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Correlation of circRNA
Correlation of circRNA
and THO/TREX for genome integrity
and THO/TREX for genome integrity
Correlation of circRNA
and THO/TREX for genome integrity
Correlation of circRNA and THO/TREX for genome integrity and THO/TREX for genome integrity
Correlation of circRNA
Chulsung Park and Junho K. Hur
Chulsung Park and Junho K. Hur
Chulsung Park and Junho K. HurChulsung Park and Junho K. Hur
Chulsung Park and Junho K. Hur
Department of Biomedical Science, Graduate school of Biomedical Science & Engineering, Hanyang University
Department of Biomedical Science, Graduate school of Biomedical Science & Engineering, Hanyang University
Department of Biomedical Science, Graduate school of Biomedical Science & Engineering, Hanyang University, 04763, , 04763,
Department of Biomedical Science, Graduate school of Biomedical Science & Engineering, Hanyang University
, 04763,
Department of Biomedical Science, Graduate school of Biomedical Science & Engineering, Hanyang University
, 04763,
, 04763,
Abstract Results
Circular RNAs (circRNAs) are a class of highly conserved non�coding A INDEL rate of thoc2 Knock Out B thoc2 mRNA expression level C thoc2 mRNA expression level
RNAs with circular forms in eukaryotic cells. They are derived from exonic
and intronic sequence by mRNA precursor back-splicing, have a covalent
structure that lacks both 5’ cap and 3’ poly(A) tail which protects from
exoribonuclease. CircRNAs have several cellular functions including miRNA
sponging, protein sponging, 5’ cap-independent translation, and DNA; RNA
hybrid formation (R-loop). Recent studies suggested that circRNAs may be
regulated by a conserved multi-subunit THO/TREX complex, which is known
to be required for the biogenesis of the export-competent mRNP. An RNAi
screen study in Drosophila cell line showed that a TREX component called
Hel25E, an RNA-dependent ATPase and RNA-DNA helicase, regulates the
export of specific circRNAs from nuclear to cytoplasm. In this study, we
sought to ask if loss of THO complex subunit would increase the formation of
R-loop which leads to genome instability. To this end, we assessed whether
depletion of THO/TREX complex disrupts circRNA export from nuclear to Figure 1. To make THOC2 Knock Out cells by using CRISPR-Cas9 system
cytoplasm and relate R-loop formation with circRNAs in the nucleus. We in Human cells.
found that several circRNAs remained in the nuclei of THO subunit K/O cells. (A) High INDEL(INsertion and DELetion) rate of thoc2 K/O from sgRNA
The patterns of R-loops depletion by THO and circRNAs were overlapped. targeted deep sequencing in transfected HEK293T cells after 3days. Depletion
Our data suggested that the regulation of circRNAs by THO/TREX complex of THO compex had severe phenotype and the servival rate of THOC2 K/O
was required for genome integrity. cells was decreased the next generation.
(B,C) thoc2 mRNA expression level was decreased about 70% in THOC2 K/O
cells against control cells. Using RT-qPCR was faster way to confirm knockout
Introduction than targeted deep sequencing.

circRNAs are highly conserved non�coding RNAs with circular forms in Figure 2. Increase the formation
eukaryotic cells. Compared with other types of RNAs, circRNAs is highly Formation of R-loop in THOC2 K/O of R-loop in THOC2 K/O cells
stable that lack both 5’ cap and 3’ poly(A) tail which protects from exoribonu- qDRIP is the fast and simple way
clease. CircRNAs have several cellular functions including miRNA spong- to check the formation of R-loop.
ing, protein sponging, and DNA; RNA hybrid formation (R-loop). The miRNA We used well-known R-loop
sponging activity of circRNAs is the best-described mode of action of targets, Actin beta(ACTB) and 45s
circRNAs. Recently, circSMARCA5, one of the exonic circRNAs, formed pre-rRNA with the location
R-loops with SMARCA5 gene to inhibit the expression in cancer cells. RIZ1(45sRIZ_1), in THOC7 knock-
THO is an evolutionary conserved stable protein complex that plays a down cells. R-loop target genes
fundamental role in the nuclear export of mRNP. Metazoan THO is were enriched in THOC2 K/O
composed of six subunits; Hpr1, TEX1, THOC5, THOC6 and THOC7. THO cells.The formation of R-loop was
with other adaptor proteins (UAP56 and ALY) constitutes a bigger complex increased in depletion of THO
termed TREX complex. Although many studies showed THO/TREX is complex.
involved in the export of mRNA in a splicing-dependent manner, it is still
unclear the features of THO/TREX-target RNAs. Depletion of THO/TREX
complex increases the formation of R-loop to lead to genome instability. Comparision of circRNA level in nuclear and cytopalsm Figure 3. The export patterns of
In Drosophila, a TREX component called Hel25E, an RNA-dependent circRNA in THOC2 K/O cells
ATPase and RNA-DNA helicase, regulates the export of specific circRNAs Expression level of exonic
from nuclear to cytoplasm. UAP56 and URH49, human orthologues of circRNAs(circHIPK3 and
Hel25E, are also required for export circRNAs(below Figure). circSMARCA5) were increased in
Former studies about depletion THO complex used siRNA for inhibition of nucleus and decreased in
THO component gene expression. In this study, we made THO disruption in cytoplasm. However intronic
Human cells by using CRISPR-Cas9 system. we showed that depletion of circRNA(ciANKRD52) showed
THO complex subunit would change the formation of R-loop and the export same pattern. This data showed
patterns of circRNAs.
THO complex was required for
export of sevral circRNA from

Summary

- THOC2 K/O by CRISPR-Cas9 was an efficient method in Human cells and
RT-qPCR was a great and fast method to confirm the gene knockout.
- qDRIP was the best way to confirm R-loop before high-throughput sequenc-
ing.
- THO/TREX complex was required for the formation of R-loop and export
Zhengguo Li et al, RNA Biology, 2019
several circRNA from nuclear to cytoplasm.