citations

                    Efficient oligonucleotide-mediated degradation of nuclear noncoding RNAs in mammalian cultured cells
                

Authors:

Ideue T, Hino K, Kitao S, Yokoi T, Hirose T

In:

Source: RNA
Publication Date: (2009)
Issue: 15(8): 1578-1587

Cells used in publication:

HeLa: Species: human; Tissue Origin: cervix

Platform:

Nucleofector® I/II/2b

Experiment

Nucleofection of chimeric siRNA to degrade at least 20 different nuclear ncRNA species in multiple mammalian cell lines with high efficiency and specificity. The ASOs (IDT) used for knockdown experiments were phosphorothioate converted at their backbones to increase stability. Five terminal nucleotides from the 59 and 39 ends were substituted with 29-O-methoxyethyl ribonucleotides. Trypsinized HeLa cells (1x 3 10^6 cells) were suspended in 100 uL of Solution R from the Cell Line Nucleofector Kit R (Amaxa Biosystems) and mixed with the ASOs (4 mM final concentration). Transfection was carried out in an electroporation cuvette using a Nucleofector instrument (Amaxa Biosystems). The transfected cells were transferred to fresh DMEM plus 10% FBS, incubated at 37°C and 5% CO2 for 24 h, and then the cells were harvested for RNA preparation. The ASOs were designed as complementary sequences to snRNA single-stranded guide sequences. The sequences of the ASOs used are provided in Table 1. For transfection of fluorescently labeled ASOs, the Cy3-labeled ASO (Cy3- aU84) was synthesized (IDT), and transfected into HeLa cells either by nucleofection or lipofection with Lipofectamine 2000 reagent (Invitrogen).

Abstract

Recent large-scale transcriptome analyses have revealed that large numbers of noncoding RNAs (ncRNAs) are transcribed from mammalian genomes. They include small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), and longer ncRNAs, many of which are localized to the nucleus, but which have remained functionally elusive. Since ncRNAs are only known to exist in mammalian species, established experimental systems, including the Xenopus oocyte system and yeast genetics, are not available for functional analysis. RNA interference (RNAi), commonly used for analysis of protein-coding genes, is effective in eliminating cytoplasmic mRNAs, but not nuclear RNAs. To circumvent this problem, we have refined the system for knockdown of nuclear ncRNAs with chemically modified chimeric antisense oligonucleotides (ASO) that were efficiently introduced into the nucleus by nucleofection. Under optimized conditions, our system appeared to degrade at least 20 different nuclear ncRNA species in multiple mammalian cell lines with high efficiency and specificity. We also confirmed that our method had greatly improved knockdown efficiency compared with that of the previously reported method in which ASOs are introduced with transfection reagents. Furthermore, we have confirmed the expected phenotypic alterations following knockdown of HBII295 snoRNA and U7 snRNA, which resulted in a loss of site-specific methylation of the artificial RNA and the appearance of abnormal polyadenylated histone mRNA species with a concomitant delay of the cell cycle S phase, respectively. In summary, we believe that our system is a powerful tool to explore the biological functions of the large number of nuclear ncRNAs with unknown function.

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