YTHDC1 delays cellular senescence and pulmonary fibrosis by activating ATR in an m6A-independent manner.

Accumulation of DNA damage in the lung induces cellular senescence and promotes age-related diseases such as idiopathic pulmonary fibrosis (IPF). Hence, understanding the mechanistic regulation of DNA damage repair is important for anti-aging therapies and disease control. Here, we identified an m6A-independent role of the RNA-binding protein YTHDC1 in counteracting stress-induced pulmonary senescence and fibrosis. YTHDC1 is primarily expressed in pulmonary alveolar epithelial type 2 (AECII) cells and its AECII expression is significantly decreased in AECIIs during fibrosis. Exogenous overexpression of YTHDC1 alleviates pulmonary senescence and fibrosis independent of its m6A-binding ability, while YTHDC1 deletion enhances disease progression in mice. Mechanistically, YTHDC1 promotes the interaction between TopBP1 and MRE11, thereby activating ATR and facilitating DNA damage repair. These findings reveal a noncanonical function of YTHDC1 in delaying cellular senescence, and suggest that enhancing YTHDC1 expression in the lung could be an effective treatment strategy for pulmonary fibrosis.

METTL3 and N6-Methyladenosine Promote Homologous Recombination-Mediated Repair of DSBs by Modulating DNA-RNA Hybrid Accumulation.

Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may lead to genome instability or cell death. Here, we report that, in response to DSBs, the RNA methyltransferase METTL3 is activated by ATM-mediated phosphorylation at S43. Phosphorylated METTL3 is then localized to DNA damage sites, where it methylates the N6 position of adenosine (m6A) in DNA damage-associated RNAs, which recruits the m6A reader protein YTHDC1 for protection. In this way, the METTL3-m6A-YTHDC1 axis modulates accumulation of DNA-RNA hybrids at DSBs sites, which then recruit RAD51 and BRCA1 for homologous recombination (HR)-mediated repair. METTL3-deficient cells display defective HR, accumulation of unrepaired DSBs, and genome instability. Accordingly, depletion of METTL3 significantly enhances the sensitivity of cancer cells and murine xenografts to DNA damage-based therapy. These findings uncover the function of METTL3 and YTHDC1 in HR-mediated DSB repair, which may have implications for cancer therapy.

UBQLN1 deficiency mediates telomere shortening and IPF through interacting with RPA1.

Premature telomere shortening is a known factor correlated to idiopathic pulmonary fibrosis (IPF) occurrence, which is a chronic, progressive, age-related disease with high mortality. The etiology of IPF is still unknown. Here, we found that UBQLN1 plays a key role in telomere length maintenance and is potentially relevant to IPF. UBQLN1 involves in DNA replication by interacting with RPA1 and shuttling it off from the replication fork. The deficiency of UBQLN1 retains RPA1 at replication fork, hinders replication and thus causes cell cycle arrest and genome instability. Especially at telomere regions of the genome, where more endogenous replication stress exists because of G rich sequences, UBQLN1 depletion leads to rapid telomere shortening in HeLa cells. It revealed that UBQLN1 depletion also shortens telomere length at mouse lung and accelerates mouse lung fibrosis. In addition, the UBQLN1 expression level in IPF patients is downregulated and correlated to poor prognosis. Altogether, these results uncover a new role of UBQLN1 in ensuring DNA replication and maintaining telomere stability, which may shed light on IPF pathogenesis and prevention.

Telomerase RNA TERC and the PI3K-AKT pathway form a positive feedback loop to regulate cell proliferation independent of telomerase activity.

The core catalytic unit of telomerase comprises telomerase reverse transcriptase (TERT) and telomerase RNA (TERC). Unlike TERT, which is predominantly expressed in cancer and stem cells, TERC is ubiquitously expressed in normal somatic cells without telomerase activity. However, the functions of TERC in these telomerase-negative cells remain elusive. Here, we reported positive feedback regulation between TERC and the PI3K-AKT pathway that controlled cell proliferation independent of telomerase activity in human fibroblasts. Mechanistically, we revealed that TERC activated the transcription of target genes from the PI3K-AKT pathway, such as PDPK1, by targeting their promoters. Overexpression of PDPK1 partially rescued the deficiency of AKT activation caused by TERC depletion. Furthermore, we found that FOXO1, a transcription factor negatively regulated by the PI3K-AKT pathway, bound to TERC promoter and suppressed its expression. Intriguingly, TERC-induced activation of the PI3K-AKT pathway also played a critical role in the proliferation of activated CD4+ T cells. Collectively, our findings identify a novel function of TERC that regulates the PI3K-AKT pathway via positive feedback to elevate cell proliferation independent of telomerase activity and provide a potential strategy to promote CD4+ T cells expansion that is responsible for enhancing adaptive immune reactions to defend against pathogens and tumor cells.

BRM-SWI/SNF chromatin remodeling complex enables functional telomeres by promoting co-expression of TRF2 and TRF1.

TRF2 and TRF1 are a key component in shelterin complex that associates with telomeric DNA and protects chromosome ends. BRM is a core ATPase subunit of SWI/SNF chromatin remodeling complex. Whether and how BRM-SWI/SNF complex is engaged in chromatin end protection by telomeres is unknown. Here, we report that depletion of BRM does not affect heterochromatin state of telomeres, but results in telomere dysfunctional phenomena including telomere uncapping and replication defect. Mechanistically, expression of TRF2 and TRF1 is jointly regulated by BRM-SWI/SNF complex, which is localized to promoter region of both genes and facilitates their transcription. BRM-deficient cells bear increased TRF2-free or TRF1-free telomeres due to insufficient expression. Importantly, BRM depletion-induced telomere uncapping or replication defect can be rescued by compensatory expression of exogenous TRF2 or TRF1, respectively. Together, these results identify a new function of BRM-SWI/SNF complex in enabling functional telomeres for maintaining genome stability.

Sonic hedgehog restrains the ubiquitin-dependent degradation of SP1 to inhibit neuronal/glial senescence associated phenotypes in chemotherapy-induced peripheral neuropathy via the TRIM25-CXCL13 axis.

INTRODUCTION: Chemotherapy-induced peripheral neuropathy (CIPN) is a common complication that affects an increasing number of cancer survivors. However, the current treatment options for CIPN are limited. Paclitaxel (PTX) is a widely used chemotherapeutic drug that induces senescence in cancer cells. While previous studies have demonstrated that Sonic hedgehog (Shh) can counteract cellular dysfunction during aging, its role in CIPN remains unknown. OBJECTIVES: Herein, the aim of this study was to investigate whether Shh activation could inhibits neuronal/glial senescence and alleviates CIPN. METHODS: We treated ND7/23 neuronal cells and RSC96 Schwann cells with two selective Shh activators (purmorphamine [PUR] and smoothened agonist [SAG]) in the presence of PTX. Additionally, we utilized a CIPN mouse model induced by PTX injection. To assess cellular senescence, we performed a senescence-associated ß-galactosidase (SA-ß-gal) assay, measured reactive oxygen species (ROS) levels, and examined the expression of P16, P21, and γH2AX. To understand the underlying mechanisms, we conducted ubiquitin assays, LC-MS/MS, H&E staining, and assessed protein expression through Western blotting and immunofluorescence staining. RESULTS: In vitro, we observed that Shh activation significantly alleviated the senescence-related decline in multiple functions included SA-ß-gal activity, expression of P16 and P21, cell viability, and ROS accumulation in DRG sensory neurons and Schwann cells after PTX exposure. Furthermore, our in vivo experiments demonstrated that Shh activation significantly reduced axonal degeneration, demyelination, and improved nerve conduction. Mechanistically, we discovered that PTX reduced the protein level of SP1, which was ubiquitinated by the E3 ligase TRIM25 at the lysine 694 (K694), leading to increased CXCL13 expression, and we found that Shh activation inhibited PTX-induced neuronal/glial senescence and CIPN through the TRIM25-SP1-CXCL13 axis. CONCLUSION: These findings provide evidence for the role of PTX-induced senescence in DRG sensory neurons and Schwann cells, suggesting that Shh could be a potential therapeutic target for CIPN.

TRF2 recruits nucleolar protein TCOF1 to coordinate telomere transcription and replication.

Telomeres are transcribed into telomeric RNA termed as TERRA. However, the transcription itself and excessive TERRA may interfere with telomere replication during S phase. The mechanism that coordinates telomere transcription and replication is unknown. Here, we report that TCOF1 leaves the nucleolus and is recruited to telomeres specifically during S phase by interacting with TRF2. Therein, TCOF1 acts to suppress telomere transcription by binding and inhibiting Pol II. Thus, TERRA is limited to low levels in S phase. Depletion of TCOF1 leads to abnormally elevated TERRA and formation of DNA/RNA hybrids (R-loops) at telomeres, which induces replication fork stalling and fragile telomeres. Importantly, telomere replication defect induced by TCOF1 deficiency can be rescued by either masking TERRA or expressing an R-loop eraser RNase H1, demonstrating a critical role of TCOF1 in coordinating telomere transcription and replication. These findings link nucleolus to telomeres and uncover a novel function of TCOF1 on ensuring telomere integrity.

RBMX is required for activation of ATR on repetitive DNAs to maintain genome stability.

ATR is a master regulator of cell response to replication stress. Adequate activation of ATR is essential for preventing genome aberrance induced by replication defect. However, the mechanism underlying ATR activation is not fully understood. Here, we identify that RBMX is an ssDNA binding protein that orchestrates a novel pathway to activate ATR. Using super-resolution STORM, we observe that RBMX and RPA bind to adjacent but nonoverlapping sites on ssDNA in response to replication stress. RBMX then binds to and facilitates positioning of TopBP1, which activates nearby ATR associated with RPA. In addition, ATR activation by ssDNA-RBMX-TopBP1 is independent of ssDNA-dsDNA junction and 9-1-1 complex. ChIP-seq analysis reveals that RBMX/RPA are highly enriched on repetitive DNAs, which are considered as fragile sites with high replication stress. RBMX depletion leads to defective localization of TopBP1 to replication stressed sites and inadequate activation of ATR. Furthermore, cells with deficient RBMX demonstrate replication defect, leading to formation of micronuclei and a high rate of sister-chromatin exchange, indicative of genome instability. Together, the results identify a new ssDNA-RBMX-TopBP1 pathway that is specifically required for activation of ATR on repetitive DNAs. Therefore, RBMX is a key factor to ensure genome stability during replication.

Quantitative serum proteomics analyses reveal shrimp responses against WSSV infection.

For the past decades, quantitative serum proteomics in mammals have been widely applied in biomarker screening. Various serum protein removal methods have been developed to effectively sequester serum abundant proteins. However, few methods have been found for the removal of arthropod serum abundant proteins. Here, gel filtration chromatography and ultracentrifugation methods were applied to remove hemocyanin from Litopeaneus vannamei serum. When shrimps were challenged with white spot syndrome virus (WSSV), a total of 486 serum proteins were identified using mass spectrometry, and 18 upregulated WSSV responsive proteins were identified with isobaric tags for relative and absolute quantification (iTRAQ). These results provide an effective method to remove hemocyanin from shrimp serum. With this method some previously unidentified WSSV responsive serum proteins were revealed, which would give us a better insight into the response of crustaceans to WSSV infection.

Ultrasensitive genotyping with target-specifically generated circular DNA templates and RNA FRET probes.

A novel RNA FRET probe that can produce target-dependent signal amplification with the catalysis of RNase H has been developed for detection of rolling circle amplification (RCA) products with greatly improved sensitivity.