TY - JOUR

T1 - Are small nucleolar RNAs "cRISPRable"? A report on box C/D small nucleolar RNA editing in human cells

AU - Filippova, Julia A.

AU - Matveeva, Anastasiya M.

AU - Zhuravlev, Evgenii S.

AU - Balakhonova, Evgenia A.

AU - Prokhorova, Daria V.

AU - Malanin, Sergey J.

AU - Mahmud, Raihan Shah

AU - Grigoryeva, Tatiana V.

AU - Anufrieva, Ksenia S.

AU - Semenov, Dmitry V.

AU - Vlassov, Valentin V.

AU - Stepanov, Grigory A.

N1 - Funding Information: The study was supported by the RFBR grant No 18-29-07073 and partially (in method development) by State Budget Program (0245-2019-0001). Publisher Copyright: © 2019 Royal Society of Chemistry. All rights reserved. Copyright: Copyright 2019 Elsevier B.V., All rights reserved.

PY - 2019/11/4

Y1 - 2019/11/4

N2 - CRISPR technologies are nowadays widely used for targeted knockout of numerous protein-coding genes and for the study of various processes and metabolic pathways in human cells. Most attention in the genome editing field is now focused on the cleavage of protein-coding genes or genes encoding long non-coding RNAs (lncRNAs), while the studies on targeted knockout of intron-encoded regulatory RNAs are sparse. Small nucleolar RNAs (snoRNAs) present a class of non-coding RNAs encoded within the introns of various host genes and involved in post-transcriptional maturation of ribosomal RNAs (rRNAs) in eukaryotic cells. Box C/D snoRNAs direct 2'-O-methylation of rRNA nucleotides. These short RNAs have specific elements in their structure, namely, boxes C and D, and a target-recognizing region. Here, we present the study devoted to CRISPR/ Cas9-mediated editing of box C/D snoRNA genes in Gas5. We obtained monoclonal cell lines carrying mutations in snoRNA genes and analyzed the levels of the mutant box C/D snoRNA as well as the 2'-O-methylation status of the target rRNA nucleotide in the obtained cells. Mutations in SNORD75 in the obtained monoclonal cell line were shown to result in aberrant splicing of Gas5 with exclusion of exons 3 to 5, which was confirmed by RT-PCR and RNA-Seq. The obtained results suggest that SNORD75 contains an element for binding of some factors regulating maturation of Gas5 pre-lncRNA. We suggest that METTL3/METTL14 is among such factors, and m6A-methylation pathways are involved in regulation of Gas5 splicing. Our results shell light on the role of SNORDs in regulating splicing of the host gene.

AB - CRISPR technologies are nowadays widely used for targeted knockout of numerous protein-coding genes and for the study of various processes and metabolic pathways in human cells. Most attention in the genome editing field is now focused on the cleavage of protein-coding genes or genes encoding long non-coding RNAs (lncRNAs), while the studies on targeted knockout of intron-encoded regulatory RNAs are sparse. Small nucleolar RNAs (snoRNAs) present a class of non-coding RNAs encoded within the introns of various host genes and involved in post-transcriptional maturation of ribosomal RNAs (rRNAs) in eukaryotic cells. Box C/D snoRNAs direct 2'-O-methylation of rRNA nucleotides. These short RNAs have specific elements in their structure, namely, boxes C and D, and a target-recognizing region. Here, we present the study devoted to CRISPR/ Cas9-mediated editing of box C/D snoRNA genes in Gas5. We obtained monoclonal cell lines carrying mutations in snoRNA genes and analyzed the levels of the mutant box C/D snoRNA as well as the 2'-O-methylation status of the target rRNA nucleotide in the obtained cells. Mutations in SNORD75 in the obtained monoclonal cell line were shown to result in aberrant splicing of Gas5 with exclusion of exons 3 to 5, which was confirmed by RT-PCR and RNA-Seq. The obtained results suggest that SNORD75 contains an element for binding of some factors regulating maturation of Gas5 pre-lncRNA. We suggest that METTL3/METTL14 is among such factors, and m6A-methylation pathways are involved in regulation of Gas5 splicing. Our results shell light on the role of SNORDs in regulating splicing of the host gene.

KW - Alternative splicing

KW - Box C/D snoRNA

KW - CRISPR/Cas9

KW - Gas5

KW - Genome editing

KW - M6A

KW - RNA modification

KW - snoRNA

KW - METHYLATION

KW - PROTEIN

KW - RIBOSOMAL-RNA

KW - MAMMALIAN GENE

KW - MODIFIED NUCLEOTIDES

KW - m(6)A

KW - genome editing

KW - CRISPR

KW - PRERIBOSOMAL RNA

KW - SNORNA

KW - alternative splicing

KW - IDENTIFICATION

KW - box C

KW - MET-DB

KW - Cas9

KW - D snoRNA

KW - REVEALS

UR - http://www.scopus.com/inward/record.url?scp=85075229203&partnerID=8YFLogxK

U2 - 10.3389/fphar.2019.01246

DO - 10.3389/fphar.2019.01246

M3 - Article

C2 - 31780925

AN - SCOPUS:85075229203

VL - 10

JO - Frontiers in Pharmacology

JF - Frontiers in Pharmacology

SN - 1663-9812

M1 - 01246

ER -