Lnc956 -TRIM28-HSP90B1 complex on replication forks promotes CMG helicase retention to ensure stem cell genomic stability and embryogenesis

Abstract

Replication stress is a major source of endogenous DNA damage. Despite the identification of numerous proteins on replication forks to modulate fork or replication machinery activities, it remains unexplored whether noncoding RNAs can localize on stalled forks and play critical regulatory roles. Here, we identify an uncharacterized long noncoding RNA NONMMUT028956 ( Lnc956 for short) predominantly expressed in mouse embryonic stem cells. Lnc956 is accumulated on replication forks to prevent fork collapse and preserve genomic stability and is essential for mouse embryogenesis. Mechanistically, it drives assembly of the Lnc956 -TRIM28-HSP90B1 complex on stalled forks in an interdependent manner downstream of ataxia telangiectasia and Rad3-related (ATR) signaling. Lnc956 -TRIM28-HSP90B1 complex physically associates with minichromosome maintenance proteins 2 (MCM2) to minichromosome maintenance proteins 7 (MCM7) hexamer via TRIM28 and directly regulates the CDC45-MCM-GINS (CMG) helicase retention on chromatin. The regulation of Lnc956 -TRIM28-HSP90B1 on CMG retention is mediated by HSP90B1’s chaperoning function. These findings reveal a player that actively regulates replisome retention to prevent fork collapse.

Characterization of long-term <i>ex vivo</i> expansion of tree shrew spermatogonial stem cells

“…To corroborate the presence of DNA damage in long-term cultured SPGs, we conducted gene expression analyses. Notably, both immunoblot assays ( Figure 5A ; Supplementary Figure S1A) and immunofluorescence staining ( Figure 5B ) revealed that levels of γH2AX, a marker of DNA DSBs ( Rogakou et al, 1998 ; Zhang et al, 2023 ), increased with culture. Alkaline comet assays, which quantify DNA breaks at the single-cell level, further verified the increase in DNA fragmentation ( Swain & Rao, 2011 ; Zhao et al, 2015 ) ( Figure 5C ).…”

Purification of Long Non-coding RNAs on Replication Forks Using iROND (Isolate RNAs on Nascent DNA)

“…PCNA and Histone H3 can be used as a fork-binding protein marker and mature chromatin marker, respectively. Please refer to Supplementary Figure 1A in our publication ( Zhang et al, 2023 ).…”

“…RNA samples can be used either for qPCR analysis or RNA sequencing ( Figures 2 and 3 ). Please refer to Figure 1B , C, and D in our publication ( Zhang et al, 2023 ).…”

“…For the preparation of mESC medium and culture and expansion of mESCs and NIH3T3, please refer to our publication ( Zhang et al, 2023 ). Before EdU labeling, incubate cells with 37 °C pre-warmed fresh mESCs medium for 2 h, with 25 mL of medium for each 15 cm diameter dish containing ~1.5 × 10 7 cells.…”

“…Using iROND, we identified a specific fork-binding LncRNA Lnc956 , in the embryonic stem cell system. Lnc956 forms a stable nucleic acid–protein complex with TRIM28 and HSP90B1, facilitating the integration of the CMG helicase at replication fork sites and ensuring replication fork stability in mESCs ( Zhang et al, 2023 ). Here, we provide a step-by-step protocol for iROND.…”