Yeast Rnt1p is required for cleavage of the pre-ribosomal RNA in the 3' ETS but not the 5' ETS

We have reexamined the role of yeast RNase III (Rnt1p) in ribosome synthesis. Analysis of pre-rRNA processing in a strain carrying a complete deletion of the RNT1 gene demonstrated that the absence of Rnt1p does not block cleavage at site A0 in the 5' external transcribed spacers (ETS), although the early pre-rRNA cleavages at sites A0, A1, and A2 are kinetically delayed. In contrast, cleavage in the 3' ETS is completely inhibited in the absence of Rnt1p, leading to the synthesis of a reduced level of a 3' extended form of the 25S rRNA. The 3' extended forms of the pre-rRNAs are consistent with the major termination at site T2 (+210). We conclude that Rnt1p is required for cleavage in the 3' ETS but not for cleavage at site A0. The sites of in vivo cleavage in the 3' ETS were mapped by primer extension. Two sites of Rnt1p-dependent cleavage were identified that lie on opposite sides of a predicted stem loop structure, at +14 and +49. These are in good agreement with the consensus Rnt1p cleavage site. Processing of the 3' end of the mature 25S rRNA sequence in wild-type cells was found to occur concomitantly with processing of the 5' end of the 5.8S rRNA, supporting previous proposals that processing in ITS1 and the 3' ETS is coupled.

Cordycepin interferes with 3' end formation in yeast independently of its potential to terminate RNA chain elongation

Cordycepin (3′ deoxyadenosine) is a biologically active compound that, when incorporated during RNA synthesis in vitro, provokes chain termination due to the absence of a 3′ hydroxyl moiety. We were interested in the effects mediated by this drug in vivo and analyzed its impact on RNA metabolism of yeast. Our results support the view that cordycepin-triphosphate (CoTP) is the toxic component that is limiting cell growth through inhibition of RNA synthesis. Unexpectedly, cordycepin treatment modulated 3′ end heterogeneity of ACT1 and ASC1 mRNAs and rapidly induced extended transcripts derived from CYH2 and NEL025c loci. Moreover, cordycepin ameliorated the growth defects of poly(A) polymerase mutants and the pap1-1 mutation neutralized the effects of the drug on gene expression. Our observations are consistent with an epistatic relationship between poly(A) polymerase function and cordycepin action and suggest that a major mode of cordycepin activity reduces 3′ end formation efficiency independently of its potential to terminate RNA chain elongation. Finally, chemical-genetic profiling revealed genome-wide pathways linked to cordycepin activity and identified novel genes involved in poly(A) homeostasis.

RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3'-end processing in Saccharomyces cerevisiae

We describe methods for obtaining a quantitative description of RNA processing at high resolution in budding yeast. As a model gene expression system, we constructed tetON (for induction studies) and tetOFF (for repression, derepression, and RNA degradation studies) yeast strains with a series of reporter genes integrated in the genome under the control of a tetO7 promoter. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were adapted to allow the determination of mRNA abundance as the average number of copies per cell in a population. Fluorescence in situ hybridization (FISH) measurements of transcript numbers in individual cells validated the RT-qPCR approach for the average copy-number determination despite the broad distribution of transcript levels within a population of cells. In addition, RT-qPCR was used to distinguish the products of the different steps in splicing of the reporter transcripts, and methods were developed to map and quantify 3′-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 3′-end maturation and degradation to be quantitatively monitored with unprecedented kinetic detail, suitable for mathematical modeling. Using this approach, we demonstrate that reporter transcripts are spliced prior to their 3′-end cleavage and polyadenylation, that is, cotranscriptionally.

Coupled RNA polymerase II transcription and 3′ end formation with yeast whole-cell extracts

 RNA polymerase II (RNAP II) transcription and pre-mRNA 3' end formation are linked through physical and functional interactions. We describe here a highly efficient yeast in vitro system that reproduces both transcription and 3' end formation in a single reaction. The system is based on simple whole-cell extracts that were supplemented with a hybrid Gal4-VP16 transcriptional activator and supercoiled plasmid DNA templates encoding G-less cassette reporters. We found that the coupling of transcription and processing in vitro enhanced pre-mRNA 3' end formation and reproduced requirements for poly(A) signals and polyadenylation factors. Unexpectedly, however, we show that in vitro transcripts lacked m⁷G-caps. Reconstitution experiments with CF IA factor assembled entirely from heterologous components suggested that the CTD interaction domain of the Pcf11 subunit was required for proper RNAP II termination but not 3' end formation. Moreover, we observed reduced termination activity associated with extracts prepared from cells carrying a mutation in the 5'-3' exonuclease Rat1 or following chemical inhibition of exonuclease activity. Thus, in vitro transcription coupled to pre-mRNA processing recapitulates hallmarks of poly(A)-dependent RNAP II termination. The in vitro transcription/processing system presented here should provide a useful tool to further define the role of factors involved in coupling.

The end of the cold war and the causes of Soviet collapse

"In 1985, Mikhail Gorbachev ascended to power in the USSR. In selecting a young reformer to the position of general secretary, the politburo had recognised the pressing need to revitalise the Soviet Union. To this end, the leadership imposed a series of reforms aimed at reinvigorating the Soviet economy and society, of which the shifts in foreign policy were the most radical and wide-ranging. Yet, the culmination of the reform process was not Soviet reinvigoration, but the rapid collapse of the USSR. The End of the Cold War and the Causes of Soviet Collapse examines the role played by this foreign policy reform process in the breakdown of Soviet power. Nick Bisley uses a historical sociological theory of the state to analyse the influence of foreign policy alongside the other domestic factors which shaped the development, functioning and failure of the Soviet state.
He concludes that the international confrontation was an important structural element of Soviet state rule and that the end of the confrontation contributed to the destabilisation of the state in the late 1980s. Moreover, he shows that international factors are fundamental to the functioning of modern states and that international and domestic orders shape one another in vital ways."--BOOK JACKET.

Detection of Arabidopsis thaliana AtRAD1 cDNA variants and assessment of function by expression in a yeast rad1 mutant

The Saccharomyces cerevisiae RAD1 and human XPF genes encode a subunit of a nucleotide excision repair endonuclease that also is implicated in some forms of homologous recombination. An Arabidopsis thaliana gene (AtRAD1) encoding the orthologous plant protein has been identified recently. Here we report the isolation of three structurally distinct AtRAD1 cDNAs from A. thaliana leaf tissue RNA. One of the isolates (AtRAD1-1) corresponds to the cDNA previously shown to encode the full-length AtRad1 protein, whereas the other two (AtRAD1-2, AtRAD1-3) differ slightly in size due to variations at the 5′ end of exon 6 or the 3′ end of exon 7, respectively. The sequence differences argue that these cDNAs were probably templated by mRNAs generated via alternative splicing. Diagnostic polymerase chain reaction pointed to the presence of the AtRAD1-1 and AtRAD1-2 but not AtRAD1-3 transcripts in bud and root tissue, and to a fourth transcript (AtRAD1-4), having both alterations identified in AtRAD1-2 and AtRAD1-3, in root tissue. However, the low frequency of detection of AtRAD1-3 and AtRAD1-4 makes the significance of these tissue-specific patterns unclear. The predicted AtRad1-2, AtRad1-3 and AtRad1-4 proteins lack part of the region likely required for endonuclease complex formation. Expression of AtRAD1-2 and AtRAD1-3 in a yeast rad1 mutant did not complement the sensitivity to ultraviolet radiation or the recombination defect associated with the rad1 mutation. These results suggest that alternative splicing may modulate the levels of functional AtRad1 protein.

Cold rolling mill thickness control using the cascade-correlation neural network

The improvements in thickness accuracy of a steel strip produced by a tandem cold-roIling mill are of substantial interest to the steel industry. In this paper, we designed a direct model-reference adaptive control (MRAC)  scheme that exploits the natural level of excitation existing in the closed-loop with a dynamically constructed cascade-correlation neural network (CCNN) as a controller for cold roIling mill thickness control. Simulation results show that the combination of a such a direct MRAC scheme and the dynamically constructed CCNN significantly improves the thickness accuracy in the presence of disturbances and noise in comparison with to the conventional PID controllers.

Fragmentation of orientation within grains of a cold-rolled interstitial-free steel

The formation of a favourable recrystallization texture in interstitial-free (IF) steels depends on the availability and activation of particular nucleation sites in the deformed microstructure. This paper presents a description of the deformed microstructure of a commercially cold-rolled IF steel, with particular emphasis on the microstructural inhomogeneities and short-range orientational variation that provide suitable nucleation sites during recrystallization. RD-fibre regions deform relatively homogeneously and exhibit little short-range orientational variation. ND-fibre regions are heavily banded and exhibit considerable short-range orientational variation associated with the bands. While the overall orientational spread of ND-fibre grains frequently is about the ND-axis, the short-range orientational variation often involves rotation about axes in the TD-ND plane that are nearer to the TD than the ND.

The influence of solute carbon in cold-rolled steels on shear band formation and recrystallization texture

Two experiments were conducted to clarify the roles of grain size, solute carbon and strain in determining the recrystallization textures of cold-rolled and annealed steels. In the first experiment, samples of coarse-grained low-carbon (LC) and interstitial-free (IF) steels were cold-rolled to a 75% reduction in thickness. One sample from each steel was polished and cold-rolled an additional 5%, while the remaining samples were annealed for various times at 650°C. In the second experiment, three samples from a commercial LC steel sheet were rolled 70% at 300°C. Two of the samples were given a further rolling reduction of 5% of the original thickness, with one of the samples being given this additional reduction at 300°C and the other at room temperature. Goss recrystallization textures are strengthened by coarse initial grain sizes, the presence of solute carbon and rolling at a temperature where dynamic strain ageing occurs, but are weakened by additional rolling beyond a reduction of 70%, especially when this extra rolling is conducted at a temperature where dynamic strain ageing does not occur. Characterization of key features of the deformed and recrystallized steels using optical microscopy, scanning electron microscopy (SEM) and electron back-scatter diffraction (EBSD) supports a rationale for these effects based on the repeated activation and deactivation of shear bands and the influence of solute carbon and dynamic strain ageing on the operating life of the bands and the accumulation of strain within them.