Modulation of long non-coding RNA Gas5 splicing using CRISPR/Cas9 editing of small nucleolar RNA genes

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Modulation of long non-coding RNA Gas5 splicing using CRISPR/Cas9 editing of small nucleolar RNA genes. / Matveeva, A.; Vinogradov, D.; Zhuravlev, E. et al.

In: FEBS Open Bio, Vol. 11, 07.2021, p. 32-32.

Research output: Contribution to journal › Meeting Abstract › peer-review

Harvard

Matveeva, A, Vinogradov, D, Zhuravlev, E, Semenov, D, Vlassov, V & Stepanov, G 2021, 'Modulation of long non-coding RNA Gas5 splicing using CRISPR/Cas9 editing of small nucleolar RNA genes', FEBS Open Bio, vol. 11, pp. 32-32.

BibTeX

@article{15a748eb008c4767b34fbda5e7b32f57,

title = "Modulation of long non-coding RNA Gas5 splicing using CRISPR/Cas9 editing of small nucleolar RNA genes",

author = "A. Matveeva and D. Vinogradov and E. Zhuravlev and D. Semenov and V. Vlassov and G. Stepanov",

note = "Small nucleolar RNAs (snoRNAs), specifically, box-C/D-snoRNAs, present an interesting and promising target for the regulation of multiple cell processes, such as ribosomal RNA maturation and splicing modulation in eukaryotic cells. These short regulatory RNAs are capable of participating in 20 -Omethylation of ribosomal RNA nucleotides due to the specific conserved elements in their structure. Current research is devoted to the CRISPR/Cas9-mediated functional analysis of box-C/DsnoRNAs encoded in Gas5 (Growth Arrest Specific 5) gene introns. 293FT-derived single cell clones were obtained with single and several simultaneous point mutations in individual boxC/D-snoRNAs, which led to the decrease of target RNA levels as well as level of 20 -O-methylation of native target rRNA nucleotides. Multiple knockouts were performed through the simultaneous transfection of two constructs, thus creating large deletions in Gas5 gene. Our results demonstrate the possibility of obtaining human cells with a suppressed level of several snoRNAs. The transcriptome of modified cell lines was examined using standard RNA-Seq of polyA fraction and small RNAs. The level of the host Gas5 transcript was shown to be decreased, in some cases the complete suppression was demonstrated. Further analysis of the host gene RNA structure demonstrated partial alterations in mature Gas5 RNA, namely, exon skipping and formation of novel isoforms. Mutation revealed in SNORD75 and SNORD81 led to changes in the sequence of the METTL3/METTL14-binding sites resulting in the deletion of the consensus motifs causing the formation of the alternative splicing products. Taken together, our data suggest that Gas5 splicing is m6A-dependent. (Previously published in: Filippova JA et al. (2019) Front. Pharmacol. 10, [01246].) This work was supported by the RFBR grant. 18-29-07073 and partially (in method development) by State Budget Program (0245-2019-0001).",

year = "2021",

month = jul,

language = "English",

volume = "11",

pages = "32--32",

journal = "FEBS Open Bio",

issn = "2211-5463",

publisher = "John Wiley & Sons Inc.",

}

RIS

TY - JOUR

T1 - Modulation of long non-coding RNA Gas5 splicing using CRISPR/Cas9 editing of small nucleolar RNA genes

AU - Matveeva, A.

AU - Vinogradov, D.

AU - Zhuravlev, E.

AU - Semenov, D.

AU - Vlassov, V.

AU - Stepanov, G.

N1 - Small nucleolar RNAs (snoRNAs), specifically, box-C/D-snoRNAs, present an interesting and promising target for the regulation of multiple cell processes, such as ribosomal RNA maturation and splicing modulation in eukaryotic cells. These short regulatory RNAs are capable of participating in 20 -Omethylation of ribosomal RNA nucleotides due to the specific conserved elements in their structure. Current research is devoted to the CRISPR/Cas9-mediated functional analysis of box-C/DsnoRNAs encoded in Gas5 (Growth Arrest Specific 5) gene introns. 293FT-derived single cell clones were obtained with single and several simultaneous point mutations in individual boxC/D-snoRNAs, which led to the decrease of target RNA levels as well as level of 20 -O-methylation of native target rRNA nucleotides. Multiple knockouts were performed through the simultaneous transfection of two constructs, thus creating large deletions in Gas5 gene. Our results demonstrate the possibility of obtaining human cells with a suppressed level of several snoRNAs. The transcriptome of modified cell lines was examined using standard RNA-Seq of polyA fraction and small RNAs. The level of the host Gas5 transcript was shown to be decreased, in some cases the complete suppression was demonstrated. Further analysis of the host gene RNA structure demonstrated partial alterations in mature Gas5 RNA, namely, exon skipping and formation of novel isoforms. Mutation revealed in SNORD75 and SNORD81 led to changes in the sequence of the METTL3/METTL14-binding sites resulting in the deletion of the consensus motifs causing the formation of the alternative splicing products. Taken together, our data suggest that Gas5 splicing is m6A-dependent. (Previously published in: Filippova JA et al. (2019) Front. Pharmacol. 10, [01246].) This work was supported by the RFBR grant. 18-29-07073 and partially (in method development) by State Budget Program (0245-2019-0001).

PY - 2021/7

Y1 - 2021/7

M3 - Meeting Abstract

VL - 11

SP - 32

EP - 32

JO - FEBS Open Bio

JF - FEBS Open Bio

SN - 2211-5463

ER -

ID: 34746568