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www.nature.com/crwww.cell-research.com Cell Research
ARTICLE
Pre-ribosomal RNA reorganizes DNA damage repair factors in
nucleus during meiotic prophase and DNA damage response
Xiaochen Gai1,2,3,4, Di Xin1,2,3,4, Duo Wu1,2,3,4, Xin Wang1,2,3,4, Linlin Chen1,2,3, Yiqing Wang1,2,3, Kai Ma1,2,3, Qilin Li1,2,3, Peng Li1,2,3 and
✉
Xiaochun Yu 1,2,3
© CEMCS, CAS 2022
In response to DNA double-strand breaks (DSBs), DNA damage repair factors are recruited to DNA lesions and form nuclear foci.
However, the underlying molecular mechanism remains largely elusive. Here, by analyzing the localization of DSB repair factors in
the XY body and DSB foci, we demonstrate that pre-ribosomal RNA (pre-rRNA) mediates the recruitment of DSB repair factors
around DNA lesions. Pre-rRNA exists in the XY body, a DSB repair hub, during meiotic prophase, and colocalizes with DSB repair
factors, such as MDC1, BRCA1 and TopBP1. Moreover, pre-rRNA-associated proteins and RNAs, such as ribosomal protein subunits,
RNase MRP and snoRNAs, also localize in the XY body. Similar to those in the XY body, pre-rRNA and ribosomal proteins also localize
at DSB foci and associate with DSB repair factors. RNA polymerase I inhibitor treatment that transiently suppresses transcription of
rDNA but does not affect global protein translation abolishes foci formation of DSB repair factors as well as DSB repair. The FHA
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domain and PST repeats of MDC1 recognize pre-rRNA and mediate phase separation of DSB repair factors, which may be the
molecular basis for the foci formation of DSB repair factors during DSB response.
Cell Research (2022) 32:254–268; https://doi.org/10.1038/s41422-021-00597-4
INTRODUCTION Thus, DSBs on the X and Y chromosomes exist for prolonged time,
Once DNA double-strand breaks (DSBs) occur, DNA repair factors which causes extensive DNA damage response. Various DNA
are recruited to DNA lesions and repair DSBs via different damage repair factors are concentrated into these two sex
pathways.1,2 These DSB repair factors are sequentially loaded chromosomes to form a phase separation-like structure, named
onto DNA lesions and form distinct condensates in nucleus, the XY body, during meiotic prophase.18
known as DNA damage-induced foci.3 This phenomenon is often This phase separation-like structure covers the whole X and Y
observed and examined when cells are treated with ionizing chromosomes. As the obvious biomarker of the XY body is γH2AX,
radiation to induce DSBs.4 Thus, it is also known as ionizing we have proposed that the XY body is by far the largest DNA
radiation-induced foci (IRIF). damage focus in human cells.18,19 Interestingly, there are two
The foci formation of DSB repair factors is maintained by distinct patterns of localization of the DSB repair factors in the XY
phosphorylation of H2AX, a variant of histone H2A, at Ser139 (aka body. DSB response factors, such as γH2AX and MDC1, cover the
γH2AX).5,6 In response to DSBs, this site-specific phosphorylation whole area of the XY body.19 In contrast, the unsynapsed region of
event is mediated by a group of PI3-like kinases, including ATM, the X and Y chromosomes is decorated by HR repair machinery
ATR and DNA-PK.3,7 γH2AX may extend to mega-base away from including RAD51, BRCA1, TOPBP1, etc.19 Since RAD51 associates
DNA lesions, and provides a platform to cluster DSB repair factors with the single-stranded DNA (ssDNA) that is processed from DSB
at vicinity of DSBs.8,9 Although the detailed molecular mechanism ends, it has been proposed that SPO11-induced DSBs exist in the
is unclear, loss of H2AX abolishes DNA damage-induced foci and unsynapsed region.20,21 And these two distinct localization
impairs DSB repair.10–12 patterns in the XY body are reminiscent to the localization of
In addition to environmental hazard-induced DSBs, DSBs also these repair factors in IRIF.18 Using super-resolution microscopy,
arise during physiologically relevant processes. A typical example those HR repair factors are always localized at the center of IRIF,
is homologous recombination (HR) before meiosis. In both whereas the DSB response factors, such as γH2AX, are extended to
autosomes and sex chromosomes of germ cells, DSBs are the peripheral of DNA lesions.22 Nevertheless, the basic molecular
generated by SPO11, a topoisomerase-like enzyme.13–17 However, mechanism of phase separation of these repair factors at DNA
unlike topoisomerases, SPO11 cannot religate DSBs. Instead, these lesions is still unclear.
DSBs are repaired by DSB repair factors to complete HR. Due to Recent studies on phase separation indicate that RNA is an
the lack of homology, the X and Y chromosomes in male germ important component in membrane-less nuclear body forma-
cells cannot use canonical HR to repair SPO11-induced DSBs.18 tion.23–27 However, the RNA species that are involved in DNA
1
Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang, China. 2School of Life Sciences, Westlake University, Hangzhou, Zhejiang, China. 3Institute of Basic
Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, Zhejiang, China. 4These authors contributed equally: Xiaochen Gai, Di Xin, Duo Wu, Xin Wang.
✉email:
Received: 12 June 2021 Accepted: 11 November 2021
Published online: 4 January 2022
, X. Gai et al.
255
damage-induced foci formation remain elusive. In particular, DNA (Supplementary information, Fig. S4b, c). These results were
damage-induced foci formation is an evolutionarily conserved further validated by real-time quantitative PCR (RT-qPCR) (Supple-
phenomenon. Although some RNA species have been proposed mentary information, Fig. S4d). Collectively, these results suggest
to regulate DNA damage-induced foci formation,28–35 none of that rRNA exists in the XY body.
these RNAs are conserved during evolution, suggesting that major
RNA component in DNA damage-induced foci has yet to be Pre-rRNAs exist in the XY body
revealed. By analyzing the XY body, the biggest DNA damage rRNA is transcribed from ribosomal DNA (rDNA) loci as 47S pre-
focus that we can observe, we found that pre-ribosomal RNAs rRNA by RNA Polymerase I (RNA pol I). Immediately following
(pre-rRNAs) were present in the XY body as well as IRIF, and may transcription, 47S pre-rRNA is processed to 45S pre-rRNA that is
act as scaffolds for the phase separation of DNA damage repair further digested into 32S and 34S pre-rRNA in murine cells, the
factors at DNA lesions. precursors of 18S, 5.8S and 28S rRNA.38 Once pre-rRNA is
transcribed, it undergoes extensive modifications such as 2’-O-
methylation and pseudouridylation.39 Moreover, protein partners
RESULTS and 5S rRNA, the smallest rRNA component transcribed by RNA
rRNA is present in the XY body Polymerase III (RNA pol III), are incorporated into pre-rRNA to form
In order to study the foci formation of DSB repair factors, we pre-ribonucleoprotein particles (pre-rRNPs).40,41 To examine the
examined the XY body, the largest DNA damage response focus in rRNA species in the XY body, we designed various probes covering
nucleus that we have observed (Fig. 1a).18 Since DNA damage- different regions of 45S and 5S rRNA (Fig. 2a). Interestingly, we
induced foci are phase separations of DSB repair factors, and could stain rRNA with probes targeting the regions of 34S, 18S,
membrane-less nuclear body phase separation is often mediated 18SE, 32S, 28S, 12S, 8S, 5.8S and 5S but not with 5’-ETS probes
by RNA components,23–26 we asked whether RNA species exist in (Fig. 2b), suggesting that the XY body contains 34S, 32S rRNA and
the XY body. Using γH2AX as the XY body marker, we stained probably their derivatives, but not 45S rRNA. We also hybridized
meiotic spreads with SYTO RNASelect Green, a specific RNA dye, different probes simultaneously and found that these rRNAs had
and found that the average level of RNA in the XY body was the same signal distribution pattern in the XY body (Supplemen-
significantly higher than that in the whole nucleus (Fig. 1b). In tary information, Fig. S5a–c). To examine the component of rRNA
contrast, DNA level in the XY body, marked by SYBR Green I, was species in the XY body, we measured the fluorescence intensity of
similar to that in the whole nucleus (Fig. 1b). Moreover, with RNase each probe in the XY body (Supplementary information, Fig. S5d).
A treatment, the RNA species were removed from meiotic spreads, Based on the relative signal intensity, we calculated and found
and RNase A treatment did not affect DNA staining in meiotic that these rRNA species mainly included 34S (18.32%), 32S
spreads (Fig. 1c). Collectively, these results indicate that RNA is (80.27%) and 5S (1.41%) rRNAs (Fig. 2c). Although probes
enriched in the XY body. targeting other regions also recognized the XY body, the staining
Earlier studies have shown that gene transcription of X and Y of other probes mainly reflected 34S and 32S pre-rRNAs.
chromosomes is suppressed following the XY body formation.36 To exclude any mature rRNA in the XY body, we used probe
Consistently, the localization of RNA polymerase II (pol II) and quenchers that could interact with adjacent 5.8S or 28S probe,
poly-A RNA was excluded from the XY body (Fig. 1d and which sufficiently abolished the fluorescence signals of 5.8S or
Supplementary information, Fig. S1), suggesting that the RNA 28S probe, suggesting that most of the 5.8S or 28S probes
species in the XY body are not mRNAs. Moreover, several long recognize 5.8S or 28S region in pre-rRNAs. Thus, the results
non-coding RNAs (lncRNAs) have been reported to participate in indicate that pre-rRNAs are dominant RNA species in the XY body
DNA damage repair.28–32 However, we did not observe these and little mature rRNA exists in the XY body (Supplementary
lncRNAs in the XY body either (Supplementary information, information, Fig. S5e).
Fig. S2). To exclude the potential R-loop in the XY body, we In the XY body, pre-rRNA localized into different patterns, and
stained the XY body with S9.6 antibody that specifically we summarized these localization patterns into four types: (1) pre-
recognizes RNA-DNA hybrids,37 and we did not detect obvious rRNA localizes at the edge of the XY body; (2) pre-rRNA forms a
staining of the XY body using this antibody (Supplementary cap on the XY body; (3) pre-rRNA partially occupies the XY body;
information, Fig. S3a). Since RNase H specifically digests RNA that (4) pre-rRNA exists in the whole XY body (Fig. 2d). Interestingly, we
forms R-loop, we also treated meiotic spreads with RNase H, and found that the localization pattern of rRNA was shifted from the
found that RNase H did not affect the RNA staining in the XY body edge of the XY body in early pachytene and gradually migrated
(Supplementary information, Fig. S3b). Thus, these results into the XY body till diplotene (Fig. 2e). Moreover, when pre-rRNA
collectively suggest that the RNA species in the XY body may entered the XY body, it was enriched at the unsynapsed axes of
not form DNA-RNA hybrid. the X and Y chromosomes (Fig. 2f, g). However, as conventional
RNA species observed in the XY body are more abundant than paraformaldehyde fixation crosslinks proteins and nucleic acids in
those in other part of nucleus, indicating that these RNA species the XY body, it may impair RNA probe hybridization. We also used
should be some of the most abundant RNAs in the cell. Since methanol/acetic acid to fix cells, in which acetic acid treatment
ribosomal RNA (rRNA) is the most abundant RNA, we examined removes proteins and exposes nucleic acids during fixation. we
rRNA localization in meiotic spreads with anti-rRNA antibody. This found that pre-RNA species formed a string pattern and might be
monoclonal antibody specifically recognizes 5.8S rRNA. Interest- enriched at the unsynapsed axes of the X and Y chromosomes
ingly, we found that rRNA was remarkably enriched in the XY body (Supplementary information, Fig. S5f). In addition, we also
(Fig. 1d). Since it could not be digested by RNase H, the rRNA in XY examined other stages of meiotic prophase before the XY body
body did not form R-loops (Supplementary information, Fig. S3c). formation, including leptotene and zygotene. However, we did
Instead, with the RNase A treatment, the rRNA staining was not observe obvious rRNA staining in these stages (Supplementary
disappeared from meiotic spreads. Using excessive rRNA to block information, Fig. S6), suggesting that pre-rRNA is highly enriched
the epitope recognized by this antibody, the rRNA in the XY body in the XY body during pachytene and diplotene.
could not be stained (Fig. 1e). Since γH2AX mainly exists in the XY
body, we harvested spermatogonia from 3-week-old male mice, Pre-rRNA-associated RNA and proteins localize in the XY body
and performed RNA sequencing (RNA-seq) following RNA- Once pre-rRNA is transcribed, it associates with other small
chromatin immunoprecipitation assay (RNA-ChIP) using anti- nucleolar RNAs (snoRNA) and nucleolar proteins to form pre-
γH2AX antibody (Supplementary information, Fig. S4a). We found rRNPs.42 Thus, we asked whether snoRNAs- and pre-rRNA-
that majorities RNA associated with γH2AX was rRNA associated proteins exist in the XY body. We found that snoRNA
Cell Research (2022) 32:254 – 268
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