scholarly journals Decoding the epitranscriptional landscape from native RNA sequences

2020 ◽  
Author(s):  
Piroon Jenjaroenpun ◽  
Thidathip Wongsurawat ◽  
Taylor D Wadley ◽  
Trudy M Wassenaar ◽  
Jun Liu ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
In Vitro Transcription ◽  
Rna Modification ◽  
Differential Analysis ◽  
Rna Sequences ◽  
Background Error ◽  
Sequencing Errors ◽  
Oxford Nanopore ◽  
The Impact

Abstract Traditional epitranscriptomics relies on capturing a single RNA modification by antibody or chemical treatment, combined with short-read sequencing to identify its transcriptomic location. This approach is labor-intensive and may introduce experimental artifacts. Direct sequencing of native RNA using Oxford Nanopore Technologies (ONT) can allow for directly detecting the RNA base modifications, although these modifications might appear as sequencing errors. The percent Error of Specific Bases (%ESB) was higher for native RNA than unmodified RNA, which enabled the detection of ribonucleotide modification sites. Based on the %ESB differences, we developed a bioinformatic tool, epitranscriptional landscape inferring from glitches of ONT signals (ELIGOS), that is based on various types of synthetic modified RNA and applied to rRNA and mRNA. ELIGOS is able to accurately predict known classes of RNA methylation sites (AUC > 0.93) in rRNAs from Escherichiacoli, yeast, and human cells, using either unmodified in vitro transcription RNA or a background error model, which mimics the systematic error of direct RNA sequencing as the reference. The well-known DRACH/RRACH motif was localized and identified, consistent with previous studies, using differential analysis of ELIGOS to study the impact of RNA m6A methyltransferase by comparing wild type and knockouts in yeast and mouse cells. Lastly, the DRACH motif could also be identified in the mRNA of three human cell lines. The mRNA modification identified by ELIGOS is at the level of individual base resolution. In summary, we have developed a bioinformatic software package to uncover native RNA modifications.

scholarly journals In vitro anticancer activity of hydrogen sulfide and nitric oxide alongside nickel nanoparticle and novel mutations in their genes in CRC patients

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zjwan Housein ◽  
Tayeb Sabir Kareem ◽  
Abbas Salihi
Keyword(s):  
Nitric Oxide ◽  
Cell Proliferation ◽  
Hydrogen Sulfide ◽  
Direct Sequencing ◽  
Scorpion Venom ◽  
Enos Gene ◽  
Cytotoxic Activities ◽  
No Donors ◽  
The Impact

AbstractThis study was carried out to assess the impact of nickel nanoparticles (NiNPs) as well as scorpion venom on colorectal cancer (CRC) cells in the presence and/or absence of 5-fluorouracil (5-FU), hydrogen sulfide (H2S), and nitric oxide (NO) donors and to determine alterations in endothelial NO synthase (eNOS) and cystathionine γ-lyase (CSE) enzyme-producing genes in CRC patients. The IC50 of both H2S and NO donors, along with NiNPs, were determined. The CRC cells were treated for 24hrs, and the cytotoxic activities were assessed using the MTT test. Moreover, the apoptosis was determined after 24hrs and 48hrs using TUNEL assay. Furthermore, the mutations in the eNOS gene (intron 4, -786T>C and 894 G>T) and CSE gene (1364GT) were determined using direct sequencing. The IC50 values for sodium disulfide (Na2S) and sodium nitroprusside (SNP) at 24hrs treatment were found to be 5 mM and 10−6 M, respectively, while the IC50 value for 5-FU was reached after 5-days of treatment in CRC cell line. Both black and yellow scorpion venoms showed no inhibition of cell proliferation after 24hrs treatment. Furthermore, Na2S showed a significant decrease in cell proliferation and an increase in apoptosis. Moreover, a co-treatment of SNP and 5-FU resulted in inhibition of the cytotoxic effect of 5-FU, while a combination treatment of NiNPs with Na2S, SNP, and 5-FU caused highly significant cytotoxicity. Direct sequencing reveals new mutations, mainly intronic variation in eNOS gene that has not previously been described in the database. These findings indicate that H2S promotes the anticancer efficiency of 5-FU in the presence of NiNPs while NO has antiapoptotic activity in CRC cell lines.

cDNA cloning and in vitro transcription of the complete brome mosaic virus genome

1984 ◽  
Vol 4 (12) ◽  
pp. 2876-2882 ◽  
Author(s):  
P Ahlquist ◽  
M Janda
Keyword(s):  
Mosaic Virus ◽  
Transcription Initiation ◽  
Direct Sequencing ◽  
In Vitro Transcription ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
In Vitro Translation ◽  
In Vitro Production ◽  
Genomic Rnas

Complete cDNA copies of each of the brome mosaic virus genomic RNAs (3.2, 2.8, and 2.1 kilobases in length) were cloned in a novel transcription vector, pPM1, designed to provide exact control of the transcription initiation site. After cleavage at a unique EcoRI site immediately downstream of the inserted cDNA, these clones can be transcribed in vitro by Escherichia coli RNA polymerase to yield complete copies of the brome mosaic virus RNAs. Dideoxy sequencing of 5' transcript cDNA runoff products and direct sequencing of 32P-3'-end-labeled transcripts show that such transcripts initiate at the same 5' position as natural viral RNA and terminate within the EcoRI runoff site after copying the entire viral RNA sequence. When synthesized in the presence of m7GpppG, the transcripts bear the natural capped 5' terminus of brome mosaic virus RNAs. Such transcripts direct the in vitro translation of proteins which coelectrophorese with the translation products of natural brome mosaic virus RNAs. pPM1 should facilitate in vitro production of other viral and nonviral RNAs.

In vitro transcription and micro-injection of Kdm4a mRNA into mouse oocytes

2020 ◽  
Author(s):  
ADITYA SANKAR
Keyword(s):  
Survival Rate ◽  
Embryo Development ◽  
In Vitro Transcription ◽  
Mouse Oocytes ◽  
The University ◽  
The Impact

Abstract This experiment describes the in vitro transcription of Kdm4a wildtype and H188A catalytic dead mRNA. This details also its subsequent injection into mouse oocytes followed my IVF to track the impact on embryo development. The procedure is technically challenging and performed by the Transgenic Core Facility at the University of Copenhagen. Oocytes have a poorer survival rate following mRNA inject as against zygotes. However the objective was to demonstrate the earliest stage of intervention to rescue developmental failure of KDM4A maternal zygotic mutant embryos

scholarly journals Inexpensive and colorimetric RNA detection by E. coli cell-free protein synthesis platform at room temperature

2021 ◽  
Author(s):  
Michela Notarangelo ◽  
Alessandro Quattrone ◽  
Massimo Pizzato ◽  
Sheref S. Mansy ◽  
O. Duhan Toparlak
Keyword(s):  
Room Temperature ◽  
Colorimetric Detection ◽  
In Vitro Transcription ◽  
Viral Rna ◽  
Rna Sequences ◽  
Rna Detection ◽  
E Coli ◽  
Free Gene ◽  
Cell Free Protein Synthesis

We report colorimetric detection of SARS-CoV-2 viral RNA by an in vitro transcription/translation assay with crude E. coli extracts at room temperature, with the aid of body heat. Clinically-relevant concentrations of viral RNA (ca. 600 copies/test) were detected from synthetic RNA samples. The activation of cell-free gene expression was achieved by toehold-switch-mediated riboregulatory elements that are specific to viral RNA sequences. The colorimetric output was generated by the α-complementation of β-galactosidase ω-fragment (LacZ-ω) with cell-free expressed LacZ-α, using an X-gal analogue as a substrate. The estimated cost of single reaction is less than 1 euro/test, which may facilitate diagnostic kit accessibility in developing countries.

scholarly journals In vitro transcription of cloned 5S RNA genes of the newt Notophthalmus.

1981 ◽  
Vol 90 (2) ◽  
pp. 323-331 ◽  
Author(s):  
B K Kay ◽  
O Schmidt ◽  
J G Gall
Keyword(s):  
Dna Sequences ◽  
Gene Segment ◽  
In Vitro Transcription ◽  
Xenopus Laevis Oocyte ◽  
Coding Region ◽  
5S Rna ◽  
Rna Sequences ◽  
Repeat Units ◽  
5S Gene

Recombinant plasmids that carried genes coding for 5S ribosomal RNA of the newt, Notophthalmus viridescens, were transcribed in vitro with extracts of Xenopus laevis oocyte nuclei. Plasmids containing multiple repeats of the 5S gene and spacer directed accurate transcription of 5S RNA (120 bases). Individual repeat units were recloned by inserting Sau 3A restriction fragments into the Bam HI site of plasmid pBR322. Because each repeat was cut by the enzyme within the coding region, the inserts had incomplete coding regions at their ends and spacer sequences in the middle. The DNA of these subclones directed synthesis of a 5S-size RNA that contained both plasmid and 5S RNA sequences. Transcription initiated in the vector, proceeded through the gene segment coding for nucleotides 41-120, and terminated at the end of the gene. The initiation of in vitro transcription required neither the original 5' flanking sequences of the spacer nor the first third of the gene. We conclude that intragenic DNA sequences control the initiation of transcription. Other subclones that include pseudogenes gave rise to some transcripts 156 nucleotides long. These long transcripts represented continuation of transcription through the 36-base-pair pseudogene that is located immediately downstream from the 5S gene. However, most transcripts of these subclones terminated at the end of the normal gene before the beginning of the pseudogene. It is probable that a run of four or more Ts serves as part of the termination signal.

scholarly journals cDNA cloning and in vitro transcription of the complete brome mosaic virus genome.

1984 ◽  
Vol 4 (12) ◽  
pp. 2876-2882 ◽  
Author(s):  
P Ahlquist ◽  
M Janda
Keyword(s):  
Mosaic Virus ◽  
Transcription Initiation ◽  
Direct Sequencing ◽  
In Vitro Transcription ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
In Vitro Translation ◽  
In Vitro Production ◽  
Genomic Rnas

Complete cDNA copies of each of the brome mosaic virus genomic RNAs (3.2, 2.8, and 2.1 kilobases in length) were cloned in a novel transcription vector, pPM1, designed to provide exact control of the transcription initiation site. After cleavage at a unique EcoRI site immediately downstream of the inserted cDNA, these clones can be transcribed in vitro by Escherichia coli RNA polymerase to yield complete copies of the brome mosaic virus RNAs. Dideoxy sequencing of 5' transcript cDNA runoff products and direct sequencing of 32P-3'-end-labeled transcripts show that such transcripts initiate at the same 5' position as natural viral RNA and terminate within the EcoRI runoff site after copying the entire viral RNA sequence. When synthesized in the presence of m7GpppG, the transcripts bear the natural capped 5' terminus of brome mosaic virus RNAs. Such transcripts direct the in vitro translation of proteins which coelectrophorese with the translation products of natural brome mosaic virus RNAs. pPM1 should facilitate in vitro production of other viral and nonviral RNAs.

scholarly journals Human OAS1 activation is highly dependent on both RNA sequence and context of activating RNA motifs

2020 ◽  
Author(s):  
Samantha L Schwartz ◽  
Esther N Park ◽  
Virginia K Vachon ◽  
Shamika Danzy ◽  
Anice C Lowen ◽  
...  
Keyword(s):  
Structural Changes ◽  
Strong Dependence ◽  
Consensus Sequence ◽  
Critical Role ◽  
A549 Cells ◽  
Rna Motifs ◽  
Rna Sequences ◽  
Dsrna Binding ◽  
The Impact

Abstract 2′-5′-Oligoadenylate synthetases (OAS) are innate immune sensors of cytosolic double-stranded RNA (dsRNA) and play a critical role in limiting viral infection. dsRNA binding induces allosteric structural changes in OAS1 that reorganize its catalytic center to promote synthesis of 2′-5′-oligoadenylate and thus activation of endoribonuclease L. Specific RNA sequences and structural motifs can also enhance activation of OAS1 through currently undefined mechanisms. To better understand these drivers of OAS activation, we tested the impact of defined sequence changes within a short dsRNA that strongly activates OAS1. Both in vitro and in human A549 cells, appending a 3′-end single-stranded pyrimidine (3′-ssPy) can strongly enhance OAS1 activation or have no effect depending on its location, suggesting that other dsRNA features are necessary for correct presentation of the motif to OAS1. Consistent with this idea, we also find that the dsRNA binding position is dictated by an established consensus sequence (WWN9WG). Unexpectedly, however, not all sequences fitting this consensus activate OAS1 equivalently, with strong dependence on the identity of both partially conserved (W) and non-conserved (N9) residues. A picture thus emerges in which both specific RNA features and the context in which they are presented dictate the ability of short dsRNAs to activate OAS1.

scholarly journals m6A-TSHub: unveiling the context-specific m6A methylation and m6A-affecting mutations in 23 human tissues

2022 ◽  
Author(s):  
Bowen Song ◽  
Daiyun Huang ◽  
Yuxin Zhang ◽  
Zhen Wei ◽  
Jionglong Su ◽  
...  
Keyword(s):  
Web Server ◽  
Rna Modification ◽  
Epigenetic Mark ◽  
Cancer Type ◽  
Human Tissues ◽  
Rna Sequences ◽  
Tissue Specific ◽  
Specific Tissue ◽  
Context Specific ◽  
The Impact

As the most pervasive epigenetic marker present on mRNA and lncRNA, N6-methyladenosine (m6A) RNA methylation has been shown to participate in essential biological processes. Recent studies revealed the distinct patterns of m6A methylome across human tissues, and a major challenge remains in elucidating the tissue-specific presence and circuitry of m6A methylation. We present here a comprehensive online platform m6A-TSHub for unveiling the context-specific m6A methylation and genetic mutations that potentially regulate m6A epigenetic mark. m6A-TSHub consists of four core components, including (1) m6A-TSDB: a comprehensive database of 184,554 functionally annotated m6A sites derived from 23 human tissues and 499,369 m6A sites from 25 tumor conditions, respectively; (2) m6A-TSFinder: a web server for high-accuracy prediction of m6A methylation sites within a specific tissue from RNA sequences, which was constructed using multi-instance deep neural networks with gated attention; (3) m6A-TSVar: a web server for assessing the impact of genetic variants on tissue-specific m6A RNA modification; and (4) m6A-CAVar: a database of 587,983 TCGA cancer mutations (derived from 27 cancer types) that were predicted to affect m6A modifications in the primary tissue of cancers. The database should make a useful resource for studying the m6A methylome and genetic factor of epitranscriptome disturbance in a specific tissue (or cancer type). m6A-TSHub is accessible at: www.xjtlu.edu.cn/biologicalsciences/m6ats.

scholarly journals Direct detection of RNA in vitro and in situ by target-primed RCA: The impact of E. coli RNase III on the detection efficiency of RNA sequences distanced far from the 3'-end

RNA ◽  
2010 ◽  
Vol 16 (8) ◽  
pp. 1508-1515 ◽  
Author(s):  
E. Merkiene ◽  
E. Gaidamaviciute ◽  
L. Riauba ◽  
A. Janulaitis ◽  
A. Lagunavicius
Keyword(s):  
Detection Efficiency ◽  
Direct Detection ◽  
Rnase Iii ◽  
Rna Sequences ◽  
E Coli ◽  
The Impact

RNA design by in vitro RNA recombination and synthesis

1987 ◽  
Vol 65 (8) ◽  
pp. 677-692 ◽  
Author(s):  
Robert Cedergren ◽  
Henri Grosjean
Keyword(s):  
In Vitro Transcription ◽  
Biologically Active ◽  
Future Research ◽  
Rna Recombination ◽  
Rna Sequences ◽  
Active Length ◽  
Rna Fragments ◽  
Rna Design ◽  
Relationship Of

The techniques of in vitro RNA synthesis and recombination are presented. These include the site-specific cleavage of RNA, the manipulation of terminal phosphates, and the ligation of RNA fragments. Areas of promising future research include the establishment of RNA cloning vectors and the use of in vitro transcription of natural or designed RNA genes. The chemical synthesis approach now offers the possibility of making large amounts of biologically active length RNAs and of incorporating modified or reporter nucleotides into RNA sequences for physical studies. The new RNA techniques taken with DNA technology will permit a new approach towards understanding the complexity of RNA metabolism and the relationship of structure to function in RNAs.

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