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DNA methylation at selective cytosine residues (5-methylcytosine (5mC)) and their removal by TET-mediated DNA demethylation are critical for setting up pluripotent states in early embryonic development. TET enzymes successively convert 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), with 5fC and 5caC subject to removal by thymine DNA glycosylase (TDG) in conjunction with base excision repair. Early reports indicate that 5fC and 5caC could be stably detected on enhancers, promoters and gene bodies, with distinct effects on gene expression, but the mechanisms have remained elusive. Here we determined the X-ray crystal structure of yeast elongating RNA polymerase II (Pol II) in complex with a DNA template containing oxidized 5mCs, revealing specific hydrogen bonds between the 5-carboxyl group of 5caC and the conserved epi-DNA recognition loop in the polymerase. This causes a positional shift for incoming nucleoside 5'-triphosphate (NTP), thus compromising nucleotide addition. To test the implication of this structural insight in vivo, we determined the global effect of increased 5fC/5caC levels on transcription, finding that such DNA modifications indeed retarded Pol II elongation on gene bodies. These results demonstrate the functional impact of oxidized 5mCs on gene expression and suggest a novel role for Pol II as a specific and direct epigenetic sensor during transcription elongation.

SignificanceCyclopurines are bulky oxidative DNA lesions that strongly block RNA polymerase II (Pol II) to interfere with gene transcription and replication in mammalian cells. Cells developed a mechanism enabling slow transcriptional bypass of the cyclopurines. Similar to translesion synthesis by DNA polymerases, lesion bypass by Pol II can be highly mutagenic, leading to transcription errors that are reminiscent of the A rule. We elucidated the mechanism and determined the domain in Pol II responsible for error-free and error-prone lesion bypass. We also identified a positive role of mammalian factor TFIIF in lesion-bypass stimulation. Strikingly, Pol II uses a similar strategy for negotiation with different DNA lesions, such as cyclopurines, pyrimidine dimers, cisplatin, and abasic sites, to reduce the burden of DNA damage on genome stability.In human cells, the oxidative DNA lesion 8,5′-cyclo-2'-deoxyadenosine (CydA) induces prolonged stalling of RNA polymerase II (Pol II) followed by transcriptional bypass, generating both error-free and mutant transcripts with AMP misincorporated immediately downstream from the lesion. Here, we present biochemical and crystallographic evidence for the mechanism of CydA recognition. Pol II stalling results from impaired loading of the template base (5′) next to CydA into the active site, leading to preferential AMP misincorporation. Such predominant AMP insertion, which also occurs at an abasic site, is unaffected by the identity of the 5′-templating base, indicating that it derives from nontemplated synthesis according to an A rule known for DNA polymerases and recently identified for Pol II bypass of pyrimidine dimers. Subsequent to AMP misincorporation, Pol II encounters a major translocation block that is slowly overcome. Thus, the translocation block combined with the poor extension of the dA.rA mispair reduce transcriptional mutagenesis. Moreover, increasing the active-site flexibility by mutation in the trigger loop, which increases the ability of Pol II to accommodate the bulky lesion, and addition of transacting factor TFIIF facilitate CydA bypass. Thus, blocking lesion entry to the active site, translesion A rule synthesis, and translocation block are common features of transcription across different bulky DNA lesions.

CSB/ERCC6 (Cockayne syndrome B protein/excision repair cross-complementation group 6), a member of a subfamily of SWI2/SNF2 (SWItch/sucrose nonfermentable)-related chromatin remodelers, plays crucial roles in gene expression and the maintenance of genome integrity. Here, we report the mechanism of the autoregulation of Rhp26, which is the homolog of CSB/ERCC6 in Schizosaccharomyces pombe. We identified a novel conserved protein motif, termed the "leucine latch," at the N terminus of Rhp26. The leucine latch motif mediates the autoinhibition of the ATPase and chromatin-remodeling activities of Rhp26 via its interaction with the core ATPase domain. Moreover, we found that the C terminus of the protein counteracts this autoinhibition and that both the N- and C-terminal regions of Rhp26 are needed for its proper function in DNA repair in vivo. The presence of the leucine latch motif in organisms ranging from yeast to humans suggests a conserved mechanism for the autoregulation of CSB/ERCC6 despite the otherwise highly divergent nature of the N- and C-terminal regions.

Simple Summary: Small extracellular vesicles (sEVs) play an important role in the occurrence and development of various diseases, exhibiting the characteristics of wide acquisition and strong specificity. Due to the difficulties in the diagnosis and treatment of urological tumors, this article mainly reviews the function of sEVs in urinary tumors and their related application in the diagnosis and treatment of such tumors. The contents herein can provide new directions for the early diagnosis and individualized treatment of urinary tumors. Extracellular vesicles (EVs) are important mediators of communication between tumor cells and normal cells. These vesicles are rich in a variety of contents such as RNA, DNA, and proteins, and can be involved in angiogenesis, epithelial-mesenchymal transition, the formation of pre-metastatic ecological niches, and the regulation of the tumor microenvironment. Small extracellular vesicles (sEVs) are a type of EVs. Currently, the main treatments for urological tumors are surgery, radiotherapy, and targeted therapy. However, urological tumors are difficult to diagnose and treat due to their high metastatic rate, tendency to develop drug resistance, and the low sensitivity of liquid biopsies. Numerous studies have shown that sEVs offer novel therapeutic options for tumor treatment, such as tumor vaccines and tumor drug carriers. sEVs have attracted a great deal of attention owing to their contribution to in intercellular communication, and as novel biomarkers, and role in the treatment of urological tumors. This article reviews the research and applications of sEVs in the diagnosis and treatment of urological tumors. [ABSTRACT FROM AUTHOR]

Cardiac electrophysiological function is under the regulatory control of the sympathetic nervous system. In addition to classical β-adrenoceptors (β-AR, including β1- and β2- subtypes), β3-AR is also expressed in human heart and shows its distinctive functions. This study is aimed to elucidate the role of β3-AR in the regulation of atrial fibrillation (AF), especially its role in rapid pacing-induced atrial electrical remodeling in rabbits. The rapid atrial pacing model was established by embedding electrodes in the right atrium pacing at a speed of 600 beats per minute. The protein level of β3-AR in the atria was found significantly upregulated by western blot. The atrial effective refractory period (AERP) and its rate adaptation were decreased after pacing which were further shortened by BRL37344, a selective β3-AR agonist, leading to the increase of AF inducibility and duration. Similarly, β3-AR activation induced time-dependent shortening of action potential duration (APD), together with decrease of L-type calcium current (ICa,L) and increase of inward rectifier potassium current (IK1) and transient outward potassium current (Ito) in rapid pacing atrial myocytes. Meanwhile, all the effects were abolished by specific β3-AR antagonist, SR59230A. In summary, our study represents that activation of β3-AR promotes the atrial electrical remodeling process by altering the balance of ion channels in atrial myocytes, which provides new insights into the pharmacological role of β3-AR in heart diseases. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is the second most common malignant tumor of the gastrointestinal tract with the second highest mortality rate and the third highest incidence rate. Early diagnosis and treatment are important measures to reduce CRC mortality. Small extracellular vesicles (sEVs) have emerged as key mediators that facilitate communication between tumor cells and various other cells, playing a significant role in the growth, invasion, and metastasis of cancer cells. Regulatory RNAs have been identified as potential biomarkers for early diagnosis and prognosis of CRC, serving as crucial factors in promoting CRC cell proliferation, invasion and metastasis, angiogenesis, drug resistance, and immune cell differentiation. This review provides a comprehensive summary of the vital role of sEVs as biomarkers in CRC diagnosis and their potential application in CRC treatment, highlighting their importance as a promising avenue for further research and clinical translation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hu, Lu, Zhou, Wang, Zhang, Mao and Chen.)

Small extracellular vesicles (sEVs) are minute vesicles secreted by various cells that are capable of transporting cargo, including microRNAs, between donor and recipient cells. MicroRNAs (miRNAs), small non-coding RNAs approximately 22 nucleotides in length, have been implicated in a wide array of biological processes, including those involved in tumorigenesis. Emerging evidence highlights the pivotal role of miRNAs encapsulated in sEVs in both the diagnosis and treatment of urological tumors, with potential implications in epithelial-mesenchymal transition, proliferation, metastasis, angiogenesis, tumor microenvironment and drug resistance. This review provides a brief overview of the biogenesis and functional mechanisms of sEVs and miRNAs, followed by a summarization of recent empirical findings on miRNAs encapsulated in sEVs from three archetypal urologic malignancies: prostate cancer, clear cell renal cell carcinoma, and bladder cancer. We conclude by underscoring the potential of sEV-enclosed miRNAs as both biomarkers and therapeutic targets, with a particular focus on their detection and analysis in biological fluids such as urine, plasma, and serum., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mao, Zhang, Wang, Lu, Hu and Chen.)