Nucleotide Modifications Decrease Innate Immune Response Induced by Synthetic Analogs of snRNAs and snoRNAs.

Grigory Stepanov,Elena Mozhaeva,Anna Nushtaeva,Marat Pavlyukov,Vladimir Koval,Irina Malyants,Elena Kuligina,Vladimir Richter,Victoria Shender,Marat Kasakin,Elena Chernolovskaya,Alexander Lomzov,Mikhail Zhorov,Vadim Govorun,Dmitry Semenov,Evgenia Balakhonova,Tatyana Kabilova,Evgenii Zhuravlev

doi:10.3390/genes9110531

Abstract

Short nuclear regulatory RNAs play a key role in the main stages of maturation of the precursors of the major RNA species. Small nuclear RNAs (snRNAs) form the core of the spliceosome and are responsible for the splicing of pre-mRNA molecules. Small nucleolar RNAs (snoRNAs) direct post-transcriptional modification of pre-rRNAs. A promising strategy for the development of non-coding RNA (ncRNAs) mimicking molecules is the introduction of modified nucleotides, which are normally present in natural ncRNAs, into the structure of synthetic RNAs. We have created a set of snoRNAs and snRNA analogs and studied the effect of base modifications, specifically, pseudouridine (Ψ) and 5-methylcytidine (m5C), on the immune-stimulating and cytotoxic properties of these RNAs. Here, we performed a whole-transcriptome study of the influence of synthetic snoRNA analogs with various modifications on gene expression in human cells. Moreover, we confirmed the role of PKR in the recognition of snoRNA and snRNA analogs using the short hairpin RNA (shRNA) technique. We believe that the data obtained will contribute to the understanding of the role of nucleotide modification in ncRNA functions, and can be useful for creating the agents for gene regulation based on the structure of natural snoRNAs and snRNAs.

Highlights

There are two major classes of short non-coding RNAs in eukaryotic cells: small nuclearRNAs, which form the core of spliceosomes and catalyze the removal of introns duringGenes 2018, 9, 531; doi:10.3390/genes9110531 www.mdpi.com/journal/genesGenes 2018, 9, 531 pre-mRNA processing, and small nucleolar RNAs, which, being a part of small nucleolar ribonucleoproteins, direct post-transcriptional modifications of rRNAs [1,2,3,4,5]
To clarify the structural effects caused by modified nucleotides, we studied the thermodynamic stability of U35a Small nucleolar RNAs (snoRNAs) and U12 Small nuclear RNAs (snRNAs) analogs
Our results showed that PKR can be activated by unmodified snoRNAs and snRNAs, and by modified RNAs without cap, with nucleotide modifications reducing and slowing down the cytotoxic and antiproliferative effects of such RNAs

Summary

Introduction

Genes 2018, 9, 531 pre-mRNA processing, and small nucleolar RNAs (snoRNAs), which, being a part of small nucleolar ribonucleoproteins (snoRNPs), direct post-transcriptional modifications of rRNAs [1,2,3,4,5]. One of the approaches commonly used to study the properties and functions of ncRNAs is the in vitro synthesis of their analogs and subsequent transfection into human or animal cells. A cascade of interferon-dependent transcription activation events in the cells results in altered expression of a large number of protein-coding and non-coding genes, making it difficult to interpret the results of the experiments aimed to elucidate the direct effect of synthetic RNAs [15,16,17,18]. The development of approaches to decrease the non-specific immune-stimulating activity of ncRNA analogs remains an important issue

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Results

Discussion

Conclusion