The CCR4-NOT Complex Physically and Functionally Interacts with TRAMP and the Nuclear Exosome

BACKGROUND: Ccr4-Not is a highly conserved multi-protein complex consisting in yeast of 9 subunits, including Not5 and the major yeast deadenylase Ccr4. It has been connected functionally in the nucleus to transcription by RNA polymerase II and in the cytoplasm to mRNA degradation. However, there has been no evidence so far that this complex is important for RNA degradation in the nucleus. METHODOLOGY/PRINCIPAL FINDINGS: In this work we point to a new role for the Ccr4-Not complex in nuclear RNA metabolism. We determine the importance of the Ccr4-Not complex for the levels of non-coding nuclear RNAs, such as mis-processed and polyadenylated snoRNAs, whose turnover depends upon the nuclear exosome and TRAMP. Consistently, mutation of both the Ccr4-Not complex and the nuclear exosome results in synthetic slow growth phenotypes. We demonstrate physical interactions between the Ccr4-Not complex and the exosome. First, Not5 co-purifies with the exosome. Second, several exosome subunits co-purify with the Ccr4-Not complex. Third, the Ccr4-Not complex is important for the integrity of large exosome-containing complexes. Finally, we reveal a connection between the Ccr4-Not complex and TRAMP through the association of the Mtr4 helicase with the Ccr4-Not complex and the importance of specific subunits of Ccr4-Not for the association of Mtr4 with the nuclear exosome subunit Rrp6. CONCLUSIONS/SIGNIFICANCE: We propose a model in which the Ccr4-Not complex may provide a platform contributing to dynamic interactions between the nuclear exosome and its co-factor TRAMP. Our findings connect for the first time the different players involved in nuclear and cytoplasmic RNA degradation

Performance Analysis of Orthogonal Pairs Designed for an Expanded Eukaryotic Genetic Code

Background: The suppression of amber stop codons with non-canonical amino acids (ncAAs) is used for the site-specific introduction of many unusual functions into proteins. Specific orthogonal aminoacyl-tRNA synthetase (o-aaRS)/amber suppressor tRNA CUA pairs (o-pairs) for the incorporation of ncAAs in S. cerevisiae were previously selected from an E. coli tyrosyl-tRNA synthetase/tRNACUA mutant library. Incorporation fidelity relies on the specificity of the o-aaRSs for their ncAAs and the ability to effectively discriminate against their natural substrate Tyr or any other canonical amino acid. Methodology/Principal Findings: We used o-pairs previously developed for ncAAs carrying reactive alkyne-, azido-, or photocrosslinker side chains to suppress an amber mutant of human superoxide dismutase 1 in S. cerevisiae. We found worse incorporation efficiencies of the alkyne- and the photocrosslinker ncAAs than reported earlier. In our hands, amber suppression with the ncAA containing the azido group did not occur at all. In addition to the incorporation experiments in S. cerevisiae, we analyzed the catalytic properties of the o-aaRSs in vitro. Surprisingly, all o-aaRSs showed much higher preference for their natural substrate Tyr than for any of the tested ncAAs. While it is unclear why efficiently recognized Tyr is not inserted at amber codons, we speculate that metabolically inert ncAAs accumulate in the cell, and for this reason they are incorporated despite being weak substrates for the o-aaRSs. Conclusions/Significance: O-pairs have been developed for a whole plethora of ncAAs. However, a systematic and detaile

Mutational analysis of the C-terminal FATC domain of Saccharomyces cerevisiae Tra1

Tra1 is a component of the Saccharomyces cerevisiae SAGA and NuA4 complexes and a member of the PIKK family, which contain a C-terminal phosphatidylinositol 3-kinase-like (PI3K) domain followed by a 35-residue FATC domain. Single residue changes of L3733A and F3744A, within the FATC domain, resulted in transcriptional changes and phenotypes that were similar but not identical to those caused by mutations in the PI3K domain or deletions of other SAGA or NuA4 components. The distinct nature of the FATC mutations was also apparent from the additive effect of tra1-L3733A with SAGA, NuA4, and tra1 PI3K domain mutations. Tra1-L3733A associates with SAGA and NuA4 components and with the Gal4 activation domain, to the same extent as wild-type Tra1; however, steady-state levels of Tra1-L3733A were reduced. We suggest that decreased stability of Tra1-L3733A accounts for the phenotypes since intragenic suppressors of tra1-L3733A restored Tra1 levels, and reducing wild-type Tra1 led to comparable growth defects. Also supporting a key role for the FATC domain in the structure/function of Tra1, addition of a C-terminal glycine residue resulted in decreased association with Spt7 and Esa1, and loss of cellular viability. These findings demonstrate the regulatory potential of mechanisms targeting the FATC domains of PIKK proteins

Concerted cutting by Spo11 illuminates meiotic DNA break mechanics

Genetic recombination arises during meiosis through the repair of DNA double-strand breaks (DSBs) that are created by Spo11, a topoisomerase-like protein1,2. Spo11 DSBs form preferentially in nucleosome-depleted regions termed hotspots3,4, yet how Spo11 engages with its DNA substrate to catalyse DNA cleavage is poorly understood. Although most recombination events are initiated by a single Spo11 cut, here we show in Saccharomyces cerevisiae that hyperlocalized, concerted Spo11 DSBs separated by 33 to more than 100 base pairs also form, which we term ‘double cuts’. Notably, the lengths of double cuts vary with a periodicity of 10.5 base pairs, which is conserved in yeast and mice. This finding suggests a model in which the orientation of adjacent Spo11 molecules is fixed relative to the DNA helix—a proposal supported by the in vitro DNA-binding properties of the Spo11 core complex. Deep sequencing of meiotic progeny identifies recombination scars that are consistent with repair initiated from gaps generated by adjacent Spo11 DSBs. Collectively, these results revise our present understanding of the mechanics of Spo11-DSB formation and expand on the original concepts of gap repair during meiosis to include DNA gaps that are generated by Spo11 itself

Design of voltage stabilization circuit for wind power generation – part I (Simulation)

Organized by Kolej Universiti Kejuruteraan Utara Malaysia (KUKUM), 18th - 19th May 2005 at Putra Palace Hotel, Kangar.There are many power stabilization systems for wind turbine application. Some uses mechanical stabilization system in which the blade of the wind turbine is forced out of the wind when high winds are blowing. Other uses electrical stabilization system. This is done by manipulating the voltage outputted from the wind generators. Usually this uses two or more converters in the design. Several previous designs are shown in this paper. The design presented does not fully stabilize the system. Some only boost the voltage and others only decrease the input voltage. To remedy this system, a new stabilization system has been designed. The system utilized both decrease of the input voltage if the input voltage is high and boost if the input voltage is low. Besides that the system also implements a circuit called consolidator circuit for system over-voltage protection. The system has been simulated and the result is discussed in the paper

Diagenetic characteristics and classification of each sample of carbonates from the Zranda section during the Palaeocene-Eocene Thermal Maximum

The Palaeocene-Eocene Thermal Maximum (PETM) is recognized as a significant climatic event in the Cenozoic era, marked by negative carbon excursions, rapid warming, and ocean acidification. However, stable isotope analysis of carbonates poses challenges due to diagenesis. This repository presents a comprehensive study of diagenetic features within the Zranda section, which represents shallow carbonates of the eastern Tethys. Through meticulous examination, this dataset facilitates the identification of minimally affected samples, enhancing their suitability for stable carbon isotope analysis. The thin sections of Zranda section were studied under polarizing Nikon Eclipse 50iPOL petrographic microscope with at 5X objective lens, a Nikon DS-Fi1 digital camera and Prior motorized stage. Different diagenetic features were in identified and samples were classified into 1) marine phreatic, 2) shallow burial or 3) shallow to deep burial diagenetic environments, using previously established petrographic criteria (Chilingar et al., 1967; Madden et al., 2017)

Stable isotope analysis of each sample of carbonates from the Zranda section during the Palaeocene-Eocene Thermal Maximum

The Palaeocene-Eocene Thermal Maximum (PETM) is recognized as a significant climatic event in the Cenozoic era, marked by negative carbon excursions, rapid warming, and ocean acidification. However, stable isotope analysis of carbonates poses challenges due to diagenesis. This repository presents a comprehensive study of diagenetic features within the Zranda section, which represents shallow carbonates of the eastern Tethys. Through meticulous examination, this dataset facilitates the identification of minimally affected samples, enhancing their suitability for stable carbon isotope analysis. Each sample was drilled with a dental drill to obtain 4 mg of powder for bulk carbonate stable isotope analysis. The best-preserved sections were targeted, avoiding visually altered areas due to diagenetic processes. Carbon and oxygen isotopes were measured using an IBEX system connected to a Thermo Fisher-MAT 253 Plus stable isotope mass spectrometer at Macquarie University's GeoAnalytical facility. The powder underwent reaction with 105% phosphoric acid at 90°C in a stirred acid bath. CO2 gas evolved from the reaction was isolated and filtered through cold traps and a sulfur-trap before being concentrated into a micro-vessel for analysis. Accuracy was monitored using an in-house Carrara marble standard and clumped isotope carbonate standards (ETH 1, ETH 2, ETH 3, and ETH 4). Isotopic values were calculated using Easotope software