Genome-wide analysis of RNA polymerase II termination at protein-coding genes.

At the end of protein-coding genes, RNA polymerase (Pol) II undergoes a concerted transition that involves 3′-processing of the pre-mRNA and transcription termination. Here, we present a genome-wide analysis of the 3′-transition in budding yeast. We find that the 3′-transition globally requires the Pol II elongation factor Spt5 and factors involved in the recognition of the polyadenylation (pA) site and in endonucleolytic RNA cleavage. Pol II release from DNA occurs in a narrow termination window downstream of the pA site and requires the “torpedo” exonuclease Rat1 (XRN2 in human). The Rat1-interacting factor Rai1 contributes to RNA degradation downstream of the pA site. Defects in the 3′-transition can result in increased transcription at downstream genes

PROSO II - a new method for protein solubility prediction.

Many fields of science and industry depend on efficient production of active protein using heterologous expression in Escherichia coli. The solubility of proteins upon expression is dependent on their amino acid sequence. Prediction of solubility from sequence is therefore highly valuable. We present a novel machine-learning-based model called PROSO II which makes use of new classification methods and growth in experimental data to improve coverage and accuracy of solubility predictions. The classification algorithm is organized as a two-layered structure in which the output of a primary Parzen window model for sequence similarity and a logistic regression classifier of amino acid k-mer composition serve as input for a second-level logistic regression classifier. Compared with previously published research our model is trained on five times more data than used by any other method before (82 000 proteins). When tested on a separate holdout set not used at any point of method development our server attained the best results in comparison with other currently available methods: accuracy 75.4%, Matthews correlation coefficient 0.39, sensitivity 0.731, specificity 0.759, gain (soluble) 2.263. In summary, due to utilization of cutting edge machine learning technologies combined with the largest currently available experimental data set the PROSO II server constitutes a substantial improvement in protein solubility predictions. PROSO II is available at

Transcriptome maps of mRNP biogenesis factors define pre-mRNA recognition.

Biogenesis of eukaryotic messenger ribonucleoprotein complexes (mRNPs) involves the synthesis, splicing, and 3' processing of pre-mRNA, and the assembly of mature mRNPs for nuclear export. We mapped 23 mRNP biogenesis factors onto the yeast transcriptome, providing 10(4)-10(6) high-confidence RNA interaction sites per factor. The data reveal how mRNP biogenesis factors recognize pre-mRNA elements in vivo. They define conserved interactions between splicing factors and pre-mRNA introns, including the recognition of intron-exon junctions and the branchpoint. They also identify a unified arrangement of 3' processing factors at pre-mRNA polyadenylation (pA) sites in yeast and human, which results from an A-U sequence bias at pA sites. Global data analysis indicates that 3' processing factors have roles in splicing and RNA surveillance, and that they couple mRNP biogenesis events to restrict nuclear export to mature mRNPs

RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases.

For transcription through chromatin, RNA polymerase (Pol) II associates with elongation factors (EFs). Here we show that many EFs crosslink to RNA emerging from transcribing Pol II in the yeast Saccharomyces cerevisiae. Most EFs crosslink preferentially to mRNAs, rather than unstable non-coding RNAs. RNA contributes to chromatin association of many EFs, including the Pol II serine 2 kinases Ctk1 and Bur1 and the histone H3 methyltransferases Set1 and Set2. The Ctk1 kinase complex binds RNA in vitro, consistent with direct EF-RNA interaction. Set1 recruitment to genes in vivo depends on its RNA recognition motifs (RRMs). These results strongly suggest that nascent RNA contributes to EF recruitment to transcribing Pol II. We propose that EF-RNA interactions facilitate assembly of the elongation complex on transcribed genes when RNA emerges from Pol II, and that loss of EF-RNA interactions upon RNA cleavage at the polyadenylation site triggers disassembly of the elongation complex

Transcriptome surveillance by selective termination of noncoding RNA synthesis

Pervasive transcription of eukaryotic genomes stems to a large extent from bidirectional promoters that synthesize mRNA and divergent noncoding RNA (ncRNA). Here, we show that ncRNA transcription in the yeast S. cerevisiae is globally restricted by early termination that relies on the essential RNA-binding factor Nrd1. Depletion of Nrd1 from the nucleus results in 1,526 Nrd1-unterminated transcripts (NUTs) that originate from nucleosome-depleted regions (NDRs) and can deregulate mRNA synthesis by antisense repression and transcription interference. Transcriptome-wide Nrd1-binding maps reveal divergent NUTs at most promoters and antisense NUTs in most 30 regions of genes. Nrd1 and its partner Nab3 preferentially bind RNA motifs that are depleted in mRNAs and enriched in ncRNAs and some mRNAs whose synthesis is controlled by transcription attenuation. These results define a global mechanism for transcriptome surveillance that selectively terminates ncRNA synthesis to provide promoter directionality and to suppress antisense transcription

Ba LIII EXAFS studies of the HTSC Y1Ba2Cu3O7−σY_1Ba_2Cu_3O_{7−\sigma}

EXAFS measurements of the HTSC $Y_1Ba_2Cu_3O_{7−σ}$ have been performed at different temperatures (RT, LNT, HeT). Sintered powder samples prepared by Haldor Topsøe A/S were used. In this paper, special emphasis is laid on the information gained from the Ba LIII-edge. For the EXAFS analysis, experimental phases and amplitudes from the peroskite BaTiO$_3$ have been used to fit the first shell (Ba-O) of the superconductors within the framework of the plane wave EXAFS theory

Minimum ion-beam exposure-dose determination for chemically amplified resist from printed dot matrices

Matrices of 90 nm dots have been printed into high-sensitivity positive resist (UVII HS, Shipley) with an ion projection system (IMS Vienna) to investigate the influence of shot noise on the printing probability of dots. Dot defect probability increases with diminishing ion dose following a Poisson distribution which demonstrates that shot noise is the dominating effect. The minimum ion numbers per dot to generate 50% defect probability are Ncrit=115 for standard resist treatment. This corresponds to N=165 for smaller defect probabilities of 10-4. Resist sensitivity was decreased with postexposure bake temperatures of 110 degrees C instead of 140 degrees C to improve the resolution capability of the resist. Under these conditions, and an additional resist top coat, Ncrit=130 ions per dot have been measured. The article demonstrates that ion projection lithography is not limited by shot noise at minimum resolution elements of 90 nm diam. The corresponding exposure doses are 0.3 mu C/cm2