The promyelocytic leukemia (PML) protein is aggregated into nuclear bodies that are associated with diverse nuclear processes. Here, we report that the distance between a locus and its nearest PML body correlates with the transcriptional activity and gene density around the locus. Genes on the active X chromosome are more significantly associated with PML bodies than their silenced homologues on the inactive X chromosome. We also found that a histone-encoding gene cluster, which is transcribed only in S-phase, is more strongly associated with PML bodies in S-phase than in G0/G1 phase of the cell cycle. However, visualization of specific RNA transcripts for several genes showed that PML bodies were not themselves sites of transcription for these genes. Furthermore, knock-down of PML bodies by RNA interference did not preferentially change the expression of genes closely associated with PML bodies. We propose that PML bodies form in nuclear compartments of high transcriptional activity, but they do not directly regulate transcription of genes in these compartments.

To investigate the function of the nucleolar protein Nop2p in Saccharomyces cerevisiae, we constructed a strain in which NOP2 is under the control of a repressible promoter. Repression of NOP2 expression lengthens the doubling time of this strain about fivefold and reduces steady-state levels of 60S ribosomal subunits, 80S ribosomes, and polysomes. Levels of 40S subunits increase as the free pool of 60S subunits is reduced. Nop2p depletion impairs processing of the 35S pre-rRNA and inhibits processing of 27S pre-rRNA, which results in lower steady-state levels of 25S rRNA and 5.8S rRNA. Processing of 20S pre-rRNA to 18S rRNA is not significantly affected. Processing at sites A 2 , A 3 , B 1L , and B 1S and the generation of 5 termini of different pre-rRNA intermediates appear to be normal after Nop2p depletion. Sequence comparisons suggest that Nop2p may function as a methyltransferase. 2-O-ribose methylation of the conserved site UmGm⌿UC 2922 is known to take place during processing of 27S pre-rRNA. Although Nop2p depletion lengthens the half-life of 27S pre-RNA, methylation of UmGm⌿UC 2922 in 27S pre-rRNA is low during Nop2p depletion. However, methylation of UmGm⌿UC 2922 in mature 25S rRNA appears normal. These findings provide evidence for a close interconnection between methylation at this conserved site and the processing step that yields the 25S rRNA.

We have identified a novel nucleolar protein, Nop5p, that is essential for growth in Saccharomyces cerevisiae. Monoclonal antibodies B47 and 37C12 recognize Nop5p, which has a predicted size of 57 kDa and possesses a KKX repeat motif at its carboxyl terminus. Truncations that removed the KKX motif were functional and localized to the nucleolus, but conferred slow growth at 37°C. Nop5p shows significant sequence homology with yeast Sik1p/Nop56p, and putative homologues in archaebacteria, plants, and human. Depletion of Nop5p in a GAL-NOP5 strain lengthened the doubling time about 5-fold, and selectively reduced steady-state levels of 40 S ribosomal subunits and 18 S rRNA relative to levels of free 60 S subunits and 25 S rRNA. Northern blotting and primer extension analyses showed that Nop5p depletion impairs processing of 35 S pre-rRNA at the A 0 and A 2 cleavage sites. Nop5p is associated with the small nucleolar RNAs U3, snR13, U14, and U18. Depletion of Nop5p caused the nucleolar protein Nop1p (yeast fibrillarin) to be localized to the nucleus and cytosol. Also, 37C12 coimmunoprecipitated Nop1p. These results suggest that Nop5p functions with Nop1p in the execution of early pre-rRNA processing steps that lead to formation of 18 S rRNA.Most of the steps of ribosome biogenesis in eukaryotic cells take place in the nucleolus. In the yeast Saccharomyces cerevisiae, a single long 35 S pre-rRNA is transcribed by RNA polymerase I and processed to 18 S, 5.8 S, and 25 S rRNAs through a series of co-and post-transcriptional steps. Ribosomal proteins imported from the cytoplasm are assembled with pre-rRNAs to form the small 40 S subunit and the large 60 S subunit. The 5 S rRNA is transcribed by RNA polymerase III from a separate transcription unit and is incorporated into the large subunit along with the 5.8 S and 25 S rRNAs, while 18 S rRNA is incorporated into the small subunit. During transcription and processing of pre-rRNA, a number of nucleotides are modified, primarily by the addition of 2Ј-O-methyl groups or by the formation of pseudouridine residues. The processing and modification of pre-rRNAs require non-ribosomal nucleolar proteins, many of which are associated with small nucleolar RNAs (snoRNAs) 1 in the form of small nucleolar ribonucleoprotein (snoRNP) complexes (reviewed in Refs. 1 and 2).The earliest processing events are those involved in the removal of the promoter proximal 5Ј-externally transcribed spacer (5Ј-ETS). Cleavage occurs at two sites within the 5Ј-ETS: at A 0 , in the middle region of the 5Ј-ETS; and at A 1 , which results in the formation of the 5Ј-end of the mature 18 S rRNA (reviewed in Ref. 3). Formation of 18 S requires processing to form its 3Ј-end, which involves processing at site A 2 in the first internally transcribed spacer (ITS1) followed by processing at site D, which yields the 3Ј-end (see Fig. 9). In yeast, many gene products are required for, or participate in, cleavage at sites A 0 , A 1 , and A 2 , attesting to the complex nature of this process. The yeast RNase III encod...

BackgroundTriple-negative breast cancers (BC) represent a heterogeneous subtype of BCs, generally associated with an aggressive clinical course and where targeted therapies are currently limited. Target validation studies for all BC subtypes have largely employed established BC cell lines, which have proven to be effective tools for drug discovery.ResultsGiven the lines of evidence suggesting that BC cell lines are effective tools for drug discovery, we assessed the similarities between triple-negative BCs and cell lines, to identify in vitro representatives, modelling the diversity within this BC subtype. 25 BC cell lines, enriched for those lacking ER, PR and HER2 expression, were subjected to transcriptomic, genomic and epigenomic profiling analyses and comparisons were made to existing knowledge of corresponding perturbations in triple-negative BCs. Transcriptional analysis segregated ER-negative BC cell lines into three groups, displaying distinctive abundances for genes involved in epithelial-mesenchymal transition, apocrine and high-grade carcinomas. DNA copy number aberrations of triple-negative BCs were well represented in cell lines and genes with coordinately altered gene expression showed similar patterns in tumours and cell lines. Methylation events in triple-negative BCs were mostly retained in epigenomes of cell lines. Combined methylation and gene expression analyses revealed a subset of genes characteristic of the Claudin-low BC subtype, exhibiting epigenetic-regulated gene expression in BC cell lines and tumours, suggesting that methylation patterns are likely to underpin subtype-specificity.ConclusionHere, we provide a comprehensive analysis of triple-negative BC features on several molecular levels in BC cell lines, thereby creating an in-depth resource to access the suitability of individual lines as experimental models for studying BC tumour biology, biomarkers and possible therapeutic targets in the context of preclinical target validation.

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A genetic screen for mutations synthetically lethal with temperature sensitive alleles of nop2 led to the identification of the nucleolar proteins Nop12p and Nop13p in Saccharomyces cerevisiae. NOP12 was identified by complementation of a synthetic lethal growth phenotype in strain YKW35, which contains a single nonsense mutation at codon 359 in an allele termed nop12-1. Database mining revealed that Nop12p was similar to a related protein, Nop13p. Nop12p and Nop13p are not essential for growth and each contains a single canonical RNA recognition motif (RRM). Both share sequence similarity with Nsr1p, a previously identified, non-essential, RRM-containing nucleolar protein. Likely orthologs of Nop12p were identified in Drosophila and Schizosaccharomyces pombe. Deletion of NOP12 resulted in a cold sensitive (cs) growth phenotype at 15 degrees C and slow growth at 20 and 25 degrees C. Growth of a nop12Delta strain at 15 and 20 degrees C resulted in impaired synthesis of 25S rRNA, but not 18S rRNA. A nop13 null strain did not produce an observable growth phenotype under the laboratory conditions examined. Epitope-tagged Nop12p, which complements the cs growth phenotype and restores normal 25S rRNA levels, was localized to the nucleolus by immunofluorescence microscopy. Epitope-tagged Nop13p was distributed primarily in the nucleolus, with a lesser portion localizing to the nucleoplasm. Thus, Nop12p is a novel nucleolar protein required for pre-25S rRNA processing and normal rates of cell growth at low temperatures.

SummaryBackgroundMeticillin-resistant Staphylococcus aureus (MRSA) is frequently endemic in healthcare settings and may be transmitted by person-to-person spread. Asymptomatic MRSA carriers are potential, unsuspected sources for transmission and some of them may be identified by admission screening.AimTo assess whether rapid point-of-care screening (POCS) for MRSA at hospital admission may be associated with a reduction in MRSA acquisition rates when compared with slower laboratory-based methods.MethodsA cluster-randomized cross-over trial was conducted in four admission wards of an acute London tertiary care hospital. Polymerase chain reaction-based POCS screening was compared with conventional culture screening. Patients were screened on ward admission and discharge, and the MRSA acquisition rate on the admission wards was calculated as the primary outcome measure.ResultsIn all, 10,017 patients were included; 4978 in the control arm, 5039 in the POCS arm. The MRSA carriage rate on admission was 1.7%. POCS reduced the median reporting time from 40.4 to 3.7 h (P < 0.001). MRSA was acquired on the admission wards by 23 (0.46%) patients in the control arm and by 24 (0.48%) in the intervention arm, acquisition rates of 5.39 and 4.60 per 1000 days respectively. After taking account of predefined confounding factors, the adjusted incidence rate ratio (IRR) for change in trend for MRSA acquisition was 0.961 (95% confidence interval: 0.766–1.206). The adjusted IRR for step change for MRSA acquisition was 0.98 (0.304–3.162).ConclusionPOCS produces a significantly faster result but has no effect on MRSA acquisition on admission wards compared with culture screening. Where compliance with infection prevention and control is high and MRSA carriage is low, POCS has no additional impact on MRSA acquisition rates over the first one to four days of admission compared with conventional culture screening.