Protocol for Transfection of Bodo saltans with SaCas9 RNP complex in conjunction with eGFP-NEO plasmid by electroporation v1

protocols.io ◽  
2021 ◽  
Author(s):  
Fatma Gomaa ◽  
Zhu-Hong Li ◽  
Roberto Docampo ◽  
Virginia Edgcomb
Keyword(s):  
Rnp Complex

scholarly journals Electronic Circular Dichroism of the Cas9 Protein and gRNA:Cas9 Ribonucleoprotein Complex

2021 ◽  
Vol 22 (6) ◽  
pp. 2937
Author(s):  
Monika Halat ◽  
Magdalena Klimek-Chodacka ◽  
Jagoda Orleanska ◽  
Malgorzata Baranska ◽  
Rafal Baranski
Keyword(s):  
Circular Dichroism ◽  
Conformational Changes ◽  
Structural Changes ◽  
Electronic Circular Dichroism ◽  
Guide Rna ◽  
Cas9 Protein ◽  
Rnp Complex ◽  
Characteristic Features ◽  
Circular Dichroïsm

The Streptococcus pyogenes Cas9 protein (SpCas9), a component of CRISPR-based immune system in microbes, has become commonly utilized for genome editing. This nuclease forms a ribonucleoprotein (RNP) complex with guide RNA (gRNA) which induces Cas9 structural changes and triggers its cleavage activity. Here, electronic circular dichroism (ECD) spectroscopy was used to confirm the RNP formation and to determine its individual components. The ECD spectra had characteristic features differentiating Cas9 and gRNA, the former showed a negative/positive profile with maxima located at 221, 209 and 196 nm, while the latter revealed positive/negative/positive/negative pattern with bands observed at 266, 242, 222 and 209 nm, respectively. For the first time, the experimental ECD spectrum of the gRNA:Cas9 RNP complex is presented. It exhibits a bisignate positive/negative ECD couplet with maxima at 273 and 235 nm, and it differs significantly from individual spectrum of each RNP components. Additionally, the Cas9 protein and RNP complex retained biological activity after ECD measurements and they were able to bind and cleave DNA in vitro. Hence, we conclude that ECD spectroscopy can be considered as a quick and non-destructive method of monitoring conformational changes of the Cas9 protein as a result of Cas9 and gRNA interaction, and identification of the gRNA:Cas9 RNP complex.

scholarly journals The catalytic activity of the Ubp3 deubiquitinating protease is required for efficient stress granule assembly inS. cerevisiae

2015 ◽  
pp. MCB.00609-15 ◽  
Author(s):  
Regina Nostramo ◽  
Sapna N. Varia ◽  
Bo Zhang ◽  
Megan M. Emerson ◽  
Paul K. Herman
Keyword(s):  
Catalytic Activity ◽  
Stress Granules ◽  
Eukaryotic Cell ◽  
Stress Granule ◽  
Resting Cells ◽  
Term Survival ◽  
General Role ◽  
Rnp Complex ◽  
Processing Body ◽  
Rnp Granules

The interior of the eukaryotic cell is a highly compartmentalized space containing both membrane-bound organelles and the recently-identified nonmembranous ribonucleoprotein (RNP) granules. This study examines inSaccharomyces cerevisiaethe assembly of one conserved type of the latter compartment, known as the stress granule. Stress granules form in response to particular environmental cues and have been linked to a variety of human diseases, including amyotrophic lateral sclerosis. To further our understanding of these structures, a candidate genetic screen was employed to identify regulators of stress granule assembly in quiescent cells. These studies identified a ubiquitin-specific protease, Ubp3, as having an essential role in the assembly of these RNP granules. This function was not shared by other members of the Ubp protease family and required Ubp3 catalytic activity as well as its interaction with the cofactor, Bre5. Interestingly, the loss of stress granules was correlated with a decrease in the long-term survival of stationary phase cells. This phenotype is similar to that observed in mutants defective for the formation of a related RNP complex, the Processing-body. Altogether, these observations raise the interesting possibility of a general role for these types of cytoplasmic RNP granules in the survival of G0-like resting cells.

scholarly journals RECAS9: Recombining wild species introgression via mitotic gene editing in barley

2020 ◽  
Author(s):  
Shelly Lazar ◽  
Manas Ranjan Prusty ◽  
Khaled Bishara ◽  
Amir Sherman ◽  
Eyal Fridman
Keyword(s):  
Wild Species ◽  
Association Studies ◽  
Digital Pcr ◽  
Crop Wild Relatives ◽  
Linkage Drag ◽  
Genome Wide Association Studies ◽  
Starting Point ◽  
Rnp Complex ◽  
Causal Genes ◽  
The Individual

AbstractGenetic loci underlying variation in traits with agronomic importance or genetic risk factors in human diseases have been identified by linkage analysis and genome-wide association studies. However, narrowing down the mapping to the individual causal genes and variations within these is much more challenging, and so is the ability to break linkage drag between beneficial and unfavourable loci in crop breeding. We developed RECAS9 as a transgene-free approach for precisely targeting recombination events by delivering CRISPR/Cas9 ribonucleotide protein (RNP) complex into heterozygous mitotic cells for the barley (Hordeum vulgare) Heat3.1 locus. A wild species (H. spontaneum) introgression in this region carries the agronomical unfavourable tough rachis phenotype (non-brittle) allele linked with a circadian clock accelerating QTL near GIGANTEA gene. We delivered RNP, which was targeted between two single nucleotide polymorphism (SNPs), to mitotic calli cells by particle bombardment. We estimated recombination events by next generation sequencing (NGS) and droplet digital PCR (ddPCR). While NGS analysis grieved from confounding effects of PCR recombination, ddPCR analysis allowed us to associate RNP treatment on heterozygous individuals with significant increase of homologous directed repair (HDR) between cultivated and wild alleles, with recombination rate ranging between zero to 57%. These results show for the first time in plants a directed and transgene free mitotic recombination driven by Cas9 RNP, and provide a starting point for precise breeding and fine scale mapping of beneficial alleles from crop wild relatives.

scholarly journals Differences in Viral RNA Synthesis but Not Budding or Entry Contribute to the In Vitro Attenuation of Reston Virus Compared to Ebola Virus

2020 ◽  
Vol 8 (8) ◽  
pp. 1215
Author(s):  
Bianca S. Bodmer ◽  
Josephin Greßler ◽  
Marie L. Schmidt ◽  
Julia Holzerland ◽  
Janine Brandt ◽  
...  
Keyword(s):  
Life Cycle ◽  
Rna Synthesis ◽  
Ebola Virus ◽  
Severe Disease ◽  
Case Fatality ◽  
Viral Rna ◽  
Pathogenic Potential ◽  
Rnp Complex ◽  
Reston Virus

Most filoviruses cause severe disease in humans. For example, Ebola virus (EBOV) is responsible for the two most extensive outbreaks of filovirus disease to date, with case fatality rates of 66% and 40%, respectively. In contrast, Reston virus (RESTV) is apparently apathogenic in humans, and while transmission of RESTV from domestic pigs to people results in seroconversion, no signs of disease have been reported in such cases. The determinants leading to these differences in pathogenicity are not well understood, but such information is needed in order to better evaluate the risks posed by the repeated spillover of RESTV into the human population and to perform risk assessments for newly emerging filoviruses with unknown pathogenic potential. Interestingly, RESTV and EBOV already show marked differences in their growth in vitro, with RESTV growing slower and reaching lower end titers. In order to understand the basis for this in vitro attenuation of RESTV, we used various life cycle modeling systems mimicking different aspects of the virus life cycle. Our results showed that viral RNA synthesis was markedly slower when using the ribonucleoprotein (RNP) components from RESTV, rather than those for EBOV. In contrast, the kinetics of budding and entry were indistinguishable between these two viruses. These data contribute to our understanding of the molecular basis for filovirus pathogenicity by showing that it is primarily differences in the robustness of RNA synthesis by the viral RNP complex that are responsible for the impaired growth of RESTV in tissue culture.

scholarly journals Genus-specific recruitment of filovirus ribonucleoprotein complexes into budding particles

2011 ◽  
Vol 92 (12) ◽  
pp. 2900-2905 ◽  
Author(s):  
Larissa Spiegelberg ◽  
Victoria Wahl-Jensen ◽  
Larissa Kolesnikova ◽  
Heinz Feldmann ◽  
Stephan Becker ◽  
...  
Keyword(s):  
Matrix Protein ◽  
Spherical Particles ◽  
Target Cells ◽  
Genome Replication ◽  
Replication Competent Virus ◽  
Ribonucleoprotein Complexes ◽  
Virus Morphogenesis ◽  
Rnp Complex ◽  
Model Virus ◽  
Transcription And Replication

The filoviral matrix protein VP40 orchestrates virus morphogenesis and budding. To do this it interacts with both the glycoprotein (GP1,2) and the ribonucleoprotein (RNP) complex components; however, these interactions are still not well understood. Here we show that for efficient VP40-driven formation of transcription and replication-competent virus-like particles (trVLPs), which contain both an RNP complex and GP1,2, the RNP components and VP40, but not GP1,2 and VP40, must be from the same genus. trVLP preparations contained both spherical and filamentous particles, but only the latter were able to infect target cells and to lead to genome replication and transcription. Interestingly, the genus specificity of the VP40–RNP interactions was specific to the formation of filamentous trVLPs, but not to spherical particles. These results not only further our understanding of VP40 interactions, but also suggest that special care is required when using trVLP or VLP systems to model virus morphogenesis.

scholarly journals A U-Rich Element in the 5′ Untranslated Region Is Necessary for the Translation of p27 mRNA

2000 ◽  
Vol 20 (16) ◽  
pp. 5947-5959 ◽  
Author(s):  
S. Sean Millard ◽  
Anxo Vidal ◽  
Maurice Markus ◽  
Andrew Koff
Keyword(s):  
Cell Cycle ◽  
Mrna Stability ◽  
Untranslated Region ◽  
Mrna Processing ◽  
Differentiated Cells ◽  
Specific Subset ◽  
Number Of Factors ◽  
Rnp Complex ◽  
Cycle Dependent

ABSTRACT Increased translation of p27 mRNA correlates with withdrawal of cells from the cell cycle. This raised the possibility that antimitogenic signals might mediate their effects on p27 expression by altering complexes that formed on p27 mRNA, regulating its translation. In this report, we identify a U-rich sequence in the 5′ untranslated region (5′UTR) of p27 mRNA that is necessary for efficient translation in proliferating and nonproliferating cells. We show that a number of factors bind to the 5′UTR in vitro in a manner dependent on the U-rich element, and their availability in the cytosol is controlled in a growth- and cell cycle-dependent fashion. One of these factors is HuR, a protein previously implicated in mRNA stability, transport, and translation. Another is hnRNP C1 and C2, proteins implicated in mRNA processing and the translation of a specific subset of mRNAs expressed in differentiated cells. In lovastatin-treated MDA468 cells, the mobility of the associated hnRNP C1 and C2 proteins changed, and this correlated with increased p27 expression. Together, these data suggest that the U-rich dependent RNP complex on the 5′UTR may regulate the translation of p27 mRNA and may be a target of antimitogenic signals.

scholarly journals Nap1 Links Transcription Elongation, Chromatin Assembly, and Messenger RNP Complex Biogenesis

2008 ◽  
Vol 28 (7) ◽  
pp. 2113-2124 ◽  
Author(s):  
Brian C. Del Rosario ◽  
Lucy F. Pemberton
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromatin Remodeling ◽  
Transcription Elongation ◽  
Mrna Export ◽  
Transcript Level ◽  
Chromatin Assembly ◽  
Regulation Of Transcription ◽  
Histone Chaperones ◽  
Coding Region ◽  
Rnp Complex

ABSTRACT Chromatin remodeling is central to the regulation of transcription elongation. We demonstrate that the conserved Saccharomyces cerevisiae histone chaperone Nap1 associates with chromatin. We show that Nap1 regulates transcription of PHO5, and the increase in transcript level and the higher phosphatase activity plateau observed for Δnap1 cells suggest that the net function of Nap1 is to facilitate nucleosome reassembly during transcription elongation. To further our understanding of histone chaperones in transcription elongation, we identified factors that regulate the function of Nap1 in this process. One factor investigated is an essential mRNA export and TREX complex component, Yra1. Nap1 interacts directly with Yra1 and genetically with other TREX complex components and the mRNA export factor Mex67. Additionally, we show that the recruitment of Nap1 to the coding region of actively transcribed genes is Yra1 dependent and that its recruitment to promoters is TREX complex independent. These observations suggest that Nap1 functions provide a new connection between transcription elongation, chromatin assembly, and messenger RNP complex biogenesis.

Trypanosoma brucei mitochondrial ribonucleoprotein complexes which contain 12S and 9S ribosomal RNAs

Parasitology ◽  
1998 ◽  
Vol 116 (2) ◽  
pp. 157-164 ◽  
Author(s):  
H. H. SHU ◽  
H. U. GÖRINGER
Keyword(s):  
Cell Membrane ◽  
Trypanosoma Brucei ◽  
Translation System ◽  
12S Rrna ◽  
Translation Inhibition ◽  
Translational Activity ◽  
Ribonucleoprotein Complexes ◽  
Rnp Complex ◽  
Synthesis Activity ◽  
Translational Apparatus

Antibiotics have been widely used to identify ribosomal activity in Trypanosoma brucei mitochondria. The validity of some of the results has been questioned because the permeability of the trypanosome cell membrane for some antibiotics was not adequately addressed. Here we describe translation inhibition experiments with digitonin-permeabilized trypanosomes to exclude diffusion barriers through the cell membrane. Using this system we were able to confirm, next to the eukaryotic and thus cycloheximide-sensitive translation system, the existence of a prokaryotic-type translational activity being cycloheximide resistant, chloramphenicol sensitive and streptomycin dependent. We interpret this observation analogous to what has been found for other eukarya as the independent protein synthesis activity of the mitochondrial organelle. We further examined the putative translational apparatus by using isokinetic density-gradient analysis of mitochondrial extracts. The 2 mitochondrially encoded rRNAs, the 9S and 12S rRNAs, were found to co-fractionate in a single RNP complex, approximately 80S in size. This complex disassembled at reduced MgCl2 concentrations into 2 unusually small complexes of 17·5S, containing the 9S rRNA, and 20S containing the 12S rRNA. A preliminary stoichiometry determination suggested a multicopy assembly of these putative subunits in a 2[ratio ]3 ratio (20S[ratio ]17·5S).

scholarly journals AID recruits the RNA exosome to degrade HIV ‐1 nascent transcripts through interaction with the Tat‐P‐ TEF b‐ TAR RNP complex

FEBS Letters ◽  
2018 ◽  
Vol 592 (2) ◽  
pp. 284-294
Author(s):  
Ruixuan Wang ◽  
Xiaowei Zhang ◽  
Haibo Ding ◽  
Ying Qiao ◽  
Xiaoxu Han ◽  
...  
Keyword(s):  
Rnp Complex ◽  
Rna Exosome ◽  
Nascent Transcripts ◽  
Hiv 1
Sign in / Sign up

Export Citation Format

Share Document