if any, might serve as a novel therapeutic against malignancies that are caused by aberrant translational control. Along these lines, we developed two RNA aptamers, aptamer 1 and aptamer 2, with high affinity for mammalian eIF4E by in vitro RNA selection-amplification. Aptamer 1 inhibits the cap binding to eIF4E more efficiently than the cap analog m7GpppN or aptamer 2. Consistently, aptamer 1 inhibits specifically cap-dependent in vitro translation while it does not inhibit cap-independent HCV IRES-directed translation initiation. The interaction between eIF4E and eIF4E-binding protein 1 (4E-BP1), however, was not inhibited by aptamer 1. Aptamer 1 is composed of 86 nucleotides, and the high affinity to eIF4E is affected by deletions at both termini. Moreover, relatively large areas in the aptamer 1 fold are protected by eIF4E as determined by ribonuclease footprinting. These findings indicate that aptamers can achieve high affinity to a specific target protein via global conformational recognition. The genetic mutation and affinity study of variant eIF4E proteins suggests that aptamer 1 binds to eIF4E adjacent to the entrance of the cap-binding slot and blocks the cap-binding pocket, thereby inhibiting translation initiation.

| S-EPMC1370693 | biostudies-literature

Translation affects mRNA stability in a codon-dependent manner in human cells.

Project description:mRNA translation decodes nucleotide into amino acid sequences. However, translation has also been shown to affect mRNA stability depending on codon composition in model organisms, although universality of this mechanism remains unclear. Here, using three independent approaches to measure exogenous and endogenous mRNA decay, we define which codons are associated with stable or unstable mRNAs in human cells. We demonstrate that the regulatory information affecting mRNA stability is encoded in codons and not in nucleotides. Stabilizing codons tend to be associated with higher tRNA levels and higher charged/total tRNA ratios. While mRNAs enriched in destabilizing codons tend to possess shorter poly(A)-tails, the poly(A)-tail is not required for the codon-mediated mRNA stability. This mechanism depends on translation; however, the number of ribosome loads into a mRNA modulates the codon-mediated effects on gene expression. This work provides definitive evidence that translation strongly affects mRNA stability in a codon-dependent manner in human cells.

| S-EPMC6529216 | biostudies-literature

Translation of Cellular Protein Localization Using Convolutional Networks.

Project description:Protein localization in cells has been analyzed by fluorescent labeling using indirect immunofluorescence and fluorescent protein tagging. However, the relationships between the localization of different proteins had not been analyzed using artificial intelligence. Here, we applied convolutional networks for the prediction of localization of the cytoskeletal proteins from the localization of the other proteins. Lamellipodia are one of the actin-dependent subcellular structures involved in cell migration and are mainly generated by the Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous protein 2 (WAVE2) and the membrane remodeling I-BAR domain protein IRSp53. Focal adhesion is another actin-based structure that contains vinculin protein and promotes lamellipodia formation and cell migration. In contrast, microtubules are not directly related to actin filaments. The convolutional network was trained using images of actin filaments paired with WAVE2, IRSp53, vinculin, and microtubules. The generated images of WAVE2, IRSp53, and vinculin were highly similar to their real images. In contrast, the microtubule images generated from actin filament images were inferior without the generation of filamentous structures, suggesting that microscopic images of actin filaments provide more information about actin-related protein localization. Collectively, this study suggests that image translation by the convolutional network can predict the localization of functionally related proteins, and the convolutional network might be used to describe the relationships between the proteins by their localization.

| S-EPMC8375474 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data