A regulatory mechanism for Rsp5, a multifunctional ubiquitin ligase in Saccharomyces cerevisiae: characterization of its interaction with a deubiquitinating enzyme

HECT E3 ubiquitin ligases are widely distributed from yeast to human cells and play important roles in many physiological processes. Rsp5, an essential HECT E3 ligase in Saccharomyces cerevisiae, is involved in many biological processes, including transcriptional activation, endocytic trafficking, mitochondrial inheritance, and RNA export pathways. Although Rsp5 has been shown to regulate multiple pathways targeting multiple substrates, mechanisms for regulating the biochemical activity of Rsp5 are largely uncharacterized (121, 199). To gain further insight into the regulation of this enzyme, I identified proteins that copurified with epitope-tagged Rsp5. Ubp2, a deubiquitinating enzyme, was a prominent copurifying protein. Rup1, a previously uncharacterized UBA domain protein, was required for binding of Rsp5 to Ubp2 both in vitro and in vivo. Biochemical and genetic evidence are consistent with a model that Ubp2and Rup1 antagonizes Rsp5-catalyzed substrate ubiquitination. In vivo and in vitro experiments showed that Rsp5 and Ubp2 display strong preferences for assembly and disassembly of K63-linked polyubiquitination, respectively. A large fraction of the K63 conjugates in ubp2∆ cells bound to Rsp5, and a proteomics approach was therefore used to identify Rsp5 substrates subject to Ubp2 regulation. Two proteins implicated in cell wall integrity, Csr2 and Ecm21, were identified and both proteins were efficiently K63- polyubiquitinated by Rsp5 and deubiquitinated by Ubp2. I have also shown that cell wall integrity is impaired in rsp5-1 cells and this can be rescued by either ubp2∆ or rup1∆ mutation, suggesting that the Ubp2/Rup1 complex negatively regulates Rsp5-mediated cell wall homeostasis. Together, these data represent a novel regulatory mechanism for Rsp5 and suggest that similar mechanisms might be utilized by its mammalian homologues. Furthermore, this work provides a basis for studying the mechanism for differential polyubiquitin chain type synthesis by HECT E3 ligases.Institute for Cellular and Molecular Biolog

Role of Deubiquitinating Enzymes in DNA Repair

Both proteolytic and nonproteolytic functions of ubiquitination are essential regulatory mechanisms for promoting DNA repair and the DNA damage response in mammalian cells. Deubiquitinating enzymes (DUBs) have emerged as key players in the maintenance of genome stability. In this minireview, we discuss the recent findings on human DUBs that participate in genome maintenance, with a focus on the role of DUBs in the modulation of DNA repair and DNA damage signaling

Role of Deubiquitinating Enzymes in DNA Repair

Both proteolytic and nonproteolytic functions of ubiquitination are essential regulatory mechanisms for promoting DNA repair and the DNA damage response in mammalian cells. Deubiquitinating enzymes (DUBs) have emerged as key players in the maintenance of genome stability. In this minireview, we discuss the recent findings on human DUBs that participate in genome maintenance, with a focus on the role of DUBs in the modulation of DNA repair and DNA damage signaling

Expanded roles of the Fanconi anemia pathway in preserving genomic stability

Studying rare human genetic diseases often leads to a better understanding of normal cellular functions. Fanconi anemia (FA), for example, has elucidated a novel DNA repair mechanism required for maintaining genomic stability and preventing cancer. The FA pathway, an essential tumor-suppressive pathway, is required for protecting the human genome from a specific type of DNA damage; namely, DNA interstrand cross-links (ICLs). In this review, we discuss the recent progress in the study of the FA pathway, such as the identification of new FANCM-binding partners and the identification of RAD51C and FAN1 (Fanconi-associated nuclease 1) as new FA pathway-related proteins. We also focus on the role of the FA pathway as a potential regulator of DNA repair choices in response to double-strand breaks, and its novel functions during the mitotic phase of the cell cycle

Inactivating UBE2M Impacts the DNA Damage Response and Genome Integrity Involving Multiple Cullin Ligases

Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle

RAD51 wrestles with SUMO

RAD51 loading onto chromatin is a key step during the homologous recombination (HR) repair. We recently reported a new mode of RAD51 regulation, which is mediated by TOPORS E3 SUMO ligase and RAD51 SUMOylation. ATM/ATR-induced phosphorylation of TOPORS is necessary for this event, revealing a new role of these master DNA damage response kinases in HR repair

Regulated degradation of FANCM in the Fanconi anemia pathway during mitosis

The 13 Fanconi anemia (FA) proteins cooperate in a common DNA repair pathway. Eight of these proteins are assembled into a multisubunit E3 ligase called the FA core complex. During S phase, the FA core complex is loaded by the FANCM protein into chromatin where it monoubiquitinates its substrates. In mitosis, the FA core complex is released from FANCM by an unknown mechanism. Here we show that FANCM is hyperphosphorylated and degraded during mitosis. β-TRCP and Plk1 are the key regulators of FANCM degradation. Nondegradable mutant forms of FANCM retain the FA core complex in the chromatin and disrupt the FA pathway. Our data provide a novel mechanism for the cell cycle-dependent regulation of the FA pathway

Splicing factor SRSF3 represses translation of p21(cip1/waf1) mRNA

Serine/arginine-rich splicing factor 3 (SRSF3) is an RNA binding protein that most often regulates gene expression at the splicing level. Although the role of SRSF3 in mRNA splicing in the nucleus is well known, its splicing-independent role outside of the nucleus is poorly understood. Here, we found that SRSF3 exerts a translational control of p21 mRNA. Depletion of SRSF3 induces cellular senescence and increases the expression of p21 independent of p53. Consistent with the expression patterns of SRSF3 and p21 mRNA in the TCGA database, SRSF3 knockdown increases the p21 mRNA level and its translation efficiency as well. SRSF3 physically associates with the 3′UTR region of p21 mRNA and the translational initiation factor, eIF4A1. Our study proposes a model in which SRSF3 regulates translation by interacting with eIF4A1 at the 3′UTR region of p21 mRNA. We also found that SRSF3 localizes to the cytoplasmic RNA granule along with eIF4A1, which may assist in translational repression therein. Thus, our results provide a new mode of regulation for p21 expression, a crucial regulator of the cell cycle and senescence, which occurs at the translational level and involves SRSF3. © 2022, The Author(s).TRU

Bathyptilones: Terpenoids from an Antarctic Sea Pen, Anthoptilum grandiflorum (Verrill, 1879)

An Antarctic coral belonging to the order Pennatulacea, collected during the 2013 austral autumn by trawl from 662 to 944 m depth, has yielded three new briarane diterpenes, bathyptilone A-C (1–3) along with a trinorditerpene, enbepeanone A (4), which bears a new carbon skeleton. Structure elucidation was facilitated by one- and two-dimensional NMR spectroscopy, mass spectrometry and confirmed by X-ray crystallography. The three compounds were screened in four cancer cell lines. Bathyptilone A displayed selective nanomolar cytotoxicity against the neurogenic mammalian cell line Ntera-2