scholarly journals Sequence-specific inhibition of reverse transcription by recombinant CRISPR/dCas13a ribonucleoprotein complexes in vitro

2021 ◽  
Author(s):  
Toshitsugu Fujita ◽  
Shoko Nagata ◽  
Miyuki Yuno ◽  
Hodaka Fujii
Keyword(s):  
Dna Sequences ◽  
Reverse Transcription ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Binding Activity ◽  
Guide Rna ◽  
Ribonucleoprotein Complexes ◽  
Specific Manner ◽  
Rnp Complex

Abstract The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for genome editing because of its ability to cleave specific DNA sequences. Recently, RNA-specific CRISPR systems have been reported. CRISPR systems, consisting of a guide RNA (gRNA) and a nuclease-dead form of Cas13a (dCas13a), can be used for RNA editing and visualization of target RNA. In this study, we examined whether a recombinant CRISPR/dCas13a ribonucleoprotein (RNP) complex could be used to inhibit reverse transcription (RT) in a sequence-specific manner in vitro. Recombinant Leptotrichia wadei dCas13a was expressed using the silkworm-baculovirus expression system and affinity-purified. We found that the CRISPR/dCas13a RNP complex, combined with a chemically-synthesized gRNA sequence, could specifically inhibit RT of EGFR and NEAT1, but not non-specific RNA. Thus, the CRISPR/dCas13a RNP complex can inhibit RT reactions in a sequence-specific manner. RT inhibition by the CRISPR/dCas13a system may be useful to assess target binding activity, to discriminate RNA species retaining target sequences of gRNA, or to suppress RT from undesirable RNA species.

scholarly journals Mitochondrial glutamate dehydrogenase from Leishmania tarentolae is a guide RNA-binding protein.

1997 ◽  
Vol 17 (7) ◽  
pp. 3915-3923 ◽  
Author(s):  
F Bringaud ◽  
R Stripecke ◽  
G C Frech ◽  
S Freedland ◽  
C Turck ◽  
...  
Keyword(s):  
Rna Binding ◽  
Leading Edge ◽  
Binding Activity ◽  
Polyclonal Antiserum ◽  
Significant Similarity ◽  
Leishmania Tarentolae ◽  
Guide Rna ◽  
Ribonucleoprotein Complexes ◽  
High Concentrations

To identify specific proteins interacting with guide RNAs (gRNAs) in mitochondrial ribonucleoprotein complexes from Leishmania tarentolae, fractionated and unfractionated mitochondrial extracts were subjected to UV cross-linking with added labeled gRNA and also with [alpha-32P]UTP-labeled endogenous RNA. An abundant 110-kDa protein (p110) localized in the T-V complex, which sediments in glycerol gradients at the leading edge of the 10S terminal uridylyltransferase peak, was found to interact with both types of labeled RNAs. The p110 protein was gel isolated and subjected to microsequence analysis, and the gene was cloned. The sequence revealed significant similarity with mitochondrial glutamate dehydrogenases. A polyclonal antiserum was raised against a recombinant fragment of the p110 gene and was used to demonstrate a stable and specific gRNA-binding activity by coimmunoprecipitation and competitive gel shift analyses. Complex formation was strongly inhibited by competition with poly(U) or by deletion or substitution of the gRNA 3' oligo(U) tail. Also, addition of a 3' oligo(U) tail to an unrelated transcript was sufficient for p110 binding. Both the gRNA-binding activity of the p110 protein and in vitro gRNA-independent and gRNA-dependent uridine insertion activities in the mitochondrial extract were inhibited by high concentrations of dinucleotides.

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 CD28-independent, TRAF2-dependent Costimulation of Resting T Cells by 4-1BB Ligand

1998 ◽  
Vol 187 (11) ◽  
pp. 1849-1862 ◽  
Author(s):  
Katina Saoulli ◽  
Soo Young Lee ◽  
Jennifer L. Cannons ◽  
Wen Chen Yeh ◽  
Angela Santana ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Dominant Negative ◽  
Soluble Form ◽  
Tnf Receptor

4-1BB ligand (4-1BBL) is a member of the tumor necrosis factor (TNF) family expressed on activated antigen-presenting cells. Its receptor, 4-1BB, is a member of the TNF receptor family expressed on activated CD4 and CD8 T cells. We have produced a soluble form of 4-1BBL using the baculovirus expression system. When coimmobilized on plastic with anti-CD3, soluble 4-1BBL induces interleukin (IL)-2 production by resting CD28+ or CD28− T cells, indicating that 4-1BBL can function independently of other cell surface molecules, including CD28, in costimulation of resting T cell activation. At low concentrations of anti-CD3, 4-1BBL is inferior to anti-CD28 in T cell activation. However, when 4-1BB ligand is provided together with strong TCR signals, then 4-1BBL and anti-CD28 are equally potent in stimulation of IL-2 production by resting T cells. We find that TNF receptor–associated factor (TRAF)1 or TRAF2 associate with a glutathione S-transferase–4-1BB cytoplasmic domain fusion protein in vitro. In T cells, we find that association of TRAF1 and TRAF2 with 4-1BB requires 4-1BB cross-linking. In support of a functional role for TRAF2 in 4-1BB signaling, we find that resting T cells isolated from TRAF2-deficient mice or from mice expressing a dominant negative form of TRAF2 fail to augment IL-2 production in response to soluble 4-1BBL. Thus 4-1BB, via the TRAF2 molecule, can provide CD28-independent costimulatory signals to resting T cells.

Identification of a cell-specific DNA-binding activity that interacts with a transcriptional activator of genes expressed in the acinar pancreas

1989 ◽  
Vol 9 (6) ◽  
pp. 2464-2476
Author(s):  
M Cockell ◽  
B J Stevenson ◽  
M Strubin ◽  
O Hagenbüchle ◽  
P K Wellauer
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Dna Interaction ◽  
Binding Activity ◽  
Alpha Amylase ◽  
Dna Binding Activity ◽  
A Cell ◽  
Flanking Regions

Footprint analysis of the 5'-flanking regions of the alpha-amylase 2, elastase 2, and trypsina genes, which are expressed in the acinar pancreas, showed multiple sites of protein-DNA interaction for each gene. Competition experiments demonstrated that a region from each 5'-flanking region interacted with the same cell-specific DNA-binding activity. We show by in vitro binding assays that this DNA-binding activity also recognizes a sequence within the 5'-flanking regions of elastase 1, chymotrypsinogen B, carboxypeptidase A, and trypsind genes. Methylation interference and protection studies showed that the DNA-binding activity recognized a bipartite motif, the subelements of which were separated by integral helical turns of DNA. The alpha-amylase 2 cognate sequence was found to enhance in vivo transcription of its own promoter in a cell-specific manner, which identified the DNA-binding activity as a transcription factor (PTF 1). The observation that PTF 1 bound to DNA sequences that have been defined as transcriptional enhancers by others suggests that this factor is involved in the coordinate expression of genes transcribed in the acinar pancreas.

scholarly journals RNA polymerase II transcription factors H4TF-1 and H4TF-2 require metal to bind specific DNA sequences.

1987 ◽  
Vol 7 (12) ◽  
pp. 4582-4584 ◽  
Author(s):  
L Dailey ◽  
S B Roberts ◽  
N Heintz
Keyword(s):  
Dna Binding ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Chelating Agents ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Rna Polymerase Ii Transcription

Specific DNA-binding and in vitro transcription activities of H4TF-1 and H4TF-2 are inactivated by chelating agents. Binding activity is restored by addition of Zn2+, and H4TF-2 is also reactivated by Fe2+. In contrast, preformed factor-DNA complexes are resistant to chelators. Therefore, metal ions are a required component of the H4TF-1 and H4TF-2 DNA-binding domains.

scholarly journals Identification of a cell-specific DNA-binding activity that interacts with a transcriptional activator of genes expressed in the acinar pancreas.

1989 ◽  
Vol 9 (6) ◽  
pp. 2464-2476 ◽  
Author(s):  
M Cockell ◽  
B J Stevenson ◽  
M Strubin ◽  
O Hagenbüchle ◽  
P K Wellauer
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Dna Interaction ◽  
Binding Activity ◽  
Alpha Amylase ◽  
Dna Binding Activity ◽  
A Cell ◽  
Flanking Regions

Footprint analysis of the 5'-flanking regions of the alpha-amylase 2, elastase 2, and trypsina genes, which are expressed in the acinar pancreas, showed multiple sites of protein-DNA interaction for each gene. Competition experiments demonstrated that a region from each 5'-flanking region interacted with the same cell-specific DNA-binding activity. We show by in vitro binding assays that this DNA-binding activity also recognizes a sequence within the 5'-flanking regions of elastase 1, chymotrypsinogen B, carboxypeptidase A, and trypsind genes. Methylation interference and protection studies showed that the DNA-binding activity recognized a bipartite motif, the subelements of which were separated by integral helical turns of DNA. The alpha-amylase 2 cognate sequence was found to enhance in vivo transcription of its own promoter in a cell-specific manner, which identified the DNA-binding activity as a transcription factor (PTF 1). The observation that PTF 1 bound to DNA sequences that have been defined as transcriptional enhancers by others suggests that this factor is involved in the coordinate expression of genes transcribed in the acinar pancreas.

scholarly journals Adenovirus E1B 55-Kilodalton Oncoprotein Inhibits p53 Acetylation by PCAF

2000 ◽  
Vol 20 (15) ◽  
pp. 5540-5553 ◽  
Author(s):  
Yue Liu ◽  
April L. Colosimo ◽  
Xiang-Jiao Yang ◽  
Daiqing Liao
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Target Genes ◽  
Binding Activity ◽  
Physical Interaction ◽  
Dna Binding Activity ◽  
C Terminus ◽  
Adenovirus E1b

ABSTRACT The adenovirus E1B 55-kDa protein binds to cellular tumor suppressor p53 and inactivates its transcriptional transactivation function. p53 transactivation activity is dependent upon its ability to bind to specific DNA sequences near the promoters of its target genes. It was shown recently that p53 is acetylated by transcriptional coactivators p300, CREB bidning protein (CBP), and PCAF and that acetylation of p53 by these proteins enhances p53 sequence-specific DNA binding. Here we show that the E1B 55-kDa protein specifically inhibits p53 acetylation by PCAF in vivo and in vitro, while acetylation of histones and PCAF autoacetylation is not affected. Furthermore, the DNA-binding activity of p53 is diminished in cells expressing the E1B 55-kDa protein. PCAF binds to the E1B 55-kDa protein and to a region near the C terminus of p53 encompassing Lys-320, the specific PCAF acetylation site. We further show that the E1B 55-kDa protein interferes with the physical interaction between PCAF and p53, suggesting that the E1B 55-kDa protein inhibits PCAF acetylase function on p53 by preventing enzyme-substrate interaction. These results underscore the importance of p53 acetylation for its function and suggest that inhibition of p53 acetylation by viral oncoproteins prevent its activation, thereby contributing to viral transformation.

Production of recombinant androgen receptor in a heterologous expression system

1993 ◽  
Vol 39 (2) ◽  
pp. 346-352 ◽  
Author(s):  
O A Jänne ◽  
J J Palvimo ◽  
P Kallio ◽  
M Mehto ◽  
Y B Xie ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Binding Activity ◽  
Full Length ◽  
Biologically Active ◽  
Receptor Protein ◽  
Cell Protein ◽  
Domain Specific

Abstract To facilitate detailed studies of androgen receptor, we have produced a full-length receptor protein and some of its deletion mutants in Spodoptera frugiperda (Sf9) insect cells, using the baculovirus expression system. Recombinant baculovirus DNA-infected Sf9 cells expressed these proteins in very high quantities, which represented as much as 30-40% of total insect cell protein at 72 h after infection. Only < 1% of the recombinant protein was soluble in low-salt buffers; the majority formed electron-dense cytoplasmic aggregates 30-40 nm in diameter. These aggregates could be solubilized in 6 mol/L guanidine HCl, and biologically active receptor was generated by diluting the guanidine HCl preparation 20- to 50-fold. The full-length receptor, expressed either in a soluble or aggregated form, had characteristics typical of a native receptor: it bound steroids with high affinity and specificity, interacted with DNA in a sequence-specific fashion, and was recognized by domain-specific receptor antibodies. Androgen-receptor protein purified to homogeneity in guanidine HCl required the presence of Zn2+ ions during the refolding to reconstitute its DNA-binding form; ZnCl2 was not, however, needed to restore the receptor's steroid-binding activity.

Sign in / Sign up

Export Citation Format

Share Document