scholarly journals In vitro capping and transcription of rhabdoviruses

Methods ◽  
2013 ◽  
Vol 59 (2) ◽  
pp. 188-198 ◽  
Author(s):  
Tomoaki Ogino
Keyword(s):  
In Vitro Capping

scholarly journals High resolution biosensor to test the capping level and integrity of mRNAs

2020 ◽  
Vol 48 (22) ◽  
pp. e129-e129
Author(s):  
Ignacio Moya-Ramírez ◽  
Clement Bouton ◽  
Cleo Kontoravdi ◽  
Karen Polizzi
Keyword(s):  
Single Molecule ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Chimeric Protein ◽  
Single Step ◽  
Quality Analysis ◽  
Eukaryotic Mrnas ◽  
In Vitro Capping

Abstract 5′ Cap structures are ubiquitous on eukaryotic mRNAs, essential for post-transcriptional processing, translation initiation and stability. Here we describe a biosensor designed to detect the presence of cap structures on mRNAs that is also sensitive to mRNA degradation, so uncapped or degraded mRNAs can be detected in a single step. The biosensor is based on a chimeric protein that combines the recognition and transduction roles in a single molecule. The main feature of this sensor is its simplicity, enabling semi-quantitative analyses of capping levels with minimal instrumentation. The biosensor was demonstrated to detect the capping level on several in vitro transcribed mRNAs. Its sensitivity and dynamic range remained constant with RNAs ranging in size from 250 nt to approximately 2700 nt and the biosensor was able to detect variations in the capping level in increments of at least 20%, with a limit of detection of 2.4 pmol. Remarkably, it also can be applied to more complex analytes, such mRNA vaccines and mRNAs transcribed in vivo. This biosensor is an innovative example of a technology able to detect analytically challenging structures such as mRNA caps. It could find application in a variety of scenarios, from quality analysis of mRNA-based products such as vaccines to optimization of in vitro capping reactions.

scholarly journals A new U6 small nuclear ribonucleoprotein-specific protein conserved between cis- and trans-splicing systems.

1991 ◽  
Vol 11 (4) ◽  
pp. 2026-2034 ◽  
Author(s):  
K Gröning ◽  
Z Palfi ◽  
S Gupta ◽  
M Cross ◽  
T Wolff ◽  
...  
Keyword(s):  
Affinity Purification ◽  
Nuclear Extract ◽  
Specific Protein ◽  
Small Nuclear Rna ◽  
Sm Proteins ◽  
Sequence Element ◽  
U6 Snrna ◽  
Small Nuclear Ribonucleoprotein ◽  
In Vitro Capping

Spliceosomal U6 small nuclear RNA (snRNA) plays a central role in the pre-mRNA splicing mechanism and is highly conserved throughout evolution. Previously, a sequence element essential for both capping and cytoplasmic-nuclear transport of U6 snRNA was mapped in the 5'-terminal domain of U6 snRNA. We have identified a protein in cytoplasmic extracts of mammalian and Trypanosoma brucei cells that binds specifically to this U6 snRNA element. Competition studies with mutant and heterologous RNAs demonstrated the conserved binding specificity of the mammalian and trypanosomal proteins. The in vitro capping analysis of mutant U6 snRNAs indicated that protein binding is required but not sufficient for capping of U6 snRNA by a gamma-monomethyl phosphate. Through RNA affinity purification of mammalian small nuclear ribonucleoproteins (snRNPs), we detected this protein also in nuclear extract as a new specific component of the U6 snRNP but surprisingly not of the U4/U6 or the U4/U5/U6 multi-snRNP. These results suggest that the U6-specific protein is involved in U6 snRNA maturation and transport and may therefore be functionally related to the Sm proteins of the other spliceosomal snRNPs.

A new U6 small nuclear ribonucleoprotein-specific protein conserved between cis- and trans-splicing systems

1991 ◽  
Vol 11 (4) ◽  
pp. 2026-2034
Author(s):  
K Gröning ◽  
Z Palfi ◽  
S Gupta ◽  
M Cross ◽  
T Wolff ◽  
...  
Keyword(s):  
Affinity Purification ◽  
Nuclear Extract ◽  
Specific Protein ◽  
Small Nuclear Rna ◽  
Sm Proteins ◽  
Sequence Element ◽  
U6 Snrna ◽  
Small Nuclear Ribonucleoprotein ◽  
In Vitro Capping

Spliceosomal U6 small nuclear RNA (snRNA) plays a central role in the pre-mRNA splicing mechanism and is highly conserved throughout evolution. Previously, a sequence element essential for both capping and cytoplasmic-nuclear transport of U6 snRNA was mapped in the 5'-terminal domain of U6 snRNA. We have identified a protein in cytoplasmic extracts of mammalian and Trypanosoma brucei cells that binds specifically to this U6 snRNA element. Competition studies with mutant and heterologous RNAs demonstrated the conserved binding specificity of the mammalian and trypanosomal proteins. The in vitro capping analysis of mutant U6 snRNAs indicated that protein binding is required but not sufficient for capping of U6 snRNA by a gamma-monomethyl phosphate. Through RNA affinity purification of mammalian small nuclear ribonucleoproteins (snRNPs), we detected this protein also in nuclear extract as a new specific component of the U6 snRNP but surprisingly not of the U4/U6 or the U4/U5/U6 multi-snRNP. These results suggest that the U6-specific protein is involved in U6 snRNA maturation and transport and may therefore be functionally related to the Sm proteins of the other spliceosomal snRNPs.

Small RNAs of the silkworm, Bombyx mori as revealed by in vitro capping and in vitro transcription

1988 ◽  
Vol 91 (2) ◽  
pp. 383-388 ◽  
Author(s):  
Haruhiko Fujiwara ◽  
Hideaki Maekawa ◽  
Naoko Takada ◽  
Naoko Miyajima ◽  
Hajime Ishikawa
Keyword(s):  
Bombyx Mori ◽  
Small Rnas ◽  
In Vitro Transcription ◽  
In Vitro Capping ◽  
Silkworm Bombyx Mori
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