Biomedical subjects

Patrick Young

Publications and source records attributed to Patrick Young.

12 recordsLinked to original sources

PubMed2006

Studies on the role of NonA in mRNA biogenesis.

The NonA protein of Drosophila melanogaster is an abundant nuclear protein that belongs to the DBHS (Drosophila behavior, human splicing) protein family. The DBHS proteins bind both DNA and RNA in vitro and have been involved in different aspects of gene expression, including pre-mRNA splicing, transcription regulation and nuclear retention of mRNA. We have used double-stranded RNA interference in Drosophila S2 cells to silence the expression of NonA and to investigate its role in mRNA biogenesis. We show that knockdown of NonA does not affect transcription nor splicing. We demonstrate that NonA forms a complex with the essential nuclear export factor NXF1 in an RNA-dependent manner. We have constructed stable S2 cell lines that express full-length and truncated NXF1 fused to GFP in order to perform fluorescence recovery after photobleaching experiments. We show that knockdown of NonA reduces the intranuclear mobility of NXF1-GFP associated with poly(A)(+) RNA in vivo, while the mobility of the truncated NXF1-GFP that does not bind RNA is not affected. Our data suggest that NonA facilitates the intranuclear mobility of mRNP particles.

Active Transport, Cell Nucleus↗

PubMed2006

Probiotic use in irritable bowel syndrome.

Over the past several years, a number of studies have addressed the role of specific strains of bacteria, or combinations thereof, to alleviate certain symptoms of irritable bowel syndrome (IBS). More importantly, the precise factors that contribute to this therapeutic effect, such as modulations in cytokine levels and alterations in colonic motility, are being clarified. This review serves to summarize the evidence for the use of probiotics in the treatment of IBS and to place this information in clinical context. Potential future developments and areas of possible research are also discussed.

Humans↗

PubMed2005

Structure of S5a bound to monoubiquitin provides a model for polyubiquitin recognition.

Ubiquitin is a key regulatory molecule in diverse cellular events. How cells determine the outcome of ubiquitylation remains unclear; however, a likely determinant is the specificity of ubiquitin receptor proteins for polyubiquitin chains of certain length and linkage. Proteasome subunit S5a contains two ubiquitin-interacting motifs (UIMs) through which it recruits ubiquitylated substrates to the proteasome for their degradation. Here, we report the structure of S5a (196-306) alone and complexed with two monoubiquitin molecules. This construct contains the two UIMs of S5a and we reveal their different ubiquitin-binding mechanisms and provide a rationale for their unique specificities for different ubiquitin-like domains. Furthermore, we provide direct evidence that S5a (196-306) binds either K63-linked or K48-linked polyubiquitin, and in both cases prefers longer chains. On the basis of these results we present a model for how S5a and other ubiquitin-binding proteins recognize polyubiquitin.

Carrier Proteins↗

PubMed2005

Identification and characterization of a Drosophila proteasome regulatory network.

Maintaining adequate proteasomal proteolytic activity is essential for eukaryotic cells. For metazoan cells, little is known about the composition of genes that are regulated in the proteasome network or the mechanisms that modulate the levels of proteasome genes. Previously, two distinct treatments have been observed to induce 26S proteasome levels in Drosophila melanogaster cell lines, RNA interference (RNAi)-mediated inhibition of the 26S proteasome subunit Rpn10/S5a and suppression of proteasome activity through treatment with active-site inhibitors. We have carried out genome array profiles from cells with decreased Rpn10/S5a levels using RNAi or from cells treated with proteasome inhibitor MG132 and have thereby identified candidate genes that are regulated as part of a metazoan proteasome network. The profiles reveal that the majority of genes that were identified to be under the control of the regulatory network consisted of 26S proteasome subunits. The 26S proteasome genes, including three new subunits, Ubp6p, Uch-L3, and Sem1p, were found to be up-regulated. A number of genes known to have proteasome-related functions, including Rad23, isopeptidase T, sequestosome, and the genes for the segregase complex TER94/VCP-Ufd1-Npl4 were also found to be up-regulated. RNAi-mediated inhibition against the segregase complex genes demonstrated pronounced stabilization of proteasome substrates throughout the Drosophila cell. Finally, transcriptional reporter assays and deletion mapping studies in Drosophila demonstrate that proteasome mRNA induction is dependent upon the 5' untranslated regions (UTRs). Transfer of the 5' UTR from the proteasome subunit Rpn1/S2 to a noninducible promoter was sufficient to confer transcriptional upregulation of the reporter mRNA after proteasome inhibition.

5' Untranslated Regions↗

PubMed2003

Drosophila proteasome regulator REGgamma: transcriptional activation by DNA replication-related factor DREF and evidence for a role in cell cycle progression.

The proteasome regulator REG (PA28gamma) is a conserved complex present in metazoan nuclei and is able to stimulate the trypsin-like activity of the proteasome in a non-ATP dependent manner. However, the in vivo function for REGgamma in metazoan cells is currently unknown. To understand the role of Drosophila REGgamma we have attempted to identify the type of promoter elements regulating its transcription. Mapping the site of the transcription initiation revealed a TATA-less promoter, and a sequence search identified elements found typically in Drosophila genes involved in cell cycle progression and DNA replication. In order to test the relevance of the motifs, REGgamma transcriptional assays were carried out with mutations in the proposed promoter. Our results indicate that a single Drosophila replication-related element sequence, DRE, is essential for REGgamma transcription. To confirm that REGgamma has a role in cell cycle progression, the effect of removing REGgamma from S2 cells was tested using RNA interference. Drosophila cells depleted of REGgamma showed partial arrests in G1/S cell cycle transition. Immuno-staining of Drosophila embryos revealed that REGgamma is typically localized to the nucleus during embryogenesis with increased levels present in invaginating cells during gastrulation. The REGgamma was found dispersed throughout the cell volume within mitotic domains undergoing cell division. Finally, database searches suggest that the DRE system may regulate key members of the proteasome system in Drosophila.

Animals↗

PubMed2003

Use of RNA interference and complementation to study the function of the Drosophila and human 26S proteasome subunit S13.

The S13 subunit (also called Pad1, Rpn11, and MPR1) is a component of the 19S complex, a regulatory complex essential for the ubiquitin-dependent proteolytic activity of the 26S proteasome. To address the functional role of S13, we combined double-stranded RNA interference (RNAi) against the Drosophila proteasome subunit DmS13 with expression of wild-type and mutant forms of the homologous human gene, HS13. These studies show that DmS13 is essential for 26S function. Loss of the S13 subunit in metazoan cells leads to increased levels of ubiquitin conjugates, cell cycle defects, DNA overreplication, and apoptosis. In vivo assays using short-lived proteasome substrates confirmed that the 26S ubiquitin-dependent degradation pathway is compromised in S13-depleted cells. In complementation experiments using Drosophila cell lines expressing HS13, wild-type HS13 was found to fully rescue the knockdown phenotype after DmS13 RNAi treatment, while an HS13 containing mutations (H113A-H115A) in the proposed isopeptidase active site was unable to rescue. A mutation within the conserved MPN/JAMM domain (C120A) abolished the ability of HS13 to rescue the Drosophila cells from apoptosis or DNA overreplication. However, the C120A mutant was found to partially restore normal levels of ubiquitin conjugates. The S13 subunit may possess multiple functions, including a deubiquitinylating activity and distinct activities essential for cell cycle progression that require the conserved C120 residue.

Adenosine Triphosphate↗

PubMed1977