Multiparameter screen optimizes immunoprecipitation.

Immunoprecipitation (IP) coupled with mass spectrometry effectively maps protein-protein interactions when genome-wide, affinity-tagged cell collections are used. Such studies have recorded significant portions of the compositions of physiological protein complexes, providing draft 'interactomes'; yet many constituents of protein complexes still remain uncharted. This gap exists partly because high-throughput approaches cannot optimize each IP. A key challenge for IP optimization is stabilizing in vivo interactions during the transfer from cells to test tubes; failure to do so leads to the loss of genuine interactions during the IP and subsequent failure to detect. Our high-content screening method explores the relationship between in vitro chemical conditions and IP outcomes, enabling rapid empirical optimization of conditions for capturing target macromolecular assemblies.

CCDC88B interacts with RASAL3 and ARHGEF2 and regulates dendritic cell function in neuroinflammation and colitis.

CCDC88B is a risk factor for several chronic inflammatory diseases in humans and its inactivation causes a migratory defect in DCs in mice. CCDC88B belongs to a family of cytoskeleton-associated scaffold proteins that feature protein:protein interaction domains. Here, we identified the Rho/Rac Guanine Nucleotide Exchange Factor 2 (ARHGEF2) and the RAS Protein Activator Like 3 (RASAL3) as CCDC88B physical and functional interactors. Mice defective in Arhgef2 or Rasal3 show dampened neuroinflammation, and display altered cellular response and susceptibility to colitis; ARHGEF2 maps to a human Chromosome 1 locus associated with susceptibility to IBD. Arhgef2 and Rasal3 mutant DCs show altered migration and motility in vitro, causing either reduced (Arhgef2) or enhanced (Rasal3) migratory properties. The CCDC88B/RASAL3/ARHGEF2 complex appears to regulate DCs migration by modulating activation of RHOA, with ARHGEF2 and RASAL3 acting in opposite regulatory fashions, providing a molecular mechanism for the involvement of these proteins in DCs immune functions.

Structures, functions and adaptations of the human LINE-1 ORF2 protein.

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.

Consequences of a telomerase-related fitness defect and chromosome substitution technology in yeast synIX strains.

We describe the complete synthesis, assembly, debugging, and characterization of a synthetic 404,963 bp chromosome, synIX (synthetic chromosome IX). Combined chromosome construction methods were used to synthesize and integrate its left arm (synIXL) into a strain containing previously described synIXR. We identified and resolved a bug affecting expression of EST3, a crucial gene for telomerase function, producing a synIX strain with near wild-type fitness. To facilitate future synthetic chromosome consolidation and increase flexibility of chromosome transfer between distinct strains, we combined chromoduction, a method to transfer a whole chromosome between two strains, with conditional centromere destabilization to substitute a chromosome of interest for its native counterpart. Both steps of this chromosome substitution method were efficient. We observed that wild-type II tended to co-transfer with synIX and was co-destabilized with wild-type IX, suggesting a potential gene dosage compensation relationship between these chromosomes.

MsPHep: An online application for low molecular weight heparin rapid characterization based on liquid chromatography-tandem mass spectrometry.

Low-molecular-weight heparins (LMWHs) are important anticoagulants widely used in clinic. Since they are comprised of complex and heterogenous glycan chains, liquid chromatography-tandem mass spectrometry (LC-MS) is commonly used for structural analysis and quality control of LMWHs to ensure their safety and efficacy. Yet, the structural complexity arising from the parent heparin macromolecules, as well as the different depolymerization methods used for preparing LMWHs, makes processing and assigning the LC-MS data of LWMHs very tedious and challenging. We therefore developed, and here report, an open-source and easy-to-use web application, MsPHep, to facilitate the LMWH analysis based on LC-MS data. MsPHep is compatible with various LMWHs and chromatographic separation methods. With the HepQual function, MsPHep is capable of annotating both the LMWH compound and its isotopic distribution from mass spectra. Moreover, the HepQuant function enables automatic quantification of LMWH compositions without prior knowledge or any database generation. To demonstrate the reliability and system stability of MsPHep, we tested various types of LMWHs that were analyzed with different chromatographic methods coupled to MS. The results show that MsPHep has its own advantages compared to another public tool GlycReSoft for LMWH analysis, and it is available online under an open-source license at https://ngrc-glycan.shinyapps.io/MsPHep.

Cancer cells co-evolve with retrotransposons to mitigate viral mimicry.

Overexpression of repetitive elements is an emerging hallmark of human cancers 1 . Diverse repeats can mimic viruses by replicating within the cancer genome through retrotransposition, or presenting pathogen-associated molecular patterns (PAMPs) to the pattern recognition receptors (PRRs) of the innate immune system 2-5 . Yet, how specific repeats affect tumor evolution and shape the tumor immune microenvironment (TME) in a pro- or anti-tumorigenic manner remains poorly defined. Here, we integrate whole genome and total transcriptome data from a unique autopsy cohort of multiregional samples collected in pancreatic ductal adenocarcinoma (PDAC) patients, into a comprehensive evolutionary analysis. We find that more recently evolved S hort I nterspersed N uclear E lements (SINE), a family of retrotransposable repeats, are more likely to form immunostimulatory double-strand RNAs (dsRNAs). Consequently, younger SINEs are strongly co-regulated with RIG-I like receptor associated type-I interferon genes but anti-correlated with pro-tumorigenic macrophage infiltration. We discover that immunostimulatory SINE expression in tumors is regulated by either L ong I nterspersed N uclear E lements 1 (LINE1/L1) mobility or ADAR1 activity in a TP53 mutation dependent manner. Moreover, L1 retrotransposition activity tracks with tumor evolution and is associated with TP53 mutation status. Altogether, our results suggest pancreatic tumors actively evolve to modulate immunogenic SINE stress and induce pro-tumorigenic inflammation. Our integrative, evolutionary analysis therefore illustrates, for the first time, how dark matter genomic repeats enable tumors to co-evolve with the TME by actively regulating viral mimicry to their selective advantage.

Expression of L1 retrotransposons in granulocytes from patients with active systemic lupus erythematosus.

BACKGROUND: Patients with systemic lupus erythematosus (SLE) have autoantibodies against the L1-encoded open-reading frame 1 protein (ORF1p). Here, we report (i) which immune cells ORF1p emanates from, (ii) which L1 loci are transcriptionally active, (iii) whether the cells express L1-dependent interferon and interferon-stimulated genes, and (iv) the effect of inhibition of L1 ORF2p by reverse transcriptase inhibitors. RESULTS: L1 ORF1p was detected by flow cytometry primarily in SLE CD66b+CD15+ regular and low-density granulocytes, but much less in other immune cell lineages. The amount of ORF1p was higher in neutrophils from patients with SLE disease activity index (SLEDAI) > 6 (p = 0.011) compared to patients with inactive disease, SLEDAI < 4. Patient neutrophils transcribed seven to twelve human-specific L1 loci (L1Hs), but only 3 that are full-length and with an intact ORF1. Besides serving as a source of detectable ORF1p, the most abundant transcript encoded a truncated ORF2p reverse transcriptase predicted to remain cytosolic, while the two other encoded an intact full-length ORF2p. A number of genes encoding proteins that influence L1 transcription positively or negatively were altered in patients, particularly those with active disease, compared to healthy controls. Components of nucleic acid sensing and interferon induction were also altered. SLE neutrophils also expressed type I interferon-inducible genes and interferon ß, which were substantially reduced after treatment of the cells with drugs known to inhibit ORF2p reverse transcriptase activity. CONCLUSIONS: We identified L1Hs loci that are transcriptionally active in SLE neutrophils, and a reduction in the epigenetic silencing mechanisms that normally counteract L1 transcription. SLE neutrophils contained L1-encoded ORF1p protein, as well as activation of the type I interferon system, which was inhibited by treatment with reverse transcriptase inhibitors. Our findings will enable a deeper analysis of L1 dysregulation and its potential role in SLE pathogenesis.

Affinity-Based Interactome Analysis of Endogenous LINE-1 Macromolecules.

During their proliferation and the host's concomitant attempts to suppress it, LINE-1 (L1) retrotransposons give rise to a collection of heterogeneous ribonucleoproteins (RNPs); their protein and RNA compositions remain poorly defined. The constituents of L1-associated macromolecules can differ depending on numerous factors, including, for example, position within the L1 life cycle, whether the macromolecule is productive or under suppression, and the cell type within which the proliferation is occurring. This chapter describes techniques that aid the capture and characterization of protein and RNA components of L1 macromolecules from tissues that natively express them. The protocols described have been applied to embryonal carcinoma cell lines that are popular model systems for L1 molecular biology (e.g., N2102Ep, NTERA-2, and PA-1 cells), as well as colorectal cancer tissues. N2102Ep cells are given as the use case for this chapter; the protocols should be applicable to essentially any tissue exhibiting endogenous L1 expression with minor modifications.

Targeting DNA topoisomerases or checkpoint kinases results in an overload of chaperone systems, triggering aggregation of a metastable subproteome.

A loss of the checkpoint kinase ataxia telangiectasia mutated (ATM) leads to impairments in the DNA damage response, and in humans causes cerebellar neurodegeneration, and an increased risk of cancer. A loss of ATM is also associated with increased protein aggregation. The relevance and characteristics of this aggregation are still incompletely understood. Moreover, it is unclear to what extent other genotoxic conditions can trigger protein aggregation as well. Here, we show that targeting ATM, but also ATR or DNA topoisomerases, results in the widespread aggregation of a metastable, disease-associated subfraction of the proteome. Aggregation-prone model substrates, including Huntingtin exon 1 containing an expanded polyglutamine repeat, aggregate faster under these conditions. This increased aggregation results from an overload of chaperone systems, which lowers the cell-intrinsic threshold for proteins to aggregate. In line with this, we find that inhibition of the HSP70 chaperone system further exacerbates the increased protein aggregation. Moreover, we identify the molecular chaperone HSPB5 as a cell-specific suppressor of it. Our findings reveal that various genotoxic conditions trigger widespread protein aggregation in a manner that is highly reminiscent of the aggregation occurring in situations of proteotoxic stress and in proteinopathies.

Cells are constantly perceiving and responding to changes in their surroundings, and challenging conditions such as extreme heat or toxic chemicals can put cells under stress. When this happens, protein production can be affected. Proteins are long chains of chemical building blocks called amino acids, and they can only perform their roles if they fold into the right shape. Some proteins fold easily and remain folded, but others can be unstable and often become misfolded. Unfolded proteins can become a problem because they stick to each other, forming large clumps called aggregates that can interfere with the normal activity of cells, causing damage. The causes of stress that have a direct effect on protein folding are called proteotoxic stresses, and include, for example, high temperatures, which make proteins more flexible and unstable, increasing their chances of becoming unfolded. To prevent proteins becoming misfolded, cells can make 'protein chaperones', a type of proteins that help other proteins fold correctly and stay folded. The production of protein chaperones often increases in response to proteotoxic stress. However, there are other types of stress too, such as genotoxic stress, which damages DNA. It is unclear what effect genotoxic stress has on protein folding. Huiting et al. studied protein folding during genotoxic stress in human cells grown in the lab. Stress was induced by either blocking the proteins that repair DNA or by 'trapping' the proteins that release DNA tension, both of which result in DNA damage. The analysis showed that, similar to the effects of proteotoxic stress, genotoxic stress increased the number of proteins that aggregate, although certain proteins formed aggregates even without stress, particularly if they were common and relatively unstable proteins. Huiting et al.'s results suggest that aggregation increases in cells under genotoxic stress because the cells fail to produce enough chaperones to effectively fold all the proteins that need it. Indeed, Huiting et al. showed that aggregates contain many proteins that rely on chaperones, and that increasing the number of chaperones in stressed cells reduced protein aggregation. This work shows that genotoxic stress can affect protein folding by limiting the availability of chaperones, which increases protein aggregation. Remarkably, there is a substantial overlap between proteins that aggregate in diseases that affect the brain ­ such as Alzheimer's disease ­ and proteins that aggregate after genotoxic stress. Therefore, further research could focus on determining whether genotoxic stress is involved in the progression of these neurological diseases.

LINE-1 expression in cancer correlates with p53 mutation, copy number alteration, and S phase checkpoint.

Retrotransposons are genomic DNA sequences that copy themselves to new genomic locations via RNA intermediates; LINE-1 is the only active and autonomous retrotransposon in the human genome. The mobility of LINE-1 is largely repressed in somatic tissues but is derepressed in many cancers, where LINE-1 retrotransposition is correlated with p53 mutation and copy number alteration (CNA). In cell lines, inducing LINE-1 expression can cause double-strand breaks (DSBs) and replication stress. Reanalyzing multiomic data from breast, ovarian, endometrial, and colon cancers, we confirmed correlations between LINE-1 expression, p53 mutation status, and CNA. We observed a consistent correlation between LINE-1 expression and the abundance of DNA replication complex components, indicating that LINE-1 may also induce replication stress in human tumors. In endometrial cancer, high-quality phosphoproteomic data allowed us to identify the DSB-induced ATM-MRN-SMC S phase checkpoint pathway as the primary DNA damage response (DDR) pathway associated with LINE-1 expression. Induction of LINE-1 expression in an in vitro model led to increased phosphorylation of MRN complex member RAD50, suggesting that LINE-1 directly activates this pathway.

IgG and IgA autoantibodies against L1 ORF1p expressed in granulocytes correlate with granulocyte consumption and disease activity in pediatric systemic lupus erythematosus.

BACKGROUND: Most patients with systemic lupus erythematosus (SLE) have IgG autoantibodies against the RNA-binding p40 (ORF1p) protein encoded by the L1 retroelement. This study tested if these autoantibodies are also present in children with pediatric SLE (pSLE) and if the p40 protein itself could be detected in immune cells. METHODS: Autoantibodies in the plasma of pSLE patients (n = 30), healthy children (n = 37), and disease controls juvenile idiopathic arthritis (JIA) (n = 32) and juvenile dermatomyositis (JDM) (n = 60), were measured by ELISA. Expression of p40 in immune cells was assessed by flow cytometry. Markers of neutrophil activation and death were quantitated by ELISA. RESULTS: IgG and IgA autoantibodies reactive with p40 were detected in the pSLE patients, but were low in healthy controls and in JIA or JDM. pSLE patients with active disease (13 of them newly diagnosed) had higher titers than the same patients after effective therapy (p = 0.0003). IgG titers correlated with SLEDAI (r = 0.65, p = 0.0001), ESR (r = 0.43, p = 0.02), and anti-dsDNA antibodies (r = 0.49, p < 0.03), and inversely with complement C3 (r = -0.55, p = 0.002) and C4 (r = -0.51, p = 0.006). p40 protein was detected in a subpopulation of CD66b+ granulocytes in pSLE, as well as in adult SLE patients. Myeloperoxidase and neutrophil elastase complexed with DNA and the neutrophil-derived S100A8/A9 were elevated in plasma from pSLE patients with active disease and correlated with anti-p40 autoantibodies and disease activity. CONCLUSIONS: Children with active SLE have elevated IgG and IgA autoantibodies against L1 p40, and this protein can be detected in circulating granulocytes in both pediatric and adult SLE patients. P40 expression and autoantibody levels correlate with disease activity. Markers of neutrophil activation and death also correlate with these autoantibodies and with disease activity, suggesting that neutrophils express L1 and are a source of p40.

Correction to: The Eukaryotic RNA Exosome.

The original version of this book was inadvertently published with the hyperlinks deleted from the reference section in the Chapters 1,5,8,11,13,15,16 and 24. These are corrected now.

NCBP3 positively impacts mRNA biogenesis.

The nuclear Cap-Binding Complex (CBC), consisting of Nuclear Cap-Binding Protein 1 (NCBP1) and 2 (NCBP2), associates with the nascent 5'cap of RNA polymerase II transcripts and impacts RNA fate decisions. Recently, the C17orf85 protein, also called NCBP3, was suggested to form an alternative CBC by replacing NCBP2. However, applying protein-protein interaction screening of NCBP1, 2 and 3, we find that the interaction profile of NCBP3 is distinct. Whereas NCBP1 and 2 identify known CBC interactors, NCBP3 primarily interacts with components of the Exon Junction Complex (EJC) and the TRanscription and EXport (TREX) complex. NCBP3-EJC association in vitro and in vivo requires EJC core integrity and the in vivo RNA binding profiles of EJC and NCBP3 overlap. We further show that NCBP3 competes with the RNA degradation factor ZC3H18 for binding CBC-bound transcripts, and that NCBP3 positively impacts the nuclear export of polyadenylated RNAs and the expression of large multi-exonic transcripts. Collectively, our results place NCBP3 with the EJC and TREX complexes in supporting mRNA expression.

Affinity proteomic dissection of the human nuclear cap-binding complex interactome.

A 5',7-methylguanosine cap is a quintessential feature of RNA polymerase II-transcribed RNAs, and a textbook aspect of co-transcriptional RNA processing. The cap is bound by the cap-binding complex (CBC), canonically consisting of nuclear cap-binding proteins 1 and 2 (NCBP1/2). Interest in the CBC has recently renewed due to its participation in RNA-fate decisions via interactions with RNA productive factors as well as with adapters of the degradative RNA exosome. A novel cap-binding protein, NCBP3, was recently proposed to form an alternative CBC together with NCBP1, and to interact with the canonical CBC along with the protein SRRT. The theme of post-transcriptional RNA fate, and how it relates to co-transcriptional ribonucleoprotein assembly, is abundant with complicated, ambiguous, and likely incomplete models. In an effort to clarify the compositions of NCBP1-, 2- and 3-related macromolecular assemblies, we have applied an affinity capture-based interactome screen where the experimental design and data processing have been modified to quantitatively identify interactome differences between targets under a range of experimental conditions. This study generated a comprehensive view of NCBP-protein interactions in the ribonucleoprotein context and demonstrates the potential of our approach to benefit the interpretation of complex biological pathways.

National Cancer Institute Think-Tank Meeting Report on Proteomic Cartography and Biomarkers at the Single-Cell Level: Interrogation of Premalignant Lesions.

A Think-Tank Meeting was convened by the National Cancer Institute (NCI) to solicit experts' opinion on the development and application of multiomic single-cell analyses, and especially single-cell proteomics, to improve the development of a new generation of biomarkers for cancer risk, early detection, diagnosis, and prognosis as well as to discuss the discovery of new targets for prevention and therapy. It is anticipated that such markers and targets will be based on cellular, subcellular, molecular, and functional aberrations within the lesion and within individual cells. Single-cell proteomic data will be essential for the establishment of new tools with searchable and scalable features that include spatial and temporal cartographies of premalignant and malignant lesions. Challenges and potential solutions that were discussed included (i) The best way/s to analyze single-cells from fresh and preserved tissue; (ii) Detection and analysis of secreted molecules and from single cells, especially from a tissue slice; (iii) Detection of new, previously undocumented cell type/s in the premalignant and early stage cancer tissue microenvironment; (iv) Multiomic integration of data to support and inform proteomic measurements; (v) Subcellular organelles-identifying abnormal structure, function, distribution, and location within individual premalignant and malignant cells; (vi) How to improve the dynamic range of single-cell proteomic measurements for discovery of differentially expressed proteins and their post-translational modifications (PTM); (vii) The depth of coverage measured concurrently using single-cell techniques; (viii) Quantitation - absolute or semiquantitative? (ix) Single methodology or multiplexed combinations? (x) Application of analytical methods for identification of biologically significant subsets; (xi) Data visualization of N-dimensional data sets; (xii) How to construct intercellular signaling networks in individual cells within premalignant tumor microenvironments (TME); (xiii) Associations between intrinsic cellular processes and extrinsic stimuli; (xiv) How to predict cellular responses to stress-inducing stimuli; (xv) Identification of new markers for prediction of progression from precursor, benign, and localized lesions to invasive cancer, based on spatial and temporal changes within individual cells; (xvi) Identification of new targets for immunoprevention or immunotherapy-identification of neoantigens and surfactome of individual cells within a lesion.

LINE-1 ORF2p expression is nearly imperceptible in human cancers.

BACKGROUND: Long interspersed element-1 (LINE-1, L1) is the major driver of mobile DNA activity in modern humans. When expressed, LINE-1 loci produce bicistronic transcripts encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Many types of human cancers are characterized by L1 promoter hypomethylation, L1 transcription, L1 ORF1p protein expression, and somatic L1 retrotransposition. ORF2p encodes the endonuclease and reverse transcriptase activities required for L1 retrotransposition. Its expression is poorly characterized in human tissues and cell lines. RESULTS: We report mass spectrometry-based tumor proteome profiling studies wherein ORF2p eludes detection. To test whether ORF2p could be detected with specific reagents, we developed and validated five rabbit monoclonal antibodies with immunoreactivity for specific epitopes on the protein. These reagents readily detect ectopic ORF2p expressed from bicistronic L1 constructs. However, endogenous ORF2p is not detected in human tumor samples or cell lines by western blot, immunoprecipitation, or immunohistochemistry despite high levels of ORF1p expression. Moreover, we report endogenous ORF1p-associated interactomes, affinity isolated from colorectal cancers, wherein we similarly fail to detect ORF2p. These samples include primary tumors harboring hundreds of somatically acquired L1 insertions. The new data are available via ProteomeXchange with identifier PXD013743. CONCLUSIONS: Although somatic retrotransposition provides unequivocal genetic evidence for the expression of ORF2p in human cancers, we are unable to directly measure its presence using several standard methods. Experimental systems have previously indicated an unequal stoichiometry between ORF1p and ORF2p, but in vivo, the expression of these two proteins may be more strikingly uncoupled. These findings are consistent with observations that ORF2p is not tolerable for cell growth.

High Prevalence and Disease Correlation of Autoantibodies Against p40 Encoded by Long Interspersed Nuclear Elements in Systemic Lupus Erythematosus.

OBJECTIVE: Long interspersed nuclear element 1 (LINE-1) encodes 2 proteins, the RNA binding protein p40 and endonuclease and reverse transcriptase (open-reading frame 2p [ORF2p]), which are both required for LINE-1 to retrotranspose. In cells expressing LINE-1, these proteins assemble with LINE-1 RNA and additional RNA binding proteins, some of which are well-known autoantigens in patients with systemic lupus erythematosus (SLE). This study was undertaken to investigate whether SLE patients also produce autoantibodies against LINE-1 p40. METHODS: Highly purified p40 protein was used to quantitate IgG autoantibodies in serum from 172 SLE patients and from disease controls and age-matched healthy subjects by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Preparations of p40 that also contained associated proteins were analyzed by immunoblotting with patient sera. RESULTS: Antibodies reactive with p40 were detected in the majority of patients and many healthy controls. Their levels were higher in patients with SLE, but not those with systemic sclerosis, compared to healthy subjects (P = 0.01). Anti-p40 reactivity was higher in patients during a flare than in patients with disease in remission (P = 0.03); correlated with the SLE Disease Activity Index score (P = 0.0002), type I interferon score (P = 0.006), decrease in complement C3 level (P = 0.0001), the presence of anti-DNA antibodies (P < 0.0001) and anti-C1q antibodies (P = 0.004), and current or past history of nephritis (P = 0.02 and P = 0.003, respectively); and correlated inversely with age (r = -0.49, P < 0.0001). SLE patient sera also reacted with p40-associated proteins. CONCLUSION: Autoantibodies reacting with LINE-1 p40 characterize a population of SLE patients with severe and active disease. These autoantibodies may represent an early immune response against LINE-1 p40 that does not yet by itself imply clinically significant autoimmunity, but may represent an early, and still reversible, step toward SLE pathogenesis.

Affinity Proteomic Analysis of the Human Exosome and Its Cofactor Complexes.

In humans, the RNA exosome consists of an enzymatically inactive nine-subunit core, with ribonucleolytic activity contributed by additional components. Several cofactor complexes also interact with the exosome-these enable the recruitment of, and specify the activity upon, diverse substrates. Affinity capture coupled with mass spectrometry has proven to be an effective means to identify the compositions of RNA exosomes and their cofactor complexes: here, we describe a general experimental strategy for proteomic characterization of macromolecular complexes, applied to the exosome and an affiliated adapter protein, ZC3H18.

Identification of RNase-sensitive LINE-1 Ribonucleoprotein Interactions by Differential Affinity Immobilization.

Long Interspersed Nuclear Element-1 (LINE-1, L1) constitutes a family of autonomous, self-replicating genetic elements known as retrotransposons. Although most are inactive, copious L1 sequences populate the human genome. L1s proliferate in a 'copy-and-paste' fashion through an RNA intermediate; a full-length L1 transcript is ~6,000 nucleotides long and functions as a bicistronic mRNA that encodes and assembles in cis with two main polypeptides, ORF1p and ORF2p, forming a ribonucleoprotein (RNP); L1 RNPs also interact with a wide range of host factors in positive and negative regulatory capacities. The following protocol describes an approach to affinity enrich ectopically expressed L1 RNPs and, using RNases, release the fraction of protein that depends upon the presence of intact RNA for retention in the immobilized macromolecules.