The Neurospora circadian oscillator comprises FREQUENCY (FRQ) and its transcription activator, the White Collar Complex (WCC). Repression of WCC's transcriptional activity by FRQ via negative feedback is indispensable for clock function. An unbiased genetic screen that targeted mutants with defects in negative feedback regulation yielded a fully viable arrhythmic strain bearing a novel allele of FRQ-interacting RNA helicase ( frh), an essential gene that encodes a putative exosome component protein. In the allele, frh R806H , clock function is completely disturbed, while roles of FRQ-interacting RNA helicase (FRH) essential for viability are left intact. FRH R806H still interacts with FRQ, but interaction between the FRQ-FRH R806H complex (FFC) and WCC is severely affected. Phosphorylation of WC-1 is reduced in the mutant leading to constantly elevated WCC activity, which breaks the negative feedback loop. WCC levels are considerably reduced in the mutant, especially those of WC-1, consistent both with loss of positive feedback (FRQ-dependent WC-1 stabilization) and with a reduced level of the FRQ-mediated WCC phosphorylation that leads to high WCC activity accompanied by rapid transcription-associated turnover. FRH overexpression promotes WC-1 accumulation, confirming that FRH together with FRQ plays a role in WC-1 stabilization. Identification of a viable allele of frh, displaying virtually complete loss of both negative and positive circadian feedback, positions FRH as a core component of the central oscillator that is permissive for rhythmicity but appears not to modulate periodicity. Moreover, the results suggest that there are clock-specific roles for FRH that are distinct from the predicted essential exosome-associated functions for the protein.
Mutations in the DYNAMIN2 (DNM2) gene are frequently detected in human acute T-cell lymphoblastic leukemia (T-ALL), although the mechanisms linking these mutations to disease pathogenesis remain unknown. Using an ENU-based forward genetic screen for mice with erythroid phenotypes, we identified a heterozygous mouse line carrying a mutation in the GTPase domain of Dnm2 (Dnm2) that induced a microcytic anemia. In vitro assays using the V265G mutant demonstrated loss of GTPase activity and impaired endocytosis that was comparable to other DNM2 mutants identified in human T-ALL. To determine the effects of DNM2 mutations in T-ALL, we bred the Dnm2 mice with the Lmo2 transgenic mouse model of T-ALL. Heterozygous Dnm2 mutants lacking the Lmo2 transgene displayed normal T-cell development, and did not develop T-ALL. In contrast, compound heterozygotes displayed an accelerated onset of T-ALL compared with mice carrying the Lmo2 oncogene alone. The leukemias from these mice exhibited a more immature immunophenotype and an expansion in leukemic stem cell numbers. Mechanistically, the Dnm2 mutation impaired clathrin-mediated endocytosis of the interleukin (IL)-7 receptor resulting in increased receptor density on the surface of leukemic stem cells. These findings suggest that DNM2 mutations cooperate with T-cell oncogenes by enhancing IL-7 signalling.
To understand the role of white collar-2 in the Neurospora circadian clock, we examined alleles of wc-2 thought to encode partially functional proteins. We found that wc-2 allele ER24 contained a conservative mutation in the zinc finger. This mutation results in reduced levels of circadian rhythm-critical clock gene products, frq mRNA and FRQ protein, and in a lengthened period of the circadian clock. In addition, this mutation altered a second canonical property of the clock, temperature compensation: as temperature increased, period length decreased substantially. This temperature compensation defect correlated with a temperature-dependent increase in overall FRQ protein levels, with the relative increase being greater in wc-2 (ER24) than in wild type, while overall frq mRNA levels were largely unaltered by temperature. We suggest that this temperaturedependent increase in FRQ levels partially rescues the lowered levels of FRQ resulting from the wc-2 (ER24) defect, yielding a shorter period at higher temperatures. Thus, normal activity of the essential clock component WC-2, a positive regulator of frq, is critical for establishing period length and temperature compensation in this circadian system. Predictable daily oscillations in environmental variables such as light and temperature have led to the evolution of circadian rhythms, endogenous programs that allow organisms to anticipate and respond appropriately to predictable environmental changes (53,68). Circadian rhythms regulate a number of different physiological and developmental processes in diverse species, ranging from the sleep-wake cycle in humans to photosynthesis in single-celled algae. These rhythms persist under constant environmental conditions with a period length of about 24 h. The daily oscillations in light and temperature found in nature provide cues to which circadian clocks are responsive; these environmental variables provide reference points that synchronize the organism's internal clock (19).Genetic and molecular analyses have identified a number of so-called clock genes involved in the generation of circadian rhythms, most notably in the fruit fly Drosophila melanogaster, the filamentous fungus Neurospora crassa, the cyanobacterium Synechococcus, and mammalian systems (8,18,26,35,45,56,66,67). Such studies have established that circadian rhythms are based in part upon transcription-translation-based negative feedback loops. In each case, oscillations in the transcript and/or protein levels of specific clock genes frequency (frq) and WC-1 (wc-1 transcript does not cycle) in fungi, kaiA and kaiBC in cyanobacteria, period, timeless (per and tim), and clk in flies, and mPer1, mPer2, mPer3, cry1, cry2, and Bmal1 in mammals appear to play a central role in the generation of rhythms. It has been shown that the proteins encoded by many of these loci negatively feed back to reduce the level of their own transcripts (18, 26, 30); thus, negative autoregulatory feedback appears to be a central process in the generation of circadian rhythms.In Ne...
Surface, secreted and transmembrane protein-encoding open reading frames, collectively the secretome, can be identified in bacterial genome sequences using bioinformatics. However, functional analysis of translated secretomes is possible only if many secretome proteins are expressed and purified individually. We have now developed and applied a phage display system for direct selection, identification, expression and purification of bacterial secretome proteins.
Visible light is thought to reset the Neurospora circadian clock by acting through heterodimers of the WHITE COLLAR-1 and WHITE COLLAR-2 proteins to induce transcription of the frequency gene. To characterize this photic entrainment we examined frq expression in constant light, under which condition the mRNA and protein of this clock gene were strongly induced. In continuous illumination FRQ accumulated in a highly phosphorylated state similar to that seen at subjective dusk, the time at which a step from constant light to darkness sets the clock. Examination of frq expression in several wc-2 mutant alleles surprisingly revealed differential regulation when frq expression was compared between constant light, following a light pulse, and darkness (clock-driven expression). Construction of a wc-2 null strain then demonstrated that WC-2 is absolutely required for both light and clock-driven frq expression, in contrast to previous expectations based on presumptive nulls containing altered Zn-finger function. Additionally, we found that frq light signal transduction differs from that of other light-regulated genes. Thus clock and light-driven frq expression is differentially regulated by, but dependent on, WC-2.
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