The BRCA1 tumor suppressor forms a heterodimer with the BARD1 protein, and the resulting complex functions as an E3 ubiquitin ligase that catalyzes the synthesis of polyubiquitin chains. In theory, polyubiquitination can occur by isopeptide bond formation at any of the seven lysine residues of ubiquitin. The isopeptide linkage of a polyubiquitin chain is a particularly important determinant of its cellular function, such that K48-linked chains commonly target proteins for proteasomal degradation, while K63 chains serve non-proteolytic roles in various signaling pathways. To determine the isopeptide linkage formed by BRCA1/BARD1-dependent polyubiquitination, we purified a full-length heterodimeric complex and compared its linkage specificity with that of E6-AP, an E3 ligase known to induce proteolysis of its cellular substrates. Using a comprehensive mutation analysis, we found that E6-AP catalyzes the synthesis of K48-linked polyubiquitin chains. In contrast, however, the BRCA1/BARD1 heterodimer directs polymerization of ubiquitin primarily through an unconventional linkage involving lysine residue K6. Although heterologous substrates of BRCA1/BARD1 are not known, BRCA1 autoubiquitination occurs principally by conjugation with K6-linked polymers. The ability of BRCA1/BARD1 to form K6-linked polyubiquitin chains suggests that it may impart unique cellular properties to its natural enzymatic substrates.The BRCA1 tumor suppressor gene has been implicated in various cellular processes that include DNA repair, transcriptional regulation, and cell cycle checkpoint control (1). Its protein product contains an NH 2 -terminal RING domain and two COOH-terminal BRCT repeats. In vivo, BRCA1 exists as a heterodimer with BARD1, a related protein that displays a similar configuration of RING and BRCT motifs (2). The BRCA1/BARD1 heterodimer is stabilized by a 4-helix bundle formed by ␣-helices that immediately flank the RING domains of both polypeptides (3). Since most cellular BRCA1 polypeptides are found in association with BARD1 (4, 5), the BRCA1/ BARD1 heterodimer is likely to be an essential mediator of BRCA1 function (6). This conclusion is strongly supported by analysis of Bard1-null mice, which display a characteristic phenotype that is essentially indistinguishable from that of Brca1-null animals (7).Recent studies have uncovered an enzymatic role for the BRCA1/BARD1 heterodimer in protein ubiquitination (8 -15). Ubiquitination occurs through a sequential process involving ubiquitin-activating enzyme (E1), 1 a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3) (16,17). In the final step of this process, a ubiquitin monomer is covalently attached to a lysine residue on the ultimate substrate. The monoubiquitinated substrate can also serve as a nucleus for assembly of a polyubiquitin chain that, in many cases, will target the substrate for proteasome-mediated degradation. A common feature of many E3 ligases is the presence of a RING motif, and recent studies have established that sequences encompassing the RI...

Post-transcriptional gene silencing mediated by microRNAs (miRNAs) modulates numerous developmental and stress response pathways. For the last two decades, HASTY (HST), the ortholog of human EX-PORTIN 5, was considered to be a candidate protein that exports plant miRNAs from the nucleus to the cytoplasm. Here, we report that HST functions in the miRNA pathway independent of its cargo-exporting activity in Arabidopsis. We found that Arabidopsis mutants with impaired HST shuttling exhibit normal subcellular distribution of miRNAs. Interestingly, protein-protein interaction and microscopy assays showed that HST directly interacts with the microprocessor core component DCL1 through its N-terminal domain. Moreover, mass spectrometry analysis revealed that HST also interacts independently of its N-terminal domain with the mediator complex subunit MED37. Further experiments revealed that HST could act as a scaffold to facilitate the recruitment of DCL1 to genomic MIRNA loci by stabilizing the DCL1-MED37 complex, which in turn promotes the transcription and proper processing of primary miRNA transcripts (pri-miRNAs). Taken together, these results suggest that HST is likely associated with the formation of the miRNA biogenesis complex at MIRNA genes, promoting the transcription and processing of pri-miRNAs rather than the direct export of processed miRNAs from the nucleus.

DNA methylation patterns in plants are dynamically regulated by DNA methylation and active DNA demethylation in response to both environmental changes and development of plant. Beginning with the removal of methylated cytosine by ROS1/DME family of 5-methylcytosine DNA glycosylases, active DNA demethylation in plants occurs through base excision repair. So far, many components involved in active DNA demethylation remain undiscovered. Through a forward genetic screening of Arabidopsis mutants showing DNA hypermethylation at the EPF2 promoter region, we identified the conserved iron-sulfur cluster assembly protein MET18. MET18 dysfunction caused DNA hypermethylation at more than 1000 loci as well as the silencing of reporter genes and some endogenous genes. MET18 can directly interact with ROS1 in vitro and in vivo. ROS1 activity was reduced in the met18 mutant plants and point mutation in the conserved Fe-S cluster binding motif of ROS1 disrupted its biological function. Interestingly, a large number of DNA hypomethylated loci, especially in the CHH context, were identified from the met18 mutants and most of the hypo-DMRs were from TE regions. Our results suggest that MET18 can regulate both active DNA demethylation and DNA methylation pathways in Arabidopsis.

The N6‐methyladenosine (m6A) RNA modification serves crucial functions in RNA metabolism; however, the molecular mechanisms underlying the regulation of m6A are not well understood. Here, we establish arginine methylation of METTL14, a component of the m6A methyltransferase complex, as a novel pathway that controls m6A deposition in mammalian cells. Specifically, protein arginine methyltransferase 1 (PRMT1) interacts with, and methylates the intrinsically disordered C terminus of METTL14, which promotes its interaction with RNA substrates, enhances its RNA methylation activity, and is crucial for its interaction with RNA polymerase II (RNAPII). Mouse embryonic stem cells (mESCs) expressing arginine methylation‐deficient METTL14 exhibit significantly reduced global m6A levels. Transcriptome‐wide m6A analysis identified 1,701 METTL14 arginine methylation‐dependent m6A sites located in 1,290 genes involved in various cellular processes, including stem cell maintenance and DNA repair. These arginine methylation‐dependent m6A sites are associated with enhanced translation of genes essential for the repair of DNA interstrand crosslinks; thus, METTL14 arginine methylation‐deficient mESCs are hypersensitive to DNA crosslinking agents. Collectively, these findings reveal important aspects of m6A regulation and new functions of arginine methylation in RNA metabolism.

R-loops, which consist of a DNA/RNA hybrid and a displaced single-stranded DNA (ssDNA), are increasingly recognized as critical regulators of chromatin biology. R-loops are particularly enriched at gene promoters, where they play important roles in regulating gene expression. However, the molecular mechanisms that control promoter-associated R-loops remain unclear. The epigenetic ‘reader’ Tudor domain-containing protein 3 (TDRD3), which recognizes methylarginine marks on histones and on the C-terminal domain of RNA polymerase II, was previously shown to recruit DNA topoisomerase 3B (TOP3B) to relax negatively supercoiled DNA and prevent R-loop formation. Here, we further characterize the function of TDRD3 in R-loop metabolism and introduce the DExH-box helicase 9 (DHX9) as a novel interaction partner of the TDRD3/TOP3B complex. TDRD3 directly interacts with DHX9 via its Tudor domain. This interaction is important for recruiting DHX9 to target gene promoters, where it resolves R-loops in a helicase activity-dependent manner to facilitate gene expression. Additionally, TDRD3 also stimulates the helicase activity of DHX9. This stimulation relies on the OB-fold of TDRD3, which likely binds the ssDNA in the R-loop structure. Thus, DHX9 functions together with TOP3B to suppress promoter-associated R-loops. Collectively, these findings reveal new functions of TDRD3 and provide important mechanistic insights into the regulation of R-loop metabolism.

Background: Methylation is one of the common forms of RNA modification, which mainly include N6methyladenosine (m6A), C5-methylcytidine (m5C), and N1-methyladenosine (m1A). Numerous studies have shown that RNA methylation is associated with tumor development. We aim to construct prognostic models of gastric cancer based on RNA methylation regulators. Methods:The transcriptome and clinical data of gastric cancer and normal samples were obtained from the National Cancer Institute Genome Data Commons (NCI-GDC). Use Least Absolute Shrinkage and Selection Operator (LASSO) Cox regression analysis to construct risk models for different types of RNA methylation. Receiver operating characteristic (ROC) curves were generated to evaluate the predictive efficiency of risk characteristics. Cluster heat maps are used to assess the correlation with clinical information. Univariate and multivariate Cox analyses were used to analyze prognostic effects of risk scores.Gene Set Enrichment Analysis (GSEA) analyzes the functional enrichment of RNA methylation genes. And make a separate analysis of the data of Asians.Results: The expression of most of the 30 RNA methylation regulators were significantly different in cancer and paracancerous tissues (P<0.05). Three methylated genes (FTO, ALKBH5, and RBM15) were screened from m6A by LASSO Cox regression analysis. Five methylated genes (FTO, ALKBH5, TRMT61B, RBM15, and YXB1) were selected from the population, and were used to construct two risk ratio models.Survival analysis showed that the survival rate of patients in the low-risk group was significantly higher than that in the high-risk group (P<0.05). All ROC curves indicated that the predictive efficiency of risk characteristics was good [area under the ROC curve (AUC): 0.6-1].Cluster analysis reveals differences in clinical data between the two groups. Univariate and multivariate Cox regression results show that the risk score has independent prognostic value. GSEA showed that pathways such as cell cycle were significantly enriched in the low-risk group, while pathways such as calcium signaling pathway were significantly enriched in the high-risk group. In addition, three methylation models that can predict the prognosis of Asian gastric cancer patients were obtained. Li et al. Prognostic model of RNA methylation in gastric cancer

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