FAX-RIC enables robust profiling of dynamic RNP complex formation in multicellular organisms in vivo.

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.

Hypo- and Hyper-Assembly Diseases of RNA-Protein Complexes.

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.

Calcium Binding by Ro 60 Multiple Antigenic Peptides on PVDF Membrane.

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.

Reduced expression of TRIM21/Ro52 predicts poor prognosis in diffuse large B-cell lymphoma patients with and without rheumatic disease.

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.

5-Fluorouracil affects assembly of stress granules based on RNA incorporation.

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.

An optimized streptavidin-binding RNA aptamer for purification of ribonucleoprotein complexes identifies novel ARE-binding proteins.

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.

A rare, human prostate oncocyte cell originates from the prostatic carcinoma (DU145) cell line.

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.

Effects of total glucosides of paeony for delaying onset of Sjogren's syndrome: an animal study.

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.

Proteomic analysis of podosome fractions from macrophages reveals similarities to spreading initiation centres.

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.

Monitoring endoplasmic reticulum stress responsive mRNAs by RNA sequencing.

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.

JAK2(V617F) negatively regulates p53 stabilization by enhancing MDM2 via La expression in myeloproliferative neoplasms.

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.

Microarray analyses of oral punch biopsies from acute myeloid leukemia (AML) patients treated with chemotherapy.

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.

Proteomic analysis for nuclear proteins related to tumour malignant progression: a comparative proteomic study between malignant progressive cells and regressive cells.

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.

PAR-CliP--a method to identify transcriptome-wide the binding sites of RNA binding proteins.

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.

Cytoskeleton-associated large RNP complexes in tobacco male gametophyte (EPPs) are associated with ribosomes and are involved in protein synthesis, processing, and localization.

The progamic phase of male gametophyte development involves activation of synthetic and catabolic processes required for the rapid growth of the pollen tube. It is well-established that both transcription and translation play an important role in global and specific gene expression patterns during pollen maturation. On the contrary, germination of many pollen species has been shown to be largely independent of transcription but vitally dependent on translation of stored mRNAs. Here, we report the first structural and proteomic data about large ribonucleoprotein particles (EPPs) in tobacco male gametophyte. These complexes are formed in immature pollen where they contain translationally silent mRNAs. Although massively activated at the early progamic phase, they also serve as a long-term storage of mRNA transported along with the translational machinery to the tip region. Moreover, EPPs were shown to contain ribosomal subunits, rRNAs and a set of mRNAs. Presented results extend our view of EPP complexes from mere RNA storage and transport compartment in particular stages of pollen development to the complex and well-organized machinery devoted to mRNA storage, transport and subsequent controlled activation resulting in protein synthesis, processing and precise localization. Such an organization is extremely useful in fast tip-growing pollen tube. There, massive and orchestrated protein synthesis, processing, and transport must take place in accurately localized regions. Moreover, presented complex role of EPPs in tobacco cytoplasmic mRNA and protein metabolism makes them likely to be active in another plant species too. Expression of vast majority of the closest orthologues of EPP proteins also in Arabidopsis male gametophyte further extends this concept from tobacco to Arabidopsis, the model species with advanced tricellular pollen.

Participation of Xenopus Elr-type proteins in vegetal mRNA localization during oogenesis.

Directional transport of specific mRNAs is of primary biological relevance. In Xenopus oocytes, mRNA localization to the vegetal pole is important for germ layer formation and germ cell development. Using a biochemical approach, we identified Xenopus Elr-type proteins, homologs of the Hu/ELAV proteins, as novel components of the vegetal mRNA localization machinery. They bind specifically to the localization elements of several different vegetally localizing Xenopus mRNAs, and they are part of one RNP together with other localization proteins, such as Vg1RBP and XStaufen 1. Blocking Elr-type protein binding by either localization element mutagenesis or antisense morpholino oligonucleotide-mediated masking of their target RNA structures, as well as overexpression of wild type and mutant ElrB proteins, interferes with vegetal localization in Xenopus oocytes.

Spliceosomal proteomics in Trypanosoma brucei reveal new RNA splicing factors.

In trypanosomatid parasites, spliced leader (SL) trans splicing is an essential nuclear mRNA maturation step which caps mRNAs posttranscriptionally and, in conjunction with polyadenylation, resolves individual mRNAs from polycistronic precursors. While all trypanosomatid mRNAs are trans spliced, intron removal by cis splicing is extremely rare and predicted to occur in only four pre-mRNAs. trans- and cis-splicing reactions are carried out by the spliceosome, which consists of U-rich small nuclear ribonucleoprotein particles (U snRNPs) and of non-snRNP factors. Mammalian and yeast spliceosome complexes are well characterized and found to be associated with up to 170 proteins. Despite the central importance of trans splicing in trypanosomatid gene expression, only the core RNP proteins and a few snRNP-specific proteins are known. To characterize the trypanosome spliceosomal protein repertoire, we conducted a proteomic analysis by tagging and tandem affinity-purifying the canonical core RNP protein SmD1 in Trypanosoma brucei and by identifying copurified proteins by mass spectrometry. The set of 47 identified proteins harbored nearly all spliceosomal snRNP factors characterized in trypanosomes thus far and 21 proteins lacking a specific annotation. A bioinformatic analysis combined with protein pull-down assays and immunofluorescence microscopy identified 10 divergent orthologues of known splicing factors, including the missing U1-specific protein U1A. In addition, a novel U5-specific, and, as we show, an essential splicing factor was identified that shares a short, highly conserved N-terminal domain with the yeast protein Cwc21p and was thus tentatively named U5-Cwc21. Together, these data strongly indicate that most of the identified proteins are components of the spliceosome.

[Salivary and lacrymal gland disorders and HTLV-1 infection]. / Atteinte des glandes salivaires et lacrymales et infection par le HTLV-1.

INTRODUCTION: The frequency and severity of salivary and lacrymal gland human T-cell lymphotropic virus type 1 (HTLV-1) infection were assessed in HTLV-1 plus patients, presenting with neurological deficit (tropical spastic paraparesis/HTLV-1 associated myelopathy [TSP/HAM]) or not. The mechanism of this deficit was investigated. MATERIAL AND METHODS: A case-control study was made from April 2002 to December 2005, in an area strongly endemic for HTLV-1. The patients were classified in three groups: group 1 with 16 patients presenting with TSP/HAM; group 2 with 67 HTLV-1 carriers and group 3 with 29 healthy volunteers. The dry syndrome was investigated by history taking and by oral and ophthalmological clinical examination. Immunological and biological screening for rhumatoid factors, antinuclear antibodies, and antibodies against soluble nuclear antigens (SSA, SSB). Peripheral blood was separated by density gradient and mononuclear cells were recovered to dose interferon-gamma and tumor necrosis factor-alpha. Patients in the three groups were assessed for salivary flow by stimulated weighing using Saxon's test. A Chi-2 test, a variance analysis (Anova), and the Spearman rank correlation test were used for the statistical analysis. RESULTS: The dry syndrome was mild and more common in group 1 patients (75%). In group 2, 22% of the patients presented with functional signs of buccal mucosa dryness comparable to those observed in group 1. No correlation was found between salivary flow and screened pro-inflammatory cytokines. DISCUSSION: Our results show that hyposialia is an important part of the disease induced by HTLV-1, even in virus carriers without neurological deficit. Its mechanism seems different than that of the Gougerot-Sjögren syndrome.

Oral manifestations of Sjögren's syndrome.

Sjögren's syndrome is a common autoimmune rheumatic disease. The most common symptoms of Sjögren's syndrome are extreme tiredness, along with dry eyes (keratoconjunctivitis sicca) and dry mouth (xerostomia). Saliva plays an essential role in numerous functions of the mouth. Xerostomia can be caused by medications, chronic diseases like Sjögren's syndrome, and medical treatments, such as radiation therapy and bone marrow transplant. Xerostomia can eventually lead to difficulty in swallowing, severe and progressive tooth decay, or oral infections. Despite having excellent oral hygiene, individuals with Sjögren's syndrome have elevated levels of dental caries, along with the loss of many teeth, early in the disease. Sjögren's syndrome alters the protein profile and brings about a change in the composition of saliva. There is an increase in the levels of lactoferrin, beta(2)-microglobulin, sodium, lysozyme C, and cystatin C, and a decrease in salivary amylase and carbonic anhydrase. Up to 90% of individuals with Sjögren's syndrome have antibodies targeting the Ro 60 and La autoantigens. Natural aging, regardless of Sjögren's syndrome, is also another factor that brings about a significant change in the composition of saliva. The most prevailing cause of xerostomia in elderly persons is the use of anticholinergic medications. Currently, there is no cure for Sjögren's syndrome, and treatment is mainly palliative.

PABPN1 polyalanine tract deletion and long expansions modify its aggregation pattern and expression.

Expansions of a (GCN)10/polyalanine tract in the Poly(A) Binding Protein Nuclear 1 (PABPN1) cause autosomal dominant oculopharyngeal muscular dystrophy (OPMD). In OPMD muscles, as in models, PABPN1 accumulates in intranuclear inclusions (INIs) whereas in other diseases caused by similar polyalanine expansions, the mutated proteins have been shown to abnormally accumulate in the cytoplasm. This study presents the impact on the subcellular localization of PABPN1 produced by large expansions or deletion of its polyalanine tract. Large tracts of more than 24 alanines result in the nuclear accumulation of PABPN1 in SFRS2-positive functional speckles and a significant decline in cell survival. These large expansions do not cause INIs formation nor do they lead to cytoplasmic accumulation. Deletion of the polyalanine tract induces the formation of aggregates that are located on either side and cross the nuclear membrane, highlighting the possible role of the N-terminal polyalanine tract in PABPN1 nucleo-cytoplasmic transport. We also show that even though five other proteins with polyalanine tracts tend to aggregate when over-expressed they do not co-aggregate with PABPN1 INIs. This study presents the first experimental evidence that there may be a relative loss of function in OPMD by decreasing the availability of PABPN1 through an INI-independent mechanism.