Artificial Box C/D RNAs Affect Pre-mRNA Maturation in Human Cells

Grigoriy A Stepanov,Dmitry V Semenov,Vladimir A Richter,Olga A Koval,Igor V Rabinov,Elena V Kuligina,Anna V Savelyeva

doi:10.1155/2013/656158

Abstract

Box C/D small nucleolar RNAs (snoRNAs) are known to guide the 2′-O-ribose methylation of nucleotides in eukaryotic ribosomal RNAs and small nuclear RNAs. Recently snoRNAs are predicted to regulate posttranscriptional modifications of pre-mRNA. To expand understanding of the role of snoRNAs in control of gene expression, in this study we tested the ability of artificial box C/D RNAs to affect the maturation of target pre-mRNA. We found that transfection of artificial box C/D snoRNA analogues directed to HSPA8 pre-mRNAs into human cells induced suppression of the target mRNA expression in a time- and dose-dependent manner. The artificial box C/D RNA directed to the branch point adenosine of the second intron, as well as the analogue directed to the last nucleotide of the second exon of the HSPA8 pre-mRNA caused the most prominent influence on the level of HSPA8 mRNAs. Neither box D nor the ability to direct 2′-O-methylation of nucleotides in target RNA was essential for the knockdown activity of artificial snoRNAs. Inasmuch as artificial box C/D RNAs decreased viability of transfected human cells, we propose that natural snoRNAs as well as their artificial analogues can influence the maturation of complementary pre-mRNA and can be effective regulators of vital cellular processes.

Highlights

Small nucleolar RNAs are commonly known to be involved in the processing of precursor ribosomal RNA and small nuclear RNAs
Analogues were constructed to direct 2󸀠-O-methylation of G1702 in 18S rRNA and of the HSPA8 pre-mRNA nucleotides that were critical to splicing the second intron of the pre-mRNA-target (Figure 1(b))
To obtain the artificial box C/D RNAs, we constructed a series of DNA templates and primers containing the T7 promoter

Summary

Introduction

Small nucleolar RNAs (snoRNA) are commonly known to be involved in the processing of precursor ribosomal RNA (pre-rRNA) and small nuclear RNAs (snRNAs). Box C/D snoRNAs direct 2󸀠-O-methylation of rRNA nucleotides, and box H/ACA snoRNAs guide the conversion of uridine to pseudouridine [1,2,3,4]. The snoRNA-dependent modifications are catalyzed by small nucleolar ribonucleoprotein particles (snoRNPs). Box C/D RNAs are associated with four snoRNP core proteins: NOP56, NOP58, fibrillarin, and 15.5 kDa [5,6,7]. Box C/D RNAs contain the following functional elements: boxes C and D, which are essential for snoRNA interaction with specific proteins, and a guide sequence that determines the nucleotide to be modified (Figure 1(a)). SnoRNAs hold two potential guide sequences [2, 4]

Methods

Results

Conclusion