Benjamin Thomas

Genetic programs expressed in resting and IL-4 alternatively activated mouse and human macrophages: similarities and differences.

The molecular repertoire of macrophages in health and disease can provide novel biomarkers for diagnosis, prognosis, and treatment. Th2-IL-4–activated macrophages (M2) have been associated with important diseases in mice, yet no specific markers are available for their detection in human tissues. Although mouse models are widely used for macrophage research, translation to the human can be problematic and the human macrophage system remains poorly described. In the present study, we analyzed and compared the transcriptome and proteome of human and murine macrophages under resting conditions (M0) and after IL-4 activation (M2). We provide a resource for tools enabling macrophage detection in human tissues by identifying a set of 87 macrophage-related genes. Furthermore, we extend current understanding of M2 activation in different species and identify Transglutaminase 2 as a conserved M2 marker that is highly expressed by human macrophages and monocytes in the prototypic Th2 pathology asthma.

U1 snRNA associates with TFIIH and regulates transcriptional initiation

Diverse classes of noncoding RNA, including small nuclear RNAs (snRNAs), play fundamental regulatory roles at many stages of gene expression. For example, recent studies have implicated 7SK RNA and components of the splicing apparatus in the regulation of transcriptional elongation. Here we present the first evidence of the involvement of an snRNA in the regulation of transcriptional initiation. We demonstrate that TFIIH, a general transcription initiation factor, specifically associates with U1 snRNA, a core-splicing component. Analysis of the TFIIH-dependent stages of transcription in a reconstituted system demonstrates that U1 stimulates the rate of formation of the first phosphodiester bond by RNA polymerase II. In addition, a promoter-proximal 5′ splice site recognized by U1 snRNA stimulates TFIIH-dependent reinitiation of productive transcription. Our results suggest that U1 snRNA functions in regulating transcription by RNA Polymerase II in addition to its role in RNA processing.

Role of Ran Binding Protein 5 in Nuclear Import and Assembly of the Influenza Virus RNA Polymerase Complex

ABSTRACT The influenza A virus RNA-dependent RNA polymerase is a heterotrimeric complex of polymerase basic protein 1 (PB1), PB2, and polymerase acidic protein (PA) subunits. It performs transcription and replication of the viral RNA genome in the nucleus of infected cells. We have identified a nuclear import factor, Ran binding protein 5 (RanBP5), also known as karyopherin β3, importin β3, or importin 5, as an interactor of the PB1 subunit. RanBP5 interacted with either PB1 alone or with a PB1-PA dimer but not with a PB1-PB2 dimer or the trimeric complex. The interaction between RanBP5 and PB1-PA was disrupted by RanGTP in vitro, allowing PB2 to bind to the PB1-PA dimer to form a functional trimeric RNA polymerase complex. We propose a model in which RanBP5 acts as an import factor for the newly synthesized polymerase by targeting the PB1-PA dimer to the nucleus. In agreement with this model, small interfering RNA (siRNA)-mediated knock-down of RanBP5 inhibited the nuclear accumulation of the PB1-PA dimer. Moreover, siRNA knock-down of RanBP5 resulted in the delayed accumulation of viral RNAs in infected cells, confirming that RanBP5 plays a biological role during the influenza virus life cycle.

Autocatalytic RNA cleavage in the human |[beta]|-globin pre-mRNA promotes transcription termination

New evidence indicates that termination of transcription is an important regulatory step, closely related to transcriptional interference and even transcriptional initiation. However, how this occurs is poorly understood. Recently, in vivo analysis of transcriptional termination for the human β-globin gene revealed a new phenomenon—co-transcriptional cleavage (CoTC). This primary cleavage event within β-globin pre-messenger RNA, downstream of the poly(A) site, is critical for efficient transcriptional termination by RNA polymerase II. Here we show that the CoTC process in the human β-globin gene involves an RNA self-cleaving activity. We characterize the autocatalytic core of the CoTC ribozyme and show its functional role in efficient termination in vivo. The identified core CoTC is highly conserved in the 3′ flanking regions of other primate β-globin genes. Functionally, it resembles the 3′ processive, self-cleaving ribozymes described for the protein-encoding genes from the myxomycetes Didymium iridis and Physarum polycephalum, indicating evolutionary conservation of this molecular process. We predict that regulated autocatalytic cleavage elements within pre-mRNAs may be a general phenomenon and that functionally it may provide the entry point for exonucleases involved in mRNA maturation, turnover and, in particular, transcriptional termination.

The myeloid 7/4‐antigen defines recently generated inflammatory macrophages and is synonymous with Ly‐6B

This study aimed to identify the inflammation‐associated 7/4‐antigen, which is highly expressed on neutrophils, inflammatory monocytes, some activated macrophages, as well as on bone marrow myeloid‐restricted progenitors. The high expression on inflammatory cells is suggestive of a role in inflammation and makes the 7/4‐antigen a potential target for the manipulation of inflammatory cells. Consistent with this, the 7/4‐antibody mediates specific depletion of 7/4‐expressing neutrophils and monocytes. We have identified the 7/4‐antigen as a 25‐ to 30‐kDa GPI‐anchored glycoprotein synonymous with the Ly‐6B.2 alloantigen. We characterized the expression of Ly‐6B during the inflammatory reaction induced by zymosan. During the later stages of an experimental, acute, self‐resolving inflammatory response, we found that Ly‐6B is differentially expressed on macrophages. Ly‐6B‐expressing macrophages also express more MHCII, CIITA, CCR2, Ly‐6C, and CD62L than the Ly‐6B‐negative macrophages, which in turn, express more of the resident tissue macrophage marker SIGN‐R1 and higher CD11b and F4/80. Ly‐6B‐expressing macrophages incorporate more BrdU than their Ly‐6B‐negative contemporaries when fed during the resolution phase of the acute inflammatory response. Thus, Ly‐6B expression on mature macrophages defines a subset of recently generated inflammatory macrophages that retain monocytic markers and is hence a surrogate marker of macrophage turnover in inflammatory lesions. The definition of the 7/4:Ly‐6B antigen will allow further characterization and specific modulation of Ly‐6B‐expressing cells in vivo.

DDX5 and its associated lncRNA Rmrp modulate TH17 cell effector functions

T helper 17 (TH17) lymphocytes protect mucosal barriers from infections, but also contribute to multiple chronic inflammatory diseases. Their differentiation is controlled by RORγt, a ligand-regulated nuclear receptor. Here we identify the RNA helicase DEAD-box protein 5 (DDX5) as a RORγt partner that coordinates transcription of selective TH17 genes, and is required for TH17-mediated inflammatory pathologies. Surprisingly, the ability of DDX5 to interact with RORγt and coactivate its targets depends on intrinsic RNA helicase activity and binding of a conserved nuclear long noncoding RNA (lncRNA), Rmrp, which is mutated in patients with cartilage-hair hypoplasia. A targeted Rmrp gene mutation in mice, corresponding to a gene mutation in cartilage-hair hypoplasia patients, altered lncRNA chromatin occupancy, and reduced the DDX5–RORγt interaction and RORγt target gene transcription. Elucidation of the link between Rmrp and the DDX5–RORγt complex reveals a role for RNA helicases and lncRNAs in tissue-specific transcriptional regulation, and provides new opportunities for therapeutic intervention in TH17-dependent diseases.

Comparative evaluation of label-free SINQ normalized spectral index quantitation in the central proteomics facilities pipeline

Normalized spectral index quantification was recently presented as an accurate method of label‐free quantitation, which improved spectral counting by incorporating the intensities of peptide MS/MS fragment ions into the calculation of protein abundance. We present SINQ, a tool implementing this method within the framework of existing analysis software, our freely available central proteomics facilities pipeline (CPFP). We demonstrate, using data sets of protein standards acquired on a variety of mass spectrometers, that SINQ can rapidly provide useful estimates of the absolute quantity of proteins present in a medium‐complexity sample. In addition, relative quantitation of standard proteins spiked into a complex lysate background and run without pre‐fractionation produces accurate results at amounts above 1 fmol on column. We compare quantitation performance to various precursor intensity‐ and identification‐based methods, including the normalized spectral abundance factor (NSAF), exponentially modified protein abundance index (emPAI), MaxQuant, and Progenesis LC‐MS. We anticipate that the SINQ tool will be a useful asset for core facilities and individual laboratories that wish to produce quantitative MS data, but lack the necessary manpower to routinely support more complicated software workflows. SINQ is freely available to obtain and use as part of the central proteomics facilities pipeline, which is released under an open‐source license.

QuaNCAT: quantitating proteome dynamics in primary cells

Here we demonstrate quantitation of stimuli-induced proteome dynamics in primary cells by combining the power of bio-orthogonal noncanonical amino acid tagging (BONCAT) and stable-isotope labeling of amino acids in cell culture (SILAC). In conjunction with nanoscale liquid chromatography–tandem mass spectrometry (nanoLC-MS/MS), quantitative noncanonical amino acid tagging (QuaNCAT) allowed us to monitor the early expression changes of >600 proteins in primary resting T cells subjected to activation stimuli.

CPFP: a central proteomics facilities pipeline

UNLABELLED The central proteomics facilities pipeline (CPFP) provides identification, validation, and quantitation of peptides and proteins from LC-MS/MS datasets through an easy to use web interface. It is the first analysis pipeline targeted specifically at the needs of proteomics core facilities, reducing the data analysis load on staff, and allowing facility clients to easily access and work with their data. Identification of peptides is performed using multiple search engines, their output combined and validated using state-of-the-art techniques for improved results. Cluster execution of jobs allows analysis capacity to be increased easily as demand grows. AVAILABILITY Released under the Common Development and Distribution License at http://cpfp.sourceforge.net/. Demonstration available at https://cpfp-master.molbiol.ox.ac.uk/cpfp_demo.

Conserved and host-specific features of influenza virion architecture

Viruses use virions to spread between hosts, and virion composition is therefore the primary determinant of viral transmissibility and immunogenicity. However, the virions of many viruses are complex and pleomorphic, making them difficult to analyse in detail. Here we address this by identifying and quantifying virion proteins with mass spectrometry, producing a complete and quantified model of the hundreds of viral and host-encoded proteins that make up the pleomorphic virions of influenza viruses. We show that a conserved influenza virion architecture is maintained across diverse combinations of virus and host. This ‘core’ architecture, which includes substantial quantities of host proteins as well as the viral protein NS1, is elaborated with abundant host-dependent features. As a result, influenza virions produced by mammalian and avian hosts have distinct protein compositions. Finally we note that influenza virions share an underlying protein composition with exosomes, suggesting that influenza virions form by subverting microvesicle production.