ABSTRACTDuring cell division and gene expression, the DNA double‐helical structure unwinds, thereby generating torsional stress. DNA topoisomerases are enzymes that relieve this stress. During this process, topoisomerases form temporary covalent bonds with the phosphate backbone of DNA, generating DNA strand breaks and relieving torsional stress. Topoisomerases then dissociate from DNA after rejoining the DNA breaks. Torsional stress associated with replication or transcription is primarily relieved by topoisomerase I (TOP1) and II (TOP2). Some anticancer drugs targeting topoisomerases, known as topoisomerase poisons, trap the topoisomerase reaction intermediates and cause DNA strand breaks bearing topoisomerase–DNA–protein crosslinks (TOP–DPCs). TOP1 poisons, such as camptothecin, cause DNA single‐strand breaks bearing TOP1–DPCs, which are converted to DNA double‐strand breaks (DSBs) when they collide with DNA replication forks. In contrast, TOP2 poisons, such as etoposide, directly induce DSBs in TOP2–DPCs. However, to elicit a DSB response, TOP2–DPC must first be removed from the DSB ends. Cells possess various pathways to remove TOP2–DPC, and these pathways are thought to function in coordination depending on the situation. This review summarizes these sophisticated TOP2–DPC removal pathways and discusses the clinical applications of TOP2 poison as an anticancer drug, as well as the related challenges.

ABSTRACTMicroRNAs (miRNAs) play a crucial role in posttranscriptional gene regulation. The biogenesis of mature miRNAs requires precise cleavage of precursor miRNAs (pre‐miRNAs) by Dicer. Several computational approaches have been developed to predict human Dicer cleavage sites; however, important limitations persist. Cleavage Pattern‐based models, which rely on short pre‐miRNA subsequences, can only identify positive patterns in which the cleavage site is centrally located. Conversely, models that do not rely on Cleavage Patterns generally exhibit suboptimal performance. These limitations highlight the need for a more accurate predictor that fully exploits sequence and structural information from pre‐miRNAs. In this study, we propose DiCleavePlus, a Cleavage Pattern‐based framework for predicting Dicer cleavage sites on pre‐miRNAs. DiCleavePlus takes an extended Cleavage Pattern together with the full‐length pre‐miRNA sequence from which it is derived as input. A Transformer‐based encoder is employed to extract features from both the pattern and the pre‐miRNA. Benchmarking experiments demonstrate that DiCleavePlus achieves accurate and robust performance in predicting human Dicer cleavage sites.

ABSTRACTTestis‐specific gene regulatory mechanisms govern the differentiation of germ cells into mature sperm. However, the molecular underpinnings are not fully elucidated. Here, we show the subunits forming Paf1C, a transcription regulator complex conserved across eukaryotes, have their individual paralogs predominantly expressed in Drosophila testes. One of these, namely, Ctr9 paralog enriched in testes (Ctr9t) was found to play a critical and nonredundant role in postmeiotic spermatid differentiation and male fertility in D. melanogaster. A proximity proteome analysis provides evidence that Ctr9t prefers the interaction between paralog members. We show endogenous Ctr9t is expressed and functional in germ cells at spermatocyte stages, accumulating in a distinct compartment within the nucleolus. There, Ctr9t co‐localizes with Spermatocyte arrest (Sa), a testis‐specific paralog of TATA‐binding protein (TBP)‐associated factor 8 (TAF8). We further demonstrate that ctr9t function is crucial for maintaining Sa in the nucleolus, but not vice versa. Transcriptome profiling reveals that Ctr9t acts as an activator for the set of male fertility genes on the Y chromosome, but it also acts as a global repressor of X chromosome genes. Collectively, our results shed light on the nucleolus‐associated, paralog‐dependent regulation of gene expression from sex chromosomes, which ensures the terminal differentiation of male germ cells.

Ganciclovir (GCV) is a clinically important drug as it is used to treat viral infections. GCV is incorporated into the DNA during replication, where it interferes with subsequent replication on GCV‐incorporated templates. However, the effects of GCV on the host genome and the mechanisms underlying cellular tolerance to GCV remain unclear. In this study, we explored these mechanisms using a collection of mutant DT40 cells. We identified RAD17/−, BRCA1−/−, and RAD18−/− cells as highly GCV‐sensitive. RAD17, a component of the alternative checkpoint‐clamp loader RAD17‐RFC, was required for the activation of the intra‐S checkpoint following GCV treatment. BRCA1, a critical factor for promoting homologous recombination (HR), was required for suppressing DNA double‐strand breaks (DSBs). Moreover, RAD18, an E3‐ligase involved in DNA repair, was critical in suppressing the aberrant ligation of broken chromosomes caused by GCV. We found that BRCA1 suppresses DSBs through HR‐mediated repair and template switching (TS)‐mediated damage bypass. Moreover, the strong GCV sensitivity of BRCA1−/− cells was rescued by the loss of 53BP1, despite the only partial restoration in the sister chromatid exchange events which are hallmarks of HR. These results indicate that BRCA1 promotes cellular tolerance to GCV through two mechanisms, TS and HR‐mediated repair.

Front cover: The year 2024, the Year of the Dragon, was opened. Mt. Fuji was quickly enveloped by colorful clustered single‐cell RNA‐seq data‐like clouds that rose from the dragon targeting the mountain. Designed by TRAIS Co., Ltd. (Kobe, Japan).image

Mitophagy is programmed selective autophagy of mitochondria and is important for mitochondrial quality control and cellular homeostasis. Mitochondrial dysfunction and impaired mitophagy are closely associated with various diseases, including heart failure and diabetes. To better understand the pathophysiological role of mitophagy, we generated doxycycline-inducible mitophagy mice using a synthetic mitophagy adaptor protein consisting of an outer mitochondrial membrane targeting sequence and an engineered LIR. To evaluate the activation of mitophagy upon doxycycline treatment, we also generated mitophagy reporter mito-QC mice in which mitochondria tandemly express mCherry and GFP, and only GFP signals are lost in acidic lysosomes subjected to mitophagy. With the ROSA26 promoter-driven rtTA, mitophagy was observed at least in heart, liver, and skeletal muscle. We investigated the relationship between mitophagy activation and pressure overload heart failure or high fat diet-induced obesity. Unexpectedly, we were unable to confirm the protective effect of mitophagy in these two pathological models. Further titration of the level of mitophagy induction is required to demonstrate the potency of the protective effects of mitophagy in disease models.

Suppressor of cancer cell invasion (SCAI) acts as a transcriptional repressor of serum response factor (SRF)-mediated gene expression by binding to megakaryoblastic leukemia (MKL)/myocardin-related transcription factor (MRTF), which is an SRF transcriptional coactivator. Growing evidence suggests that SCAI is a negative regulator of neuronal morphology, whereas MKL2/MRTFB is a positive regulator. The mRNA expression of SCAI is downregulated during brain development, suggesting that a reduction in SCAI contributes to the reduced suppression of SRF-mediated gene induction, thus increasing dendritic complexity and developing neuronal circuits. In the present study, we hypothesized that brain-derived neurotrophic factor (BDNF), which is important for neuronal plasticity and development, might alter SCAI mRNA levels. We therefore investigated the effects of BDNF on SCAI mRNA levels in primary cultured cortical neurons. Furthermore, because alternative splicing generates several SCAI variants in the brain, we measured SCAI variant mRNA after BDNF stimulation. Both SCAI variant 1 and total SCAI mRNA expression levels were downregulated by BDNF. Moreover, the extracellular signal-regulated protein kinase/mitogen-activated protein kinase (ERK/MAPK) pathway was involved in the BDNF-mediated decrease in SCAI mRNA expression. Our findings provide insights into the molecular mechanism underlying a neurotrophic factor switch for the repressive transcriptional complex that includes SCAI.

Front cover: An awake mouse is pulling to move a soundly sleeping mouse by the tail. The tail of the awake mouse is coiled just like the 3D structure of orexin‐A, a hormone that regulates arousal. Designed by TRAIS Co., Ltd. (Kobe, Japan). image