R‐loops mediate transcription‐associated formation of human rDNA secondary constrictions

Abstract

The ribosomal gene DNA (rDNA) often forms secondary constrictions in the chromosome; however, the molecular mechanism involved remains poorly understood. Here, we report that occurrence of rDNA constriction was increased in the chromosomes in human cancer cell lines compared with normal cells and that decondensed rDNA was significantly enhanced after partial inhibition of rDNA transcription. rDNA transcription was found during the S phase when replication occurred, and thus, DNA replication inhibitors caused constriction formation through hindering rDNA transcription. Inhibition of ataxia ATR (telangiectasia-mutated and RAD3-related) induced rDNA constriction formation. Replication stress or transcription inhibition increased R-loop formation. Topoisomerase I and RNase H1 suppressed secondary constriction formation. These data demonstrate that transcription stress causes the accumulation of stable R-loops (RNA-DNA hybrid) and subsequent constriction formation in the chromosomes.

The G9a/CHCHD2/Sirt1 Regulatory Module Acts on RNase H1 to Control R-Loop Formation at rDNA Sites

“…Clearly, there is a causal relationship between RNase H1-deletion mediated R-loop enrichment and rRNA transcription elongation arrest. In addition, our previous studies suggested that the decreased expression of RNase H1 triggered the formation of multiple nucleoli (Zhou et al, 2020) and RNase H1 is implicated in bigger rDNA constriction formation (Zhou et al, 2021). Those above results further confirmed that loss of RNase H1 causes RNAP I transcription-associated R-loop accumulation along with suppressing rRNA transcription to trigger disordered and fragmented nucleoli.…”

“…The secondary antibodies were the horseradish peroxidase (HRP) labeled goat anti-mouse IgG (A0126, Beyotime, Shanghai, China,) and the HRP labeled goat anti-rabbit IgG (A3327, Beyotime, Shanghai, China). Immunoreactivity was determined using the ECL method (K-12045-D50, advansta, California, USA) according to the manufacturer's instructions (Zhou et al, 2021).…”

Translesion DNA synthesis polymerase κ is essential to a carcinogen-induced nucleolar stress response

“…R-loops are transient, reversible structures that form in many regions of the genome. Predictors of R-loop presence include high transcriptional activity, open DNA and high GC content, all present in rDNA 66, 67 . R-loops participate in the regulation of transcription, but they also represent a potential source of damage, and their presence must be carefully regulated 68 .…”

Fragile sites, chromosomal lesions, tandem repeats, and disease

“…The tandem arrays of rDNA have been observed as fragile sites and other complex macro-structures ( Ferraro et al, 1977 ; Warmerdam and Wolthuis, 2019 ; Zhou et al, 2021 ). Fragility induction at rDNA arrays by aphidicolin and actinomycin-D was recently demonstrated ( Zhou et al, 2021 ).…”

“…The tandem arrays of rDNA have been observed as fragile sites and other complex macro-structures ( Ferraro et al, 1977 ; Warmerdam and Wolthuis, 2019 ; Zhou et al, 2021 ). Fragility induction at rDNA arrays by aphidicolin and actinomycin-D was recently demonstrated ( Zhou et al, 2021 ). The multiple clusters of tandem ribosomal DNA (rDNA) repeat arrays reside in the short arms of five of the 10 human acrocentric chromosomes, 13, 14, 15, 21, and 22 ( Boisvert et al, 2007 ; McStay, 2016 ).…”