scholarly journals Custom-designed, degradation-resistant messenger RNAs in yeast

2020 ◽  
Author(s):  
Ana L. Franklin ◽  
Andrea Macfadden ◽  
Jeffrey S. Kieft ◽  
Jay R. Hesselberth ◽  
Erich G. Chapman
Keyword(s):  
Internal Ribosome Entry Site ◽  
Fold Increase ◽  
Messenger Rnas ◽  
Rna Structures ◽  
Lacz Reporter ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Rna Transcripts ◽  
Luciferase Reporters ◽  
Independent Translation

ABSTRACTStructured RNA elements that protect RNA transcripts from 5’→3’ degradation are becoming useful research tools. Here we show that exonuclease-resistant RNA structures (xrRNAs) from Flaviviruses can be used to protect heterologous messenger RNAs (mRNAs) from 5’→3’ degradation in budding yeast. Installation of xrRNAs ahead of a downstream internal ribosome entry site (IRES) leads to the accumulation of partially-degraded mRNAs that are substrates for cap-independent translation of a LacZ reporter, yielding a 30-fold increase in measured β-galactosidase activity. Additionally, by monitoring the translation of dual-luciferase reporters we show that xrRNA sequences do not interfere with the progression of an elongating ribosome. Combined these data indicate that xrRNA elements can be used in creative ways to stabilize RNAs with potentially useful applications.

scholarly journals Out-of-Plane Aptamer Functionalization of RNA Three-Helix Tiles

Nanomaterials ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 507 ◽  
Author(s):  
Aradhana Chopra ◽  
Sandra Sagredo ◽  
Guido Grossi ◽  
Ebbe Andersen ◽  
Friedrich Simmel
Keyword(s):  
Small Molecules ◽  
Internal Ribosome Entry Site ◽  
Hybrid Nanostructures ◽  
Three Dimensions ◽  
Rna Structures ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Double Helices ◽  
Out Of Plane ◽  
Rna Motif

Co-transcriptionally folding RNA nanostructures have great potential as biomolecular scaffolds, which can be used to organize small molecules or proteins into spatially ordered assemblies. Here, we develop an RNA tile composed of three parallel RNA double helices, which can associate into small hexagonal assemblies via kissing loop interactions between its two outer helices. The inner RNA helix is modified with an RNA motif found in the internal ribosome entry site (IRES) of the hepatitis C virus (HCV), which provides a 90° bend. This modification is used to functionalize the RNA structures with aptamers pointing perpendicularly away from the tile plane. We demonstrate modifications with the fluorogenic malachite green and Spinach aptamers as well with the protein-binding PP7 and streptavidin aptamers. The modified structures retain the ability to associate into larger assemblies, representing a step towards RNA hybrid nanostructures extending in three dimensions.

scholarly journals Insights into the secondary and tertiary structure of the Bovine Viral Diarrhea Virus Internal Ribosome Entry Site

2021 ◽  
Author(s):  
Devadatta Gosavi ◽  
Iwona Wower ◽  
Irene Beckmann ◽  
Ivo L. Hofacker ◽  
Jacek Wower ◽  
...  
Keyword(s):  
Internal Ribosome Entry Site ◽  
Tertiary Structure ◽  
Bovine Viral Diarrhea ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus ◽  
Insight Into ◽  
Independent Translation

The Internal Ribosome Entry Site (IRES) RNA of Bovine viral diarrhea virus (BVDV), an economically significant Pestivirus, is required for the cap-independent translation of viral genomic RNA. Thus, it is essential for viral replication and pathogenesis. We applied a combination of high-throughput biochemical RNA structure probing (SHAPE-MaP) and in silico modeling approaches to gain insight into the secondary and tertiary structures of BVDV IRES RNA. Our study demonstrated that BVDV IRES RNA forms in solution a modular architecture composed of three distinct structural domains (I-III). Two regions within domain III are engaged in tertiary interactions to form an H-type pseudoknot. Computational modeling of the pseudoknot motif provided a fine-grained picture of the tertiary structure and local arrangement of helices in the BVDV IRES. Furthermore, comparative genomics and consensus structure predictions revealed that the pseudoknot is evolutionarily conserved among many Pestivirus species. These studies provide detailed insight into the structural arrangement of BVDV IRES RNA H-type pseudoknot and encompassing motifs that likely contribute to the optimal functionality of viral cap-independent translation element.

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