Investigating the regulation of the human decapping enzyme Dcp2

.Decapping is a critical step in several mRNA decay pathways that are important for the regulation of gene expression. A major decapping complex consists of the decapping enzyme Dcp2 as well as several decapping enhancers. Although several interactions between decapping factors have been mapped, little is known about how Dcp2 activity is controlled to target the appropriate mRNAs for decapping or how these enhancers regulate this activity. Through a deletion analysis, I identified an important autoregulatory domain that is responsible for Dcp2 protein turnover. I demonstrated that the metazoan specific decapping enhancer, Hedls, modulates Dcp2 stability and requires the autoregulatory domain of Dcp2 for interaction. Furthermore, Hedls depletion reduces Dcp2 cellular activity independent of protein stability. Thus Hedls promotes Dcp2 mediated decapping by at least two mechanisms: controlling Dcp2 stability and enhancing Dcp2 catalytic activity. This could provide a way to modulate Dcp2 activity according to cellular demand, thereby preventing decapping of inappropriate targets. Hedls is required for Dcp2 concentration in mRNP granules called processing bodies (PBs), which contain mRNA substrates targeted for decay and the associated mRNA decay factors. Because Hedls also modulates Dcp2 stability, I hypothesized that Dcp2 might be stabilized when associated with mRNA substrates as a mechanism for controlling Dcp2 levels according to cellular substrate levels. To test this, PBs were used as a tool to determine whether Dcp2 stability increases when concentrated with mRNPs. Through a deletion analysis, I found that the C-terminus of Hedls is required for concentration in PBs. Nevertheless, its deletion did not affect Dcp2 association or stabilization. I also examined a catalytically inactive Dcp2 mutant predicted to trap Dcp2 with mRNPs, but I did not find an increase in stability. Finally, I co-transfected an mRNA reporter containing a stem loop from Rrp41 mRNA for which Dcp2 has a high affinity and did not find stabilization. Therefore, exactly what dictates Dcp2 stability remains unknown and is an important topic for further investigation. In sum, my studies revealed an unexpected mechanism of Dcp2 regulation that could serve to prevent promiscuous Dcp2 activity thereby ensuring decapping of only mRNAs targeted for deca

Investigating the regulation of the human decapping enzyme Dcp2

.Decapping is a critical step in several mRNA decay pathways that are important for the regulation of gene expression. A major decapping complex consists of the decapping enzyme Dcp2 as well as several decapping enhancers. Although several interactions between decapping factors have been mapped, little is known about how Dcp2 activity is controlled to target the appropriate mRNAs for decapping or how these enhancers regulate this activity. Through a deletion analysis, I identified an important autoregulatory domain that is responsible for Dcp2 protein turnover. I demonstrated that the metazoan specific decapping enhancer, Hedls, modulates Dcp2 stability and requires the autoregulatory domain of Dcp2 for interaction. Furthermore, Hedls depletion reduces Dcp2 cellular activity independent of protein stability. Thus Hedls promotes Dcp2 mediated decapping by at least two mechanisms: controlling Dcp2 stability and enhancing Dcp2 catalytic activity. This could provide a way to modulate Dcp2 activity according to cellular demand, thereby preventing decapping of inappropriate targets. Hedls is required for Dcp2 concentration in mRNP granules called processing bodies (PBs), which contain mRNA substrates targeted for decay and the associated mRNA decay factors. Because Hedls also modulates Dcp2 stability, I hypothesized that Dcp2 might be stabilized when associated with mRNA substrates as a mechanism for controlling Dcp2 levels according to cellular substrate levels. To test this, PBs were used as a tool to determine whether Dcp2 stability increases when concentrated with mRNPs. Through a deletion analysis, I found that the C-terminus of Hedls is required for concentration in PBs. Nevertheless, its deletion did not affect Dcp2 association or stabilization. I also examined a catalytically inactive Dcp2 mutant predicted to trap Dcp2 with mRNPs, but I did not find an increase in stability. Finally, I co-transfected an mRNA reporter containing a stem loop from Rrp41 mRNA for which Dcp2 has a high affinity and did not find stabilization. Therefore, exactly what dictates Dcp2 stability remains unknown and is an important topic for further investigation. In sum, my studies revealed an unexpected mechanism of Dcp2 regulation that could serve to prevent promiscuous Dcp2 activity thereby ensuring decapping of only mRNAs targeted for deca

Competition between Decapping Complex Formation and Ubiquitin-Mediated Proteasomal Degradation Controls Human Dcp2 Decapping Activity

mRNA decapping is a central step in eukaryotic mRNA decay that simultaneously shuts down translation initiation and activates mRNA degradation. A major complex responsible for decapping consists of the decapping enzyme Dcp2 in association with decapping enhancers. An important question is how the activity and accumulation of Dcp2 are regulated at the cellular level to ensure the specificity and fidelity of the Dcp2 decapping complex. Here, we show that human Dcp2 levels and activity are controlled by a competition between decapping complex assembly and Dcp2 degradation. This is mediated by a regulatory domain in the Dcp2 C terminus, which, on the one hand, promotes Dcp2 activation via decapping complex formation mediated by the decapping enhancer Hedls and, on the other hand, targets Dcp2 for ubiquitin-mediated proteasomal degradation in the absence of Hedls association. This competition between Dcp2 activation and degradation restricts the accumulation and activity of uncomplexed Dcp2, which may be important for preventing uncontrolled decapping or for regulating Dcp2 levels and activity according to cellular needs

Competition between Decapping Complex Formation and Ubiquitin-Mediated Proteasomal Degradation Controls Human Dcp2 Decapping Activity

mRNA decapping is a central step in eukaryotic mRNA decay that simultaneously shuts down translation initiation and activates mRNA degradation. A major complex responsible for decapping consists of the decapping enzyme Dcp2 in association with decapping enhancers. An important question is how the activity and accumulation of Dcp2 are regulated at the cellular level to ensure the specificity and fidelity of the Dcp2 decapping complex. Here, we show that human Dcp2 levels and activity are controlled by a competition between decapping complex assembly and Dcp2 degradation. This is mediated by a regulatory domain in the Dcp2 C terminus, which, on the one hand, promotes Dcp2 activation via decapping complex formation mediated by the decapping enhancer Hedls and, on the other hand, targets Dcp2 for ubiquitin-mediated proteasomal degradation in the absence of Hedls association. This competition between Dcp2 activation and degradation restricts the accumulation and activity of uncomplexed Dcp2, which may be important for preventing uncontrolled decapping or for regulating Dcp2 levels and activity according to cellular needs

A mitotic topoisomerase II checkpoint in budding yeast is required for genome stability but acts independently of Pds1/securin

Topoisomerase II (Topo II) performs topological modifications on double-stranded DNA molecules that are essential for chromosome condensation, resolution, and segregation. In mammals, G2 and metaphase cell cycle delays induced by Topo II poisons have been proposed to be the result of checkpoint activation in response to the catenation state of DNA. However, the apparent lack of such controls in model organisms has excluded genetic proof that Topo II checkpoints exist and are separable from the conventional DNA damage checkpoint controls. But here, we define a Topo II-dependent G2/M checkpoint in a genetically amenable eukaryote, budding yeast, and demonstrate that this checkpoint enhances cell survival. Conversely, a lack of the checkpoint results in aneuploidy. Neither DNA damage-responsive pathways nor Pds1/securin are needed for this checkpoint. Unusually, spindle assembly checkpoint components are required for the Topo II checkpoint, but checkpoint activation is not the result of failed chromosome biorientation or a lack of spindle tension. Thus, compromised Topo II function activates a yeast checkpoint system that operates by a novel mechanism

Guidelines for Sexual Health Care for Prostate Cancer Patients:Recommendations of an International Panel

Background: Patients with prostate cancer suffer significant sexual dysfunction after treatment which negatively affects them and their partners psychologically, and strain their relationships. Aim: We convened an international panel with the aim of developing guidelines that will inform clinicians, patients and partners about the impact of prostate cancer therapies (PCT) on patients’ and partners’ sexual health, their relationships, and about biopsychosocial rehabilitation in prostate cancer (PC) survivorship. Methods: The guidelines panel included international expert researchers and clinicians, and a guideline methodologist. A systematic review of the literature, using the Ovid MEDLINE, Scopus, CINAHL, PsychINFO, LGBT Life, and Embase databases was conducted (1995–2022) according to the Cochrane Handbook for Systematic Reviews of Interventions. Study selection was based on Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Each statement was assigned an evidence strength (A-C) and a recommendation level (strong, moderate, conditional) based on benefit/risk assessment. Data synthesis included meta-analyses of studies deemed of sufficient quality (3), using A Measurement Tool to Assess Systematic Reviews (AMSTAR). Outcomes: Guidelines for sexual health care for patients with prostate cancer were developed, based on available evidence and the expertise of the international panel. Results: The guidelines account for patients’ cultural, ethnic, and racial diversity. They attend to the unique needs of individuals with diverse sexual orientations and gender identities. The guidelines are based on literature review, a theoretical model of sexual recovery after PCT, and 6 principles that promote clinician-initiated discussion of realistic expectations of sexual outcomes and mitigation of sexual side-effects through biopsychosocial rehabilitation. Forty-seven statements address the psychosexual, relationship, and functional domains in addition to statements on lifestyle modification, assessment, provider education, and systemic challenges to providing sexual health care in PC survivorship. Clinical Implications: The guidelines provide clinicians with a comprehensive approach to sexual health care for patients with prostate cancer. Strengths & Limitations: The strength of the study is the comprehensive evaluation of existing evidence on sexual dysfunction and rehabilitation in prostate cancer that can, along with available expert knowledge, best undergird clinical practice. Limitation is the variation in the evidence supporting interventions and the lack of research on issues facing patients with prostate cancer in low and middle-income countries. Conclusion: The guidelines document the distressing sexual sequelae of PCT, provide evidence-based recommendations for sexual rehabilitation and outline areas for future research. Wittmann D, Mehta A, McCaughan E, et al. Guidelines for Sexual Health Care for Prostate Cancer Patients: Recommendations of an International Panel. J Sex Med 2022;19:1655–1669