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There is a long experimental history supporting the principle that RNA is essential for normal nuclear and chromatin architecture. Most of the genome is transcribed into RNA but only 2% of the sequence codes for proteins. In the nucleus, most non-coding RNA, packaged in proteins, is bound into structures including chromatin and a non-chromatin scaffolding, the nuclear matrix, which was first observed by electron microscopy. Removing nuclear RNA or inhibiting its transcription causes the condensation of chromatin, showing the importance of RNA in spatially and functionally organizing the genome. Today, powerful techniques for the molecular characterization of RNA and for mapping its spatial organization in the nucleus have provided molecular detail to these principles., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The germline is a key feature of sexual animals and the ways in which it separates from the soma differ widely across Metazoa. However, at least at some point during germline differentiation, some cytoplasmic supramolecular structures (collectively called germ plasm-related structures) are present and involved in its specification and/or differentiation. The factors involved in the assembly of these granular structures are various and non-ubiquitous among animals, even if some functional patterns and the presence of certain domains appear to be shared among some. For instance, the LOTUS domain is shared by Oskar, the Holometabola germ plasm master regulator, and some Tudor-family proteins assessed as being involved in the proper assembly of germ granules of different animals. Here, we looked for the presence of LOTUS-containing proteins in the transcriptome of Ruditapes philippinarum (Bivalvia). Such species is of particular interest because it displays annual renewal of gonads, sided by the renewal of germline differentiation pathways. Moreover, previous works have identified in its early germ cells cytoplasmic granules containing germline determinants. We selected the orthologue of TDRD7 as a candidate involved in the early steps of germline differentiation through bioinformatic predictions and immunohistological patterning (immunohistochemistry and immunofluorescence). We observed the expression of the protein in putative precursors of germline cells, upstream to the germline marker Vasa. This, added to the fact that orthologues of this protein are involved in the assembly of germ granules in mouse, zebrafish, and fly, makes it a worthy study unit for investigations on the formation of such structures in bivalves., (© 2021. The Author(s).)

RNA-protein interaction is central to post-transcriptional gene regulation. Identification of RNA-binding proteins relies mainly on UV-induced crosslinking (UVX) followed by the enrichment of RNA-protein conjugates and LC-MS/MS analysis. However, UVX has limited applicability in tissues of multicellular organisms due to its low penetration depth. Here, we introduce formaldehyde crosslinking (FAX) as an alternative chemical crosslinking for RNA interactome capture (RIC). Mild FAX captures RNA-protein interaction with high specificity and efficiency in cell culture. Unlike UVX-RIC, FAX-RIC robustly detects proteins that bind to structured RNAs or uracil-poor RNAs (e.g. AGO1, STAU1, UPF1, NCBP2, EIF4E, YTHDF proteins and PABP), broadening the coverage. Applied to Xenopus laevis oocytes and embryos, FAX-RIC provided comprehensive and unbiased RNA interactome, revealing dynamic remodeling of RNA-protein complexes. Notably, translation machinery changes during oocyte-to-embryo transition, for instance, from canonical eIF4E to noncanonical eIF4E3. Furthermore, using Mus musculus liver, we demonstrate that FAX-RIC is applicable to mammalian tissue samples. Taken together, we report that FAX can extend the RNA interactome profiling into multicellular organisms., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Stable and transient interactions between molecules are determinant for cell function. Among those, numerous proteins contact coding and noncoding RNAs to modulate their fate and promote their activity. The identification of such interactions as well as the cellular and molecular conditions of these interactions represent key information for the characterization of the role of each partner. RNA immunoprecipitation (RIP) is the leading technique to detect in vivo the association of individual proteins with RNA species. Two main approaches exist: native RIP is largely used to identify and quantify RNA interactions, while crosslinked RIP (CLIP) may inform about direct interactions as well as their extent in the unaltered cellular condition, i.e., before cell lysis. In this chapter, both techniques applied to mammalian cells are described with a series of precautions regarding their design.

Objective: Complement components, including C4d, can be found on activated platelets, a process associated with vascular disease in SLE. We investigated whether platelet C4d (PC4d) adds additional value to traditional and known lupus-associated risk factors when identifying SLE patients with vascular disease., Methods: This cross-sectional study included 308 well-characterized SLE patients and 308 matched general population controls. PC4d deposition was analysed using flow cytometry. Values >95% of controls were considered as PC4d positive (+). aPL were determined by Luminex, and the LA test was performed by DRVVT. History of vascular disease (composite and as separate outcomes) was defined at inclusion., Results: SLE patients had increased PC4d deposition as compared with population controls (50 vs 5%, P < 0.0001). PC4d+ positively associated with any vascular events, and separately with venous and cerebrovascular events, and also with all investigated aPL profiles. The association for any vascular event remained statistically significant after adjustment for traditional and SLE-associated risk factors (odds ratio: 2.3, 95% CI: 1.3, 4.3, P = 0.008). Compared with patients negative for both PC4d and LA, patients with double positivity were more likely to have vascular disease (odds ratio: 12.3, 95% CI: 5.4, 29.3; attributable proportion due to interaction 0.8, 95% CI: 0.4, 1.1)., Conclusion: PC4d+ is associated with vascular events in SLE, independently of traditional and SLE-associated risk factors. Concurrent presence of PC4d and LA seem to interact to further increase the odds for vascular events. Prospective studies should examine whether the aPL/PC4d combination can improve prediction of vascular events in SLE and/or APS., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Ro60, also known as SS-A or TROVE2, is an evolutionarily conserved RNA-binding protein that is found in most animal cells, approximately 5% of sequenced prokaryotic genomes and some archaea. Ro60 is present in cells as both a free protein and as a component of a ribonucleoprotein complex, where its best-known partners are members of a class of noncoding RNAs called Y RNAs. Structural and biochemical analyses have revealed that Ro60 is a ring-shaped protein that binds Y RNAs on its outer surface. In addition to Y RNAs, Ro60 binds misfolded and aberrant noncoding RNAs in some animal cell nuclei. Although the fate of these defective Ro60-bound noncoding RNAs in animal cells is not well-defined, a bacterial Ro60 ortholog functions with 3' to 5' exoribonucleases to assist structured RNA degradation. Studies of Y RNAs have revealed that these RNAs regulate the subcellular localization of Ro60, tether Ro60 to effector proteins and regulate the access of other RNAs to its central cavity. As both mammalian cells and bacteria lacking Ro60 are sensitized to ultraviolet irradiation, Ro60 function may be important during exposure to some environmental stressors. Here we summarize the current knowledge regarding the functions of Ro60 and Y RNAs in animal cells and bacteria. Because the Ro60 RNP is a clinically important target of autoantibodies in patients with rheumatic diseases such as Sjogren's syndrome, systemic lupus erythematosus, and neonatal lupus, we also discuss potential roles for Ro60 RNPs in the initiation and pathogenesis of systemic autoimmune rheumatic disease.

Long Interspersed Nuclear Element-1 (LINE-1, L1) is a mobile genetic element active in human genomes. L1-encoded ORF1 and ORF2 proteins bind L1 RNAs, forming ribonucleoproteins (RNPs). These RNPs interact with diverse host proteins, some repressive and others required for the L1 lifecycle. Using differential affinity purifications, quantitative mass spectrometry, and next generation RNA sequencing, we have characterized the proteins and nucleic acids associated with distinctive, enzymatically active L1 macromolecular complexes. Among them, we describe a cytoplasmic intermediate that we hypothesize to be the canonical ORF1p/ORF2p/L1 - RNA-containing RNP, and we describe a nuclear population containing ORF2p, but lacking ORF1p, which likely contains host factors participating in target-primed reverse transcription., Competing Interests: MT, IA, KM, PM, HJ, EA, AW, SB, DI, GE, KB, DF, BC, DA, MR, JB, JL No competing interests declared, (© 2018, Taylor et al.)

The etiology of Behçet's disease (BD), a chronic, multisystemic autoinflammatory and autoimmune disease, remains unknown; however, researchers have postulated that infectious agents, such as herpes simplex virus, are significant triggering factors of BD. Tripartite motif-containing (TRIM) proteins exhibit antiviral properties, mediating antiviral defense mechanisms. The purpose of this study was to investigate TRIM21 protein expression in the monocytes of BD patients and to identify the role of TRIM21 in immune dysregulation in BD. In this study, the expression of TRIM21 and related molecules, including interferon regulatory factor 8 (IRF8), was analyzed in monocytes from BD patients. Functional analyses using small interfering RNA and co-culture with responder T cells were performed to examine the pathological role of TRIM21 in BD. Peripheral blood monocytes from BD patients showed increased TRIM21 expression and decreased IRF8 expression compared with that in monocytes from healthy controls. TRIM21 was found to decrease IRF8 expression. BD monocytes facilitated Th1 and Th17 differentiation of co-cultured T cells, and knock-down of TRIM21 expression by small interfering RNA inhibited this differentiation. In conclusion, TRIM21 played a pivotal role in regulating the secretion of proinflammatory cytokines in monocytes of BD patients.

Regulation of gene expression is key determinant to cell structure and function. RNA localization, where specific mRNAs are transported to subcellular regions and then translated, is highly conserved in eukaryotes ranging from yeast to extremely specialized and polarized cells such as neurons. Messenger RNA and associated proteins (mRNP) move from the site of transcription in the nucleus to their final destination in the cytoplasm both passively through diffusion and actively via directed transport. Dysfunction of RNA localization, transport and translation machinery can lead to pathology. Single-molecule live-cell imaging techniques have revealed unique features of this journey with unprecedented resolution. In this review, we highlight key recent findings that have been made using these approaches and possible implications for spatial control of gene function., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A key aspect of cellular function is the proper assembly and utilization of ribonucleoproteins (RNPs). Recent studies have shown that hyper- or hypo-assembly of various RNPs can lead to human diseases. Defects in the formation of RNPs lead to 'RNP hypo-assembly diseases', which can be caused by RNA degradation outcompeting RNP assembly. By contrast, excess RNP assembly, either in higher order RNP granules, or due to the expression of repeat-containing RNAs, can lead to 'RNP hyper-assembly diseases'. Here, we discuss the most recent advances in understanding the cause of disease onset, as well as potential therapies from the aspect of modulating RNP assembly in the cell, which presents a novel route to the treatment of these diseases., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

All decisions affecting the life cycle of human immunodeficiency virus (HIV-1) RNA are executed by ribonucleoprotein complexes (RNPs). HIV-1 RNA cycles through a progression of host RNPs composed of RNA-binding proteins regulating all stages of synthesis, processing, nuclear transport, translation, decay, and co-localization with assembling virions. RNA affinity chromatography is a versatile method to identify RNA-binding proteins to investigate the molecular basis of viral and cellular posttranscriptional control of gene expression. The bait is a HIV-1 RNA motif immobilized on a solid support, typically magnetic or Sepharose beads. The prey is pre-formed RNPs admixed in lysate from cells or concentrated virus particles. The methodology distinguishes high-affinity RNA-protein interactions from low-affinity complexes by increases in ionic strength during progressive elution cycles. Here, we describe RNA affinity chromatography of the 5' untranslated region of HIV-1, obtaining mixtures of high-affinity RNA binding proteins suitable for mass spectrometry and proteome identification.

Novel antiviral drugs, which are less prone to resistance development, are desirable alternatives to the currently approved drugs for the treatment of potentially serious influenza virus infections. The viral polymerase is highly conserved and serves as an attractive target for antiviral drugs since potent inhibitors would directly stop viral replication at an early stage. Recent structural studies on the functional domains of the heterotrimeric influenza polymerase, which comprises subunits PA, PB1, and PB2, opened the way to a structure-based approach for optimizing inhibitors of viral replication. These strategies, however, are limited by the use of isolated protein fragments instead of employing the entire ribonucleoprotein complex (RNP), which represents the functional form of the influenza polymerase in infected cells. In this study, we have established a screening assay for efficient and reliable analysis of potential influenza polymerase inhibitors of various molecular targets such as monoselective polymerase inhibitors targeting the endonuclease site, the cap-binding domain, and the polymerase active site, respectively. By utilizing whole viral RNPs and a radioactivity-free endpoint detection with the capability for efficient compound screening while offering high-content information on potential inhibitors to drive medicinal chemistry program in a reliable manner, this biochemical assay provides significant advantages over the currently available conventional assays. We propose that this assay can eventually be adapted for coinstantaneous analysis and subsequent optimization of two or more different chemical scaffold classes targeting multiple active sites within the polymerase complex, thus enabling the evaluation of drug combinations and characterization of molecules with dual functionality.

RNP granules are ribonucleoprotein assemblies that regulate the post-transcriptional fate of mRNAs in all eukaryotes. Their exact function remains poorly understood, one reason for this is that RNP granule purification has not yet been achieved. We have exploited a unique feature of trypanosomes to prepare a cellular fraction highly enriched in starvation stress granules. First, granules remain trapped within the cage-like, subpellicular microtubule array of the trypanosome cytoskeleton while soluble proteins are washed away. Second, the microtubules are depolymerized and the granules are released. RNA sequencing combined with single molecule mRNA FISH identified the short and highly abundant mRNAs encoding ribosomal mRNAs as being excluded from granules. By mass spectrometry we have identified 463 stress granule candidate proteins. For 17/49 proteins tested by eYFP tagging we have confirmed the localization to granules, including one phosphatase, one methyltransferase and two proteins with a function in trypanosome life-cycle regulation. The novel method presented here enables the unbiased identification of novel RNP granule components, paving the way towards an understanding of RNP granule function., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objective: TRIM21 (also known as Ro52) is an autoantigen in rheumatic disease and is predominantly expressed in leucocytes. Overexpression is associated with decreased proliferation, and the TRIM21 gene maps to a tumour suppressor locus. We therefore investigated the expression of TRIM21 in patients with diffuse large B-cell lymphoma (DLBCL) and its potential usefulness as a prognostic biomarker., Materials and Methods: TRIM21 expression levels were assessed by immunohistochemistry in lymphoma biopsies from three cohorts of patients with DLBCL: 42 patients with rheumatic disease treated with a cyclophosphamide, vincristine, doxorubicin and prednisone (CHOP)-like regimen, 76 CHOP-treated and 196 rituximab-CHOP-treated nonrheumatic patients. Expression was correlated with clinical and biomedical parameters. TRIM21 expression was assessed in relation to lymphocyte proliferation by quantitative PCR and correlated with (3) H-thymidine incorporation and propidium iodine staining., Results: TRIM21 expression levels differed in the lymphomas compared to normal lymphoid tissue, with reduced expression correlating with shorter overall survival in all three cohorts. In the two larger cohorts, progression-free survival was assessed and was also found to correlate with TRIM21 expression. The association was independent of commonly used clinical prognostic scores, lymphoma subtype and several previously reported prognostic biomarkers. In agreement with this clinical observation, we noted an inverse correlation between TRIM21 expression and proliferation of leucocytes in vitro., Conclusions: We show that loss of TRIM21 expression is associated with more aggressive lymphoma and increased proliferation, whereas maintenance of TRIM21 expression is associated with better prognosis in patients with DLBCL. Based on our findings, we suggest that TRIM21 should be considered as a novel biomarker for lymphoma characterization and for predicting patient survival., (© 2015 The Association for the Publication of the Journal of Internal Medicine.)

Large macromolecular complexes such as the spliceosomal small nuclear ribonucleoproteins (snRNPs) play a variety of roles within the cell. Despite their biological importance, biochemical studies of snRNPs and other machines are often thwarted by practical difficulties in the isolation of sufficient amounts of material. Studies of the snRNPs as well as other macromolecular machines would be greatly facilitated by new approaches that enable their isolation and biochemical characterization. One such approach is single-molecule pull-down (SiMPull) that combines in situ immunopurification of complexes from cell lysates with subsequent single-molecule fluorescence microscopy experiments. We report the development of a new method, called SNAP-SiMPull, that can readily be applied to studies of splicing factors and snRNPs isolated from whole-cell lysates. SNAP-SiMPull overcomes many of the limitations imposed by conventional SiMPull strategies that rely on fluorescent proteins. We have used SNAP-SiMPull to study the yeast branchpoint bridging protein (BBP) as well as the U1 and U6 snRNPs. SNAP-SiMPull will likely find broad use for rapidly isolating complex cellular machines for single-molecule fluorescence colocalization experiments., (© 2015 Rodgers et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Noncoding RNAs (ncRNAs) function with associated proteins to effect complex structural and regulatory outcomes. To reveal the composition and dynamics of specific noncoding RNA-protein complexes (RNPs) in vivo, we developed comprehensive identification of RNA binding proteins by mass spectrometry (ChIRP-MS). ChIRP-MS analysis of four ncRNAs captures key protein interactors, including a U1-specific link to the 3' RNA processing machinery. Xist, an essential lncRNA for X chromosome inactivation (XCI), interacts with 81 proteins from chromatin modification, nuclear matrix, and RNA remodeling pathways. The Xist RNA-protein particle assembles in two steps coupled with the transition from pluripotency to differentiation. Specific interactors include HnrnpK, which participates in Xist-mediated gene silencing and histone modifications but not Xist localization, and Drosophila Split ends homolog Spen, which interacts via the A-repeat domain of Xist and is required for gene silencing. Thus, Xist lncRNA engages with proteins in a modular and developmentally controlled manner to coordinate chromatin spreading and silencing., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The introduction of direct detection devices in cryo-EM has shown that specimens present beam-induced motion (BIM). Consequently, in this work, we develop a BIM correction method at the image level, resulting in an integrated image in which the in-plane BIM blurring is compensated prior to particle picking. The methodology is based on a robust Optical Flow (OF) approach that can efficiently correct for local movements in a rapid manner. The OF works particularly well if the BIM pattern presents a substantial degree of local movements, which occurs in our data sets for Falcon II data. However, for those cases in which the BIM pattern corresponds to global movements, we have found it advantageous to first run a global motion correction approach and to subsequently apply OF. Additionally, spatial analysis of the Optical Flow allows for quantitative analysis of the BIM pattern. The software that incorporates the new approach is available in XMIPP (http://xmipp.cnb.csic.es)., (Copyright © 2015. Published by Elsevier Inc.)

Nanogold-conjugated immunodetection systems are now widely and commercially available for use in a number of research applications including electron microscopy, light microscopy, and western blotting. Nanogold clusters are small, uniform in size, and stable, unlike gold colloids historically used in protein detection. Covalent linkage of nanogold particles to secondary antibodies prevents dissociation of the gold particles during the staining process, making protein detection reliable, antigen specific, and highly sensitive. Nanogold labeling is extremely versatile and can be used in conjunction with other staining methodologies including Alexa Fluor immunofluorescence detection to perform coupled staining procedures. Silver enhancement increases the limits of sensitivity for nanogold staining, thus improving detection signals for antigens with reduced expression levels. Herein, we describe the use of nanogold-silver detection as an immunodetection system for standard western blotting of autoantigens.

Minor impurities in tryptic peptide digests can affect the signal obtained in matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Therefore, it becomes necessary to purify the digests, especially those that fail to yield good mass spectra. Here, we describe a simple protocol using polyvinylidene difluoride membrane for purifying tryptic peptides prior to mass spectrometric analysis. The tryptic digest is spotted on a polyvinylidene difluoride membrane, air-dried, and washed. The membrane is then extracted with trifluoroacetic acid/acetonitrile and the extract is then subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This method enabled us to identify a cross-reactive D1 autoantigen on the surface of neutrophils that bound antibodies targeting Ro 60 autoantigen in systemic lupus erythematosus.

Antibodies directed against ribonucleoprotein (RNP) particles are observed in systemic lupus erythematosus. Ro RNP particle is one such target. It is composed of a 60 kDa protein (Ro 60 or SS-A) that is non-covalently associated with at least one of the four short uridine-rich RNAs (the hY RNAs). Previously, we showed that multiple antigenic peptides (MAPs) made from the sequence of the Ro 60 autoantigen could be used, using double-immunodiffusion studies, enzyme-linked immunosorbant assay, affinity chromatography, and surface plasmon resonance, to show intramolecular and intermolecular protein-protein interaction within the Ro 60 RNP particle. We also observed that calcium is important in mediating this interaction. We hypothesized, therefore, that 60 kDa Ro is a calcium-binding protein. To investigate this, we electrophoresed 60 kDa Ro MAPs, transferred them to PVDF membrane, and assayed calcium binding using the Quin-2 system. Several Ro 60 MAPs were found to bind calcium using this assay, as well as bovine serum albumin, another calcium-binding protein. However, a MAP constructed from the Sm autoantigen did not bind to calcium. These data, along with our observation regarding the involvement of calcium in protein-protein interaction occurring between Ro 60 antigen and Ro 60 MAPs, makes us propose that Ro 60 antigen is a calcium-binding protein.

Development of immunoblots is commonly performed using enzyme-labeled antibodies which convert soluble substrates into insoluble colored products. A simple, rapid, and sensitive alternative method which produces low background and allows a rapid quantitative evaluation is the use of radiolabeled antibodies or protein A conjugates. Here we describe the use of iodinated secondary antibodies for immunodetection of an autoantigen during HPLC purification.

Sera of patients with systemic autoimmune diseases frequently contain autoantibodies to nuclear autoantigens. Immunoblotting of recombinant and native autoantigens is a commonly used technique for the identification and characterization of autoantibody specificities. Here we describe an easy procedure which facilitates the comparison of antibody specificities by reusing the same immunoblot for at least three times in order to detect an abundantly expressed autoantigen in total cellular extracts.

Calcium plays an important role in many biochemical processes in different cell types. This divalent cation interacts with specific calcium-binding proteins that serve as calcium sensors and regulatory proteins to mediate its function. Previously, we found that calcium was involved in the protein-protein interaction observed between Ro60 multiple antigenic peptides and Ro60 autoantigen. Since calcium bound Ro60 multiple antigenic peptides, we hypothesized that it would renature human recombinant Ro60 on a protein blot. We found that anti-Ro60 antibodies bound significantly higher to the recombinant Ro60 antigen that was incubated with calcium compared to that incubated without calcium on a polyvinylidene fluoride (PVDF) blot. Since the immunological epitopes of Ro60 are mainly conformational, we believe that calcium induced a more native tertiary structure in recombinant Ro60 autoantigen following blotting to a PVDF membrane.

Processing of transcribed precursor ribosomal RNA (pre-rRNA) to a mature state is a conserved aspect of ribosome biogenesis in vivo. We developed an affinity-purification system to isolate and analyze in vivo-formed pre-rRNA-containing ribonucleoprotein (RNP) particles (rRNPs) from wild-type E. coli. We observed that the first processing intermediate of pre-small subunit (pre-SSU) rRNA is a platform for biogenesis. These pre-SSU-containing RNPs have differing ribosomal-protein and auxiliary factor association and rRNA folding. Each RNP lacks the proper architecture in functional regions, thus suggesting that checkpoints preclude immature subunits from entering the translational cycle. This work offers in vivo snapshots of SSU biogenesis and reveals that multiple pathways exist for the entire SSU biogenesis process in wild-type E. coli. These findings have implications for understanding SSU biogenesis in vivo and offer a general strategy for analysis of RNP biogenesis.

We have previously identified 55 nonribosomal proteins in PAB1-mRNP complexes in Saccharomyces cerevisiae using mass spectrometric analysis. Because one of the inherent limitations of mass spectrometry is that it does not inform as to the size or type of complexes in which the proteins are present, we consequently used analytical ultracentrifugation with fluorescent detection system (AU-FDS) to determine which proteins are present in the 77S monosomal translation complex that contains minimally the closed-loop structure components (eIF4E, eIF4G, and PAB1), mRNA, and the 40S and 60S ribosomes. We assayed by AU-FDS analysis 33 additional PAB1-mRNP factors but found that only five of these proteins were present in the 77S translation complex: eRF1, SLF1, SSD1, PUB1, and SBP1. eRF1 is involved in translation termination, SBP1 is a translational repressor, and SLF1, SSD1, and PUB1 are known mRNA binding proteins. Many of the known P body/stress granule proteins that associate with the PAB1-mRNP were not present in the 77S translation complex, implying that P body/stress granules result from significant protein additions after translational cessation. These data inform that AU-FDS can clarify protein complex identification that remains undetermined after typical immunoprecipitation and mass spectrometric analyses., (© 2014 The Protein Society.)

The antimetabolite 5-fluorouracil is a widely used chemotherapeutic for the treatment of several solid cancers. However, resistance to 5-fluorouracil remains a major drawback in its clinical use. In this study we report that treatment of HeLa cells with 5-fluorouracil resulted in de novo assembly of stress granules. Moreover, we revealed that stress granule assembly under stress conditions as well as disassembly is altered in cells treated with 5-fluorouracil. Notably, we discovered that RACK1, a protein mediating cell survival and apoptosis, is a component of 5-fluorouracil-induced stress granules. To explore the mode of action of 5-fluorouracil accountable for de novo stress granule assembly, we analyzed 5-fluorouracil metabolites and noticed that stress granule assembly is caused by RNA, not DNA incorporating 5-fluorouracil metabolites. Interestingly, we observed that other RNA incorporating drugs also cause assembly of stress granules. Thus, our results suggest that incorporation of chemotherapeutics into RNA may result in stress granule assembly with potential significance in chemoresistance., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly and fate is important to understand the cellular response to stress. The deficiency in key factors of SGs like G3BP (RasGAP SH3 domain Binding Protein) leads to developmental defects in mice and alterations of the Central Nervous System. To study the dynamics of SGs in cells from an organism, one can culture primary cells and follow the localization of a transfected tagged component of SGs. We describe time-lapse experiment to observe G3BP1-containing SGs in Mouse Embryonic Fibroblasts (MEFs). This technique can also be used to study G3BP-containing SGs in live neurons, which is crucial as it was recently shown that these SGs are formed at the onset of neurodegenerative diseases like Alzheimer's disease. This approach can be adapted to any other cellular body and granule protein component, and performed with transgenic animals, allowing the live study of granules dynamics for example in the absence of a specific factor of these granules.

Determining the composition of messenger ribonucleoprotein (mRNP) particles is essential for a comprehensive understanding of the complex mechanisms underlying mRNA regulation, but is technically challenging. Here we present an RNA-based method to identify RNP components using a modified streptavidin (SA)-binding RNA aptamer termed S1m. By optimizing the RNA aptamer S1 in structure and repeat conformation, we improved its affinity for SA and found a 4-fold repeat of S1m (4×S1m) to be more efficient than the established MS2 and PP7 systems from bacteriophages. We then attached the AU-rich element (ARE) of tumor necrosis factor alpha (TNFα), a well-known RNA motif that induces mRNA degradation, via 4×S1m to a SA matrix, and used the resulting RNA affinity column to purify ARE-binding proteins (BPs) from cellular extracts. By quantitative mass spectrometry using differential dimethyl labeling, we identified the majority of established ARE-BPs and detected several RNA-BPs that had previously not been associated with AREs. For two of these proteins, Rbms1 and Roxan, we confirmed specific binding to the TNFα ARE. The optimized 4×S1m aptamer, therefore, provides a powerful tool for the discovery of mRNP components in a single affinity purification step.

DU145 human prostate carcinoma cells are typically poorly differentiated and contain only scantily distributed organelles. However, among numerous tumor cells randomly examined by electron microscopy out of in vitro cultivation, a peculiar, rare oncocyte-like cell type has been observed whose nucleus appears to be of small dimension and with a cytoplasm almost entirely filled with often distorted mitochondria. A few small, dispersed lysosomal bodies, small cisterns of the endoplasmic reticulum and a few glycogen patches can be found among highly osmiophilic contrasted, cytosolic spaces filled by innumerable ribonucleoproteins. The excessive population of mitochondria may have arisen from a more populated tumor cell type wherein the altered mitochondria are found to appear burgeoning into a spherical-like size progeny crowding the tumor cells. Literature cited between 1950 and the present suggests that this rare, oncocytic, benign prostatic tumor cell type is likely appear epigenetically, stemming from an original secretory cell, which is confirmed by the origin of the cell line originally maintained as cell line out of a brain metastatic, adenocarcinoma niche.

LINE-1s are active human DNA parasites that are agents of genome dynamics in evolution and disease. These streamlined elements require host factors to complete their life cycles, whereas hosts have developed mechanisms to combat retrotransposition's mutagenic effects. As such, endogenous L1 expression levels are extremely low, creating a roadblock for detailed interactomic analyses. Here, we describe a system to express and purify highly active L1 RNP complexes from human suspension cell culture and characterize the copurified proteome, identifying 37 high-confidence candidate interactors. These data sets include known interactors PABPC1 and MOV10 and, with in-cell imaging studies, suggest existence of at least three types of compositionally and functionally distinct L1 RNPs. Among the findings, UPF1, a key nonsense-mediated decay factor, and PCNA, the polymerase-delta-associated sliding DNA clamp, were identified and validated. PCNA interacts with ORF2p via a PIP box motif; mechanistic studies suggest that this occurs during or immediately after target-primed reverse transcription., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: To investigate the effectiveness of total glucosides of paeony (TGP) on Sjogren's syndrome (SS) using non-obese diabetic (NOD) mice model., Study Design: Twenty-seven 8-week-old female NOD mice were assigned into TGP group, hydroxychloroquine (HCQ) group and normal saline (NS) group, receiving corresponding drugs respectively and sacrificed at 24-week-old. Saliva flow rate (SFR), ration of regulatory T cells, level of anti-SSA/SSB, histological changes in submandibular glands (SMG) and microarray analysis were assessed. The data were analyzed using SPSS., Results: Compared to NS group, in TGP group, SFR, SMG index and the ration of regulatory T cells were significantly higher, while anti-SSA/SSB and lymphocytic foci were significantly lower. HCQ group demonstrated similar results except SMG index. Altered gene expression was found in 10.71% of TGP and 13.09% of HCQ of the profile., Conclusion: TGP demonstrated a similar effectiveness as HCQ in delaying the onset of SS-like disease in NOD mice., (Copyright © 2012 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

We report Rhizomelic Chondrodysplasia Punctata (RDCP), a rare, autosomal recessive disorder with rhizomelic shortening of limbs, congenital cataracts and seizures but without any biochemical abnormality. The mother of the baby developed Systemic Lupus Erythromatosus (SLE) with Ro/SSA antibodies 11 months after delivery. Ro/SSA antibodies may generate calreticulin antibodies causing characteristic skeletal changes.

We demonstrate the development of a mass spectrometry-based epitope-mapping procedure in combination with Western blot analysis that works also with antigens that are insoluble in nondenaturing buffers consuming minute amounts of antigen (approximately 200 pmol) and antibody (approximately 15 pmol), respectively. A polyclonal anti-TRIM21 rabbit antibody serum is applied as a model serum for future patient analyses to set up the system. The major epitope that is recognized by the anti-TRIM21 serum spans the central TRIM21 region LQ-ELEKDEREQLRILGE-KE, showing that immunization with a 139-amino acid residue long peptide resulted in a 'monospecific' polyclonal antibody repertoire. Protein structure investigations, secondary structure predictions, and surface area calculations revealed that the best matching partial sequence to fulfill all primary and secondary structure requirements was the four amino acid spanning motif 'L-E-Q-L', which is present in both the sequential and the α-helical peptide conformation. Peptide chip analyses confirmed the mass spectrometric results and showed that the peptide chip platform is an appropriate method for displaying secondary structure-relying epitope conformations. As the same secondary structures are present in vivo, patient antibody screening, e.g., to identify subgroups of patients according to distinct epitope antibody reactivities, is feasible., (Copyright © 2013 John Wiley & Sons, Ltd.)

Cytoplasmic inclusions containing TDP-43 are a pathological hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. TDP-43 is an RNA binding protein involved in gene regulation through control of RNA transcription, splicing and transport. However, the function of TDP-43 in the nervous system is largely unknown and its role in the pathogenesis of ALS is unclear. The aim of this study was to identify genes in the central nervous system that are regulated by TDP-43. RNA-immunoprecipitation with anti-TDP-43 antibody, followed by microarray analysis (RIP-chip), was used to isolate and identify RNA bound to TDP-43 protein from mouse brain. This analysis produced a list of 1839 potential TDP-43 gene targets, many of which overlap with previous studies and whose functions include RNA processing and synaptic function. Immunohistochemistry demonstrated that the TDP-43 protein could be found at the presynaptic membrane of axon terminals in the neuromuscular junction in mice. In conclusion, the finding that TDP-43 binds to RNA that codes for genes related to synaptic function, together with the localization of TDP-43 protein at axon terminals, suggests a role for TDP-43 in the transport of synaptic mRNAs into distal processes.

Once the rabies virus has spread through the central nervous system (CNS), the virus is also transported centrifugally along axons, especially of the autonomic nervous system, to a wide range of organs including the heart. In this case report of a 49-year-old man who had been bitten by a dog in Asia, the rabies infection of cardiac nerves and cardiac muscle fibres is shown by immunohistochemistry. The neuritis cordis and rabies myocarditis can have important clinical effects on the heart rate and myocardial function and lead to blood pressure crises which are typical for the clinical course of rabies in humans.

The purpose of this protocol is to identify 'footprints' of protein on RNA. However, it can also be used to analyze the secondary structure of RNA. This protocol is optimized for large RNA molecules; however, it can be adapted for the study of small RNAs., (© 2013 Elsevier Inc. All rights reserved.)

Gene expression patterns change dramatically in aging and age-related events. The DNA microarray is now recognized as a useful device in molecular biology and widely used to identify the molecular mechanisms of aging and the biological effects of drugs for therapeutic purpose in age-related diseases. Recently, numerous technological advantages have led to the evolution of DNA microarrays and microarray-based techniques, revealing the genomic modification and all transcriptional activity. Here, we show the step-by-step methods currently used in our lab to handling the oligonucleotide microarray and miRNA microarray. Moreover, we introduce the protocols of ribonucleoprotein [RNP] immunoprecipitation followed by microarray analysis (RIP-chip) which reveal the target mRNA of age-related RNA-binding proteins.

Sjögren's syndrome (SjS) is one of the most common autoimmune rheumatic diseases, clinically characterized by xerostomia and keratoconjunctivitis sicca. We investigated the following controversial topics: (i) Do we have reliable ways of assessing saliva production? (ii) How important are the quantity and quality of saliva? (iii) Are only anti-SSA/Ro and anti-SSB/La relevant for the diagnosis of SjS? (iv) Are the American-European Consensus criteria (AECC) the best way to diagnose SjS? Results from literature searches suggested the following: (i) Despite the fact that numerous tests are available to assess salivation rates, direct comparisons among them are scarce with little evidence to suggest one best test. (ii) Recent developments highlight the importance of investigating the composition of saliva. However, more research is needed to standardize the methods of analysis and collection and refine the quality of the accumulating data. (iii) In addition to anti-Ro/La autoantibodies, anti α-fodrin IgA and anti-MR3 autoantibodies seem to be promising diagnostic markers of SjS, but more studies are warranted to test their sensitivity and specificity. (iv) AECC are classification, not diagnostic criteria. Moreover, recent innovations have not been incorporated into these criteria. Consequently, treatment directed to patients diagnosed using the AECC might exclude a significant proportion of patients with SjS., (© 2012 John Wiley & Sons A/S.)

Podosomes are multifunctional organelles of invasive cells that combine several key abilities, including adhesion, matrix degradation and mechanosensing. The necessary spatiotemporal fine-tuning of podosome structure, turnover and function implies the existence of an intricate network of proteins, comparable to other integrin-based adhesions. However, no systematic effort has yet been made to map the podosome proteome. Here, we describe the purification of podosome-enriched fractions from primary human macrophages, labelled with isotopically stable amino acids, and the subsequent mass spectrometric analysis of these fractions. We present a consensus list of 203 proteins, comprising 33 known podosome proteins and 170 potential novel components. We also present second-level analyses of the podosome proteome, as well as proof-of-principle experiments by showing that the newly identified components WDR1/AIP-1 and hnRNP-K localise to the core structure of macrophage podosomes. Comparisons with other adhesion structure proteomes confirm that the podosome proteome shares components with focal adhesions and invadopodia, but also reveal an extensive overlap with spreading initiation centres (SICs). We suggest that the consensus list comprises a significant part of the podosome proteome and will be helpful for future studies on podosome structure, composition and function, and also for detailed classification of adhesion structure subtypes., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Long non-coding RNAs (lncRNAs), once relegated to junk products of the genome, are becoming better appreciated for the myriad functions they play in cellular processes. It is clear that for most of the cases studied, lncRNAs carry out their functions at least in part through interactions with proteins. Here we present two complementary biochemical methods for the analysis of lncRNA-containing ribonucleoprotein complexes, hereafter referred to as RNPs. The first strategy offers users the ability to purify RNPs based on a protein component and to analyze the spectrum of lncRNAs, other proteins, and, if present, other types of RNAs that are bound to it. The second makes use of a bacteriophage MS2 binding-site affinity-handle grafted onto an lncRNA of interest to investigate the proteins and RNAs that co-purify with the tagged RNA., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Gene expression profile upon endoplasmic reticulum (ER) stress was analyzed by deep shotgun sequencing of mRNAs (DSSR) using RNAs from polysomes or cytoplasm of the HT29 cell. Two time points, 4h after tunicamycin treatment when IRE1α signaling pathway is active and 16h after the treatment when it is inactive, were used. There was a transient decrease in the proportion of shorter mRNA species (<1000bp) in polysome, while it increased transiently in the cytoplasm. Despite such an overall change and decrease in total amount of polysomes, the majority of the 6966 genes analyzed had less than 2 fold change in their expressions. We searched for the genes whose expression was elevated by 2 folds or more in both polysome and cytoplasm and confirmed the results with RT-PCR. There were 7 genes elevated only at 4h (Group I), 20 genes only at 16h (Group II) and 7 genes both at 4 and 16h (Group III). There were 3 genes involved in ribosomal RNA biogenesis in Group I and 2 genes involved mTOR control in Group III. This was consistent with the concept that the ribosome is the essential site for managing ER stress. DSSR is a useful tool for the search of candidates of ER stress responsive genes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

oskar RNA localization to the posterior pole of the Drosophila melanogaster oocyte requires splicing of the first intron and the exon junction complex (EJC) core proteins. The functional link between splicing, EJC deposition and oskar localization has been unclear. Here we demonstrate that the EJC associates with oskar mRNA upon splicing in vitro and that Drosophila EJC deposition is constitutive and conserved. Our in vivo analysis reveals that splicing creates the spliced oskar localization element (SOLE), whose structural integrity is crucial for ribonucleoprotein motility and localization in the oocyte. Splicing thus has a dual role in oskar mRNA localization: assembling the SOLE and depositing the EJC required for mRNA transport. The SOLE complements the EJC in formation of a functional unit that, together with the oskar 3' UTR, maintains proper kinesin-based motility of oskar mRNPs and posterior mRNA targeting.

JAK2(V617F) is a gain of function mutation that promotes cytokine-independent growth of myeloid cells and accounts for a majority of myeloproliferative neoplasms (MPN). Mutations in p53 are rarely found in these diseases before acute leukemia transformation, but this does not rule out a role for p53 deregulation in disease progression. Using Ba/F3-EPOR cells and ex vivo cultured CD34(+) cells from MPN patients, we demonstrate that expression of JAK2(V617F) affected the p53 response to DNA damage. We show that E3 ubiquitin ligase MDM2 accumulated in these cells, due to an increased translation of MDM2 mRNA. Accumulation of the La autoantigen, which interacts with MDM2 mRNA and promotes its translation, was responsible for the increase in MDM2 protein level and the subsequent degradation of p53 after DNA damage. Downregulation of La protein or cell treatment with nutlin-3, a MDM2 antagonist, restored the p53 response to DNA damage and the cytokine-dependence of Ba/F3-EPOR-JAK2(V617F) cells. Altogether, these data indicate that the JAK2(V617F) mutation affects p53 response to DNA damage through the upregulation of La antigen and accumulation of MDM2. They also suggest that p53 functional inactivation accounts for the cytokine hypersensitivity of JAK2(V617F) MPN and might have a role in disease progression.

Objective: Understanding the pathogenesis of chemotherapy-induced oral mucositis (CIOM) is vital to develop therapies for this common, dose-limiting side effect of cancer treatment. We investigated molecular events in CIOM from buccal mucosa tissue collected before and 2 days after chemotherapy from patients with acute myeloid leukemia (AML) and healthy controls by microarray analysis., Methods: Microarray analysis was performed using Human Genome U133 Plus 2.0 Array on buccal mucosa punch biopsies from patients with AML before (n = 4) or after chemotherapy (n = 4), and from healthy controls (n = 3). Following Robust Multichip Average (RMA) normalization, we applied Linear Models for Microarray data (LIMMA) and Significance Analysis of Microarrays (SAM) for data analysis using the TM4/TMeV v4.5.1 program., Results: LIMMA and SAM identified genes potentially affected by the presence of AML, including homeodomain-interacting protein kinase 1 (HIPK1), mex-3 homolog D (MEX3D), and genes potentially affected by chemotherapy, including argininosuccinate synthase 1 (ASS1), notch homolog 1 (NOTCH1), zinc transporter ZIP6 (SLC39A6), and TP53-regulated inhibitor of apoptosis 1 (TRIAP1). The expression of 2 genes with potential biological significance in oral mucositis, ASS1 and SLC39A6 (alias LIV-1), was confirmed by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR)., Conclusions: Our results suggest that AML-specific deregulated immune responses and inflammatory tissue damage to the oral mucosa caused by chemotherapy may not be overcome by the natural cellular repair processes and therefore contribute to CIOM., (Copyright © 2011 Mosby, Inc. All rights reserved.)

Cytoplasmic ribonucleoprotein granules, known as processing bodies (P-bodies), contain a common set of conserved RNA-processing enzymes, and mRNAs with AU-rich elements (AREs) are delivered to P-bodies for translational silencing. Although the dynamics of P-bodies is physically linked to cytoskeletal network, it is unclear how small GTPases are involved in the P-body regulation and the ARE-mRNA metabolism. We found here that glucose depletion activates RhoA GTPase and alters the P-body dynamics in HeLa cells. These glucose-depleted effects are reproduced by the overexpression of the RhoA-subfamily GTPases and conversely abolished by the inhibition of RhoA activation. Interestingly, both RhoA activation and glucose depletion inhibit the mRNA accumulation and degradation. These findings indicate that RhoA participates in the stress-induced rearrangement of P-bodies and the release of nucleated ARE-mRNAs for their stabilization.

Simple and non-invasive saliva-based diagnostics may be useful for the identification, understanding, and monitoring of autoimmune and infectious diseases. Previously, Luciferase Immunoprecipitation Systems (LIPS) were used for sensitive detection of patient serum autoantibodies in Sjögren's Syndrome (SjS), a chronic autoimmune disease affecting the salivary and lacrimal glands. Here we explored the ability of LIPS to diagnose SjS based on IgG autoantibodies in patient saliva. From LIPS testing, anti-Ro60 autoantibodies were detected in the saliva of 70% (19/27) of SjS patients with 96% specificity. Positive anti-Ro60 autoantibodies were also found in 70% of the matched serum samples (96% specificity). LIPS detected Ro52 autoantibodies in the saliva and serum of 67% of SjS patients with 100% specificity. Overall, the autoantibody titers in saliva were approximately 4000-fold lower by volume than serum, but still distinguished seropositive patients from controls. These results suggest that LIPS salivary-based testing for SjS autoantibodies is a practical alternative to serum and compatible with point-of-care testing.

RNA molecules rarely function alone in cells. For most RNAs, their function requires formation of various ribonucleoprotein (RNP) complexes. For example, mRNP composition can determine mRNA localization, translational repression, level of translation or mRNA stability. RNPs are usually studied by biochemical methods. However, biochemical approaches are unsuitable for some model systems, such as mammalian oocytes and early embryos, due to the small amounts that can be obtained for experimental analysis. In such cases, microscopic techniques are often used to learn about RNPs. Here, we present a review of immunostaining, fluorescence in situ hybridization with subcellular resolution and a combination of both, with emphasis on the mouse oocyte and early embryos models. Application of these techniques to whole-mount fixed oocytes and early embryos can provide information about RNP composition and localization with three-dimensional resolution., (Copyright © 2010 Elsevier Inc. All rights reserved.)

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary. A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip) defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking with immunoprecipitation followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP). CLIP was used to identify targets of a number of RBPs. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample. We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs. Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.

Tumour development and progression consists a series of multiple changes in gene expression. Progressive tumour cells acquire more aggressive properties manifested by rapid growth, invasiveness and metastatic ability, as well as increased genetic instability leading to multiple genetic alterations. Therefore, it is crucial to identify the possible intracellular and extracellular molecular mechanisms that accelerate tumour progression, in particular to identify nuclear proteins which interact with DNA. Nuclear proteomics provides an opportunity to qualitatively and quantitatively examine protein effectors that contribute to cellular phenotype. This study performed a differential display analysis for the expression of nuclear proteome between regressive tumour cell clone QR-32 and malignant progressive tumour cell clone QRsP-11 using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). Eight nuclear proteins whose expressions were different between QR-32 and QRsP-11 cells were identified. Seven of those protein spots, zinc finger protein ZXDC, lamin-A/C, far upstream clement-binding protein 1, heterogeneous nuclear ribonucleoprotein K, heterogeneous nuclear ribonucleoprotein A/B and guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, were down-regulated in QRsP-11, while one protein, nucleolin, was up-regulated in QRsP-11.