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The dihydrolipoamide acetyltransferase subunit DLA2 of the chloroplast pyruvate dehydrogenase complex (cpPDC) in the green alga Chlamydomonas reinhardtii has previously been shown to possess moonlighting activity in chloroplast gene expression. Under mixotrophic growth conditions, DLA2 forms part of a ribonucleoprotein particle (RNP) with the psbA mRNA that encodes the D1 protein of the photosystem II (PSII) reaction center. Here, we report on the characterization of the molecular switch that regulates shuttling of DLA2 between its functions in carbon metabolism and D1 synthesis. Determination of RNA-binding affinities by microscale thermophoresis demonstrated that the E3-binding domain (E3BD) of DLA2 mediates psbA-specific RNA recognition. Analyses of cpPDC formation and activity, as well as RNP complex formation, showed that acetylation of a single lysine residue (K197) in E3BD induces the release of DLA2 from the cpPDC, and its functional shift towards RNA binding. Moreover, Förster resonance energy transfer microscopy revealed that psbA mRNA/DLA2 complexes localize around the chloroplast's pyrenoid. Pulse labeling and D1 re-accumulation after induced PSII degradation strongly suggest that DLA2 is important for D1 synthesis during de novo PSII biogenesis.

MCTS1 Re-Initiation and Release Factor (MCTS1) has been characterised as an oncoprotein in some cancers. In this study, we explored the expression of MCTS1 in laryngeal squamous cell carcinoma (LSCC) and its regulatory effects on the proliferation and cell-cycle progression of tumour cells, as well as the underlying mechanisms. The data from the Cancer Genome Atlas was used to analyse MCTS1 expression and its correlation with survival outcomes in LSCC patients. Subsequent in vitro cellular and molecular studies were performed based on representative LSCC cell lines. Results showed that the upregulation of MCTS1 in LSCC is linked to poor progression-free survival (PFS) and disease-specific survival (DSS). In TU177 and AMC-HN-8 cells, MCTS1 exerted positive regulations on cell viability, colony formation, cell cycle progression, and the expression of CDK1, CDK2, cyclin A2, and cyclin B1. Co-IP assay confirmed mutual interaction between MCTS1 and LARP7, mainly in the cytoplasm. Cycloheximide (CHX) chase and co-IP assay of ubiquitination showed that MCTS1 could increase LARP7 protein half-life and reduce its poly-ubiquitination. LARP7 overexpression enhanced the viability and colony formation of LSCC cells and also elevated the expression of CDK1, CDK2, cyclin A2, and cyclin B1. In addition, its overexpression partly reversed the negative influence of MCTS1 knockdown. In summary, this study confirmed that the expression of MCTS1 might be an indicator of unfavourable prognosis for patients with LSCC. Mechanically, it promotes LSCC cell viability and proliferation via interacting with LARP7 and reducing its proteasomal-mediated degradation.

Cardiomyocyte dysfunction and apoptosis induced by ischemia-hypoxia are common features of many acute and chronic heart diseases. WW domain-containing E3 ubiquitin ligase (WWP2) has been identified as an important regulator in pathogenesis of some health-threatening diseases. Although a couple of recent reports prompted the potential role of WWP2 in heart dysfunction, however, its exact role and how its expression was regulated in ischemic-hypoxic cardiomyocytes are still elusive. Here, we found that WWP2 protein level was induced in anoxia/reoxygenation (A/R) treated cardiomyocytes in a time-dependent manner, accompanied by synchronous expression of LINC01588 and HNRNPL. Knockdown of LINC01588 increased cardiomyocyte apoptosis, the level of oxidative stress, and expression of pro-inflammatory cytokine genes, down-regulated the expression of WWP2 and promoted expression of SEPT4 gene that contributed to cardiomyocyte dysfunction and was a target gene of WWP2. LINC01588 overexpression improved the functions of A/R treated cardiomyocytes, up-regulated WWP2 and reduced SEPT4 expression. In the mechanism exploration, we found that LINC01588 could directly bind with HNRNPL protein that could interact with WWP2, suggesting that WWP2 was involved in the regulation of LINC01588 in A/R treated cardiomyocytes. Moreover, WWP2 inhibition declined the protective role of LINC01588 in cardiomyocyte dysfunction induced by A/R. Finally, we demonstrated that LINC01588 overexpression improved acute myocardial infarction in mice in vivo. In conclusion, LINC01588 improved A/R-induced cardiomyocyte dysfunction by interacting with HNRNPL and promoting WWP2-mediated degradation of SEPT4.

Persistent intranuclear infection is an uncommon infection strategy among RNA viruses. However, Borna disease virus 1 (BoDV-1), a nonsegmented, negative-strand RNA virus, maintains viral infection in the cell nucleus by forming structured aggregates of viral ribonucleoproteins (vRNPs), and by tethering these vRNPs onto the host chromosomes. To better understand the nuclear infection strategy of BoDV-1, we determined the host protein interactors of the BoDV-1 large (L) protein. By proximity-dependent biotinylation, we identified several nuclear host proteins interacting with BoDV-1 L, one of which is TRMT112, a partner of several RNA methyltransferases (MTase). TRMT112 binds with BoDV-1 L at the RNA-dependent RNA polymerase domain, together with BUD23, an 18S rRNA MTase and 40S ribosomal maturation factor. We then discovered that BUD23-TRMT112 mediates the chromosomal tethering of BoDV-1 vRNPs, and that the MTase activity is necessary in the tethering process. These findings provide us a better understanding on how nuclear host proteins assist the chromosomal tethering of BoDV-1, as well as new prospects of host-viral interactions for intranuclear infection strategy of orthobornaviruses. This article is protected by copyright. All rights reserved.

Effective and precise gene editing of T lymphocytes is critical for advancing the understanding of T cell biology and the development of next-generation cellular therapies. Although methods for effective CRISPR/Cas9-mediated gene knock-out in primary human T cells have been developed, complementary techniques for nonviral gene knock-in can be cumbersome and inefficient. Here, we report a simple and efficient method for nonviral CRISPR/Cas9-based gene knock-in utilizing plasmid-based donor DNA templates. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Purification of human CD4

Although most gastrointestinal stromal tumors (GISTs) exhibit activating mutations in either KIT or PDGFRA, rare cases have shown to be driven by gene fusions involving kinases, mainly involving NTRK3, and rarely BRAF or FGFR1. BRAF gene rearrangements have been described in only 2 patients to date, as separate case reports. In addition, BRAF V600E mutation is an uncommon but established oncogenic pathway in GIST. In this report, we describe two new GIST cases harboring novel BRAF fusion genes, arising in 2 young-adult women (37 and 40 years of age) in the small bowel and distal esophagus, both with a spindle cell phenotype. The small bowel GIST measured 2.8 cm and showed a high cellularity and a mitotic rate of 20/50 HPFs, while the esophageal lesion measured 7 cm and 1/50 HPFs. Immunohistochemically, both tumors showed diffuse reactivity for DOG1, while KIT/CD117 was weakly positive in the small bowel GIST and completely negative in the esophageal tumor. Based on these findings, the latter case was misinterpreted as a low grade myxoid leiomyosarcoma, as it showed a myxoid stroma, reactivity for SMA and focal positivity for desmin. Archer FusionPlex revealed a fusion between BRAF with either AGAP3 or MKRN1 gene partners. Moreover, MSK-IMPACT DNA targeted sequencing confirmed both fusions but did not identify additional mutations. In one case with available material, the BRAF gene rearrangement was also validated by FISH. The recognition of BRAF fusion-positive GISTs is critical as it may be associated with a low level of KIT expression and may result in diagnostic challenges with significant impact on therapeutic management. The clinical benefit with KIT inhibitors, such as imatinib, remains to be determined. This article is protected by copyright. All rights reserved.

Clinically, patients with urothelial carcinoma of the bladder (UCB) with tumor metastasis are incurable. To find new therapeutic strategies, the mechanisms underlying UCB invasion and metastasis should be further investigated. In this study, zinc finger and homeobox 3 (ZHX3) was first screened as a critical oncogenic factor associated with poor prognosis in a UCB dataset from The Cancer Genome Atlas (TCGA). These results were also confirmed in a large cohort of clinical UCB clinical samples. Next, we found that ZHX3 could promote the migration and invasion capacities of UCB cells both in vitro and in vivo. Mechanistically, coimmunoprecipitation (coIP) and mass spectrometry (MS) analysis indicated that ZHX3 was a target of tripartite motif 21 (TRIM21), which mediates its ubiquitination, and subsequent degradation. Notably, RNA‐seq analysis showed that ZHX3 repressed the expression of regulator of G protein signaling 2 (RGS2). Generally, our results suggest that ZHX3 plays an oncogenic role in UCB pathogenesis and might serve as a novel therapeutic target for UCB., ZHX3 promotes UCB cell invasive and metastatic capacities. ZHX3 could activate RhoA by repressing RGS2. TRIM21 promotes ZHX3 ubiquitination and degradation in UCB cells.

Processing bodies (PBs) are 100-300 nm cytoplasmic mRNP granules that regulate eukaryotic gene expression. These cytoplasmic compartments harbor mRNAs and several proteins involved in mRNA decay, miRNA silencing, nonsense-mediated mRNA decay, and splicing. Though membrane-less, PB structures are maintained by RNA-protein and protein-protein interactions. PB proteins have intrinsically disordered regions and low complexity domains, which account for its liquid to liquid phase separation. In addition to being dynamic and actively involved in the exchange of materials with other mRNPs and organelles, they undergo changes on various cellular cues and environmental stresses, including viral infections. Interestingly, several PB proteins are individually implicated in cancer development, and no study has addressed the effects on PB dynamics after epigenetic modifications of cancer-associated PB genes. In the current review, we summarise modulations undergone by P bodies or P body components upon viral infections. Furthermore, we discuss the selective and widely investigated PB proteins that undergo methylation changes in cancer and their potential as biomarkers. This article is protected by copyright. All rights reserved.

Objective The aim of this study is to describe 4 of the most common autoantibodies against components of the Th/To complex: human POP1 (hPOP1), RPP25, RPP30, and RPP40. We report their prevalence and clinical characteristics in a systemic sclerosis (SSc) population, and determine whether these specificities are associated with cancer. Methods A case-control study was performed using data from the Johns Hopkins Scleroderma Center Cohort. A total of 804 adult patients with SSc were included; 401 SSc patients with no history of cancer after at least 5 years of disease were compared to 403 SSc patients who ever had a history of cancer. Antibodies against hPOP1, RPP25, RPP30, and RPP40 were assayed by immunoprecipitation of 35 S-methionine-labeled proteins generated by in vitro transcription/translation. Demographic and clinical characteristics were compared between groups. Results Of 804 patients, 67 (8.3%) had antibodies against any component of the Th/To complex. Patients with antibodies to any component were significantly more likely to have limited cutaneous disease, less likely to have tendon friction rubs, and more likely to have findings consistent with interstitial lung disease or pulmonary hypertension. Patients with antibodies against hPOP1, RPP25, RPP30, and/or RPP40 were significantly less likely to develop cancer within 2 years of SSc onset (0% versus 11% of antibody-negative patients; P = 0.009). Conclusion SSc patients who produce autoantibodies to components of the Th/To complex have a clinical phenotype characterized by limited cutaneous disease and pulmonary involvement. Our findings show that the presence of any Th/To autoantibody may have a protective effect against contemporaneous cancer.

Fragile X syndrome and other fragile X-associated disorders are caused by the full-mutation (200 copies) and premutation (55 to 200 copies) expansion, respectively, of the CGG short tandem repeat in the fragile X messenger ribonucleoprotein 1 (FMR1) gene. Clinical diagnostic laboratories use Southern blot analysis and polymerase chain reaction (PCR)-based tests to detect and/or size the FMR1 CGG repeats. The development of sensitive and high-throughput triplet-primed PCR (TP-PCR) assays has diminished the need to subject all samples to Southern blot analysis, which is both labor- and time-intensive. In this article, we describe two direct TP-PCR (dTP-PCR) assays for the detection of FMR1 CGG repeat expansions. We outline a protocol that is based on melting curve analysis of dTP-PCR amplicons for a rapid and cost-effective first-tier screening and identification of individuals with premutation and full-mutation expansions. We also describe a protocol that employs capillary electrophoresis to resolve the dTP-PCR amplicon fragments and to estimate the repeat sizes of normal (5 to 44 copies), intermediate (45 to 54 copies), and premutation alleles, as well as to detect full mutations and determine the structure of the FMR1 alleles. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Direct triplet-primed PCR master mix preparation and amplification of the FMR1 CGG repeat locus for melting curve analysis Basic Protocol 2: Melting curve analysis of direct triplet-primed PCR amplicons on the Rotor-Gene Q MD × 5plex high-resolution melt platform Alternate Protocol: Melting curve analysis of direct triplet-primed PCR amplicons on the LightCycler 480 system Basic Protocol 3: Generation of direct triplet-primed PCR melting curve analysis profiles Basic Protocol 4: Direct triplet-primed PCR master mix preparation and amplification of the FMR1 CGG repeat locus for capillary electrophoresis Basic Protocol 5: Generation of control FMR1 plasmids for direct triplet-primed PCR melting curve analysis Basic Protocol 6: Sanger sequencing assay to verify FMR1 CGG repeat size and structure of plasmid DNA controls.

The sorting of RNAs to specific regions of the cell for local translation represents an important mechanism directing protein distribution and cell compartmentalization. While significant progress has been made in understanding the mechanisms underlying the transport and localization of mRNAs, the mechanisms governing ribosome mobilization are less well understood. Ribosomes present in the cytoplasm of multiple cell types can form ribonucleoprotein complexes that also contain myosin-Va (Myo5a), a processive, actin-dependent molecular motor. Here, we report that Myo5a can be disassociated from ribosomes when ribonucleoprotein complexes are exposed to calcium, both in vitro and in vivo. We suggest that Myo5a may act as a molecular switch able to anchor or release ribosomes from the actin cytoskeleton in response to intracellular signaling.

SUMMARY In recent years, research has increasingly focused on the key role of post‐transcriptional regulation of messenger ribonucleoprotein (mRNP) function and turnover. As a result of the complexity and dynamic nature of mRNPs, the full composition of a single mRNP complex remains unrevealed and mRNPs are poorly described in plants. Here we identify canonical Sm proteins as part of the cytoplasmic mRNP complex, indicating their function in the post‐transcriptional regulation of gene expression in plants. Sm proteins comprise an evolutionarily ancient family of small RNA‐binding proteins involved in pre‐mRNA splicing. The latest research indicates that Sm could also impact on mRNA at subsequent stages of its life cycle. In this work we show that in the microsporocyte cytoplasm of Larix decidua, the European larch, Sm proteins accumulate within distinct cytoplasmic bodies, also containing polyadenylated RNA. To date, several types of cytoplasmic bodies involved in the post‐transcriptional regulation of gene expression have been described, mainly in animal cells. Their role and molecular composition in plants remain less well established, however. A total of 222 mRNA transcripts have been identified as cytoplasmic partners for Sm proteins. The specific colocalization of these mRNAs with Sm proteins within cytoplasmic bodies has been confirmed via microscopic analysis. The results from this work support the hypothesis, that evolutionarily conserved Sm proteins have been adapted to perform a whole repertoire of functions related to the post‐transcriptional regulation of gene expression in Eukaryota. This adaptation presumably enabled them to coordinate the interdependent processes of splicing element assembly, mRNA maturation and processing, and mRNA translation regulation, and its degradation., Significance Statement The study reports the cyclic occurrence of cytoplasmic mRNP accumulations enriched in canonical Sm proteins but not in other spliceosomal components in Larix decidua microsporocytes. Based on transcriptomic analysis, 222 mRNAs were identified as cytoplasmic partners for Sm proteins, which were linked to many gene ontology terms. The results presented show that S‐bodies constitute newly described cytoplasmic domains involved in the post‐transcriptional regulation of highly expressed transcripts, particularly in cells in which mRNA synthesis occurs in transcriptional bursts.

Objective The present study investigates the peripheral neuropathy in Primary Sjögren's syndrome (pSS) using the nerve excitability test to further elucidate how peripheral nerves are affected by the autoantibodies. Methods Each patient received clinical evaluation, examination for anti‐SSA/Ro and anti‐SSB/La antibodies titer, paired motor and sensory nerve excitability test, thermal quantitative sensory test (QST), and nerve conduction study (NCS). Results A total of 40 pSS patients wasenrolled. Motor axonal study of the pSS with positive anti‐SSA/Ro or anti‐SSB/La antibodies (n = 28) was found to have increased stimulus for 50% compound muscle action potential (CMAP) (P

Our previous studies have indicated that long noncoding RNA (lncRNA) SPRY4 intronic transcript 1 (SPRY4-IT1) was highly expressed in hepatocellular carcinoma (HCC). However, it still remained unclear how SPRY4-IT1 worked in tumorgenesis in HCC. In this study, we tested the overexpression of SPRY4-IT1 in HCC tissues and cells through a quantitative real-time polymerase chain reaction. Statistical analyses showed that the upregulation had an association with the tumor node metastasis stage, thrombin time, and alkaline phosphatase. Furthermore, SPRY4-IT1 could be involved in cell proliferation, metastasis, and the epithelial-to-mesenchymal transition (EMT) process in HCC in vitro and in vivo. RNA-sequencing and transcriptome analysis were carried out to explore the mechanism of SPRY4-IT1 in HCC. With SPRY4-IT1 being knocked down or overexpressed, the level of proteins in the tumor necrosis factor (TNF) signaling pathway changed. We detected the RNA binding protein heterogeneous nuclear ribonucleoprotein L (HNRNPL) as a SPRY4-IT1 interacting protein through RNA pull-down assay and liquid chromatography-mass spectrometry, then verified through RNA immunoprecipitation. Downregulation of HNRNPL induced the change of proteins observed on SPRY4-IT1 downregulation revealing the SPRY4-IT1: HNRNPL complex in the TNF signaling pathway and EMT process in HCC. In general, our experimental data and analysis demonstrated the role of SPRY4-IT1 in promoting progress and metastasis of HCC by the TNF signaling pathway.

Osteosarcoma (OS) is the most frequent type of cancer that starts in the bones, with a rather high tendency to metastasize to other bones at the early stages. Although many types of research have demonstrated that long noncoding RNAs commonly take part in the development of various cancers, the modulating mechanism of LEF1-AS1 in OS was unknown yet. In this study, our results disclosed that LEF1-AS1, as well as LEF1, had higher expression levels in OS cells than that in normal bone cells. LEF1-AS1 knockdown dramatically inhibited the proliferation, migration, as well as invasion in OS, which proved that LEF1-AS1 contributed to the growth of OS. Furthermore, HNRNPL knockdown suppressed the expression of LEF1. LEF1-AS1 was confirmed to sponge HNRNPL and HNRNPL could bind with LEF1. Both LEF1-AS1 and HNRNPL could enhance the stability of LEF1 mRNA. LEF1-AS1 acted as a promoter in stimulating the Wnt signaling pathway in OS. In rescue experiments, overexpression of LEF1 partially offset the inhibition LEF1-AS1 knockdown brought in the proliferation, migration as well as invasion of OS cells. Collectively, this study had investigated that LEF1-AS1 bound with HNRNPL to promote OS cell proliferation, migration as well as invasion by enhancing the messenger RNA stability of LEF1.

Background Parkinson's disease (PD) is a common movement disorder for which diagnosis mainly depends on the medical history and clinical symptoms. Exosomes are now considered an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids, and genetic material. Long noncoding (lnc) RNA in exosomes plays a critical role in many diseases, including neurodegenerative disease. Aim To study expression differences for lncRNAs in peripheral blood exosomes of PD patients compared with healthy individuals and to look for lncRNAs that might be related to the pathogenesis of PD. Materials and Methods We recruited PD patients along with age‐ and sex‐matched healthy individuals as healthy controls and evaluated levels of lncRNAs extracted from exosomes in plasma samples via next‐generation sequencing and real‐time quantitative PCR. Correlation analysis was conducted for the clinical characteristics of PD patients and the expression of selected lncRNAs. Results We found 15 upregulated and 24 downregulated exosomal lncRNAs in the PD group. According to bioinformatics analyses, we chose lnc‐MKRN2‐42:1 for further study. Interestingly, lnc‐MKRN2‐42:1 was positively correlated with the MDS‐UPDRS III score for PD patients. Conclusion Our study suggested that lnc‐MKRN2‐42:1 may be involved in the occurrence and development of PD.

Telomerase has an established role in telomere maintenance in eukaryotes. However, recent studies have begun to implicate telomerase in cellular roles beyond telomere maintenance. Specifically, evidence is emerging of cross-talks between telomerase mediated telomere homeostasis and DNA repair pathways. Telomere shortening due to the end replication problem is a constant threat to genome integrity in eukaryotic cells. This poses a particular problem in unicellular parasitic protists because their major virulence genes are located at the subtelomeric loci. Although telomerase is the major regulator of telomere lengthening in eukaryotes, it is less studied in the ancient eukaryotes, including clinically important human pathogens. Recent research is highlighting interplay between telomerase and the DNA damage response in human parasites. The importance of this interplay in pathogen virulence is only beginning to be illuminated, including the potential to highlight novel developmental regulation of telomerase in parasites who transition between multiple developmental stages throughout their life cycle. In this review, we will discuss the telomerase ribonucleoprotein enzyme and DNA repair pathways with emerging views in human parasites to give a broader perspective of the possible connection of telomere, telomerase, and DNA repair pathways across eukaryotic lineages and highlight their potential role in pathogen virulence. This article is categorized under: RNA Structure and DynamicsInfluence of RNA Structure in Biological Systems RNA Evolution and GenomicsRNA and Ribonucleoprotein Evolution RNA Interactions with Proteins and Other MoleculesProtein-RNA Interactions: Functional Implications.

Background Previous studies have demonstrated that Ro60 and Ro52 have different clinical implications, and anti‐Ro52 antibodies are an independent serum marker of systemic autoimmune diseases, including Sjögren's syndrome. Many different assays have been adopted to detect anti‐Sjögren's syndrome antigen A (SSA)/Ro antibodies, while to date no specific approach has been recommended as optimal for anti‐SSA/Ro antibody testing. Herein, we performed a multi‐center study to explore the current clinical utility of different strategies for anti‐SSA/Ro antibody testing in China. Methods Twenty‐one tertiary care centers were included in this questionnaire‐based study. The self‐administered questionnaire mainly includes testing methods for anti‐SSA/Ro antibodies, reporting system of results, and interpretation of results by clinicians. Results Six different methods were applied to detect anti‐SSA/Ro antibodies in the 21 centers. Line immunoassay (eight different commercial kits) was the most frequently adopted method (21/21, 100%), with different cutoff values and strategies for intensity stratification. There were two reporting systems: One was reported as “anti‐SSA antibodies” and “anti‐Ro52 antibodies” (12/21, 57%), while the other was “anti‐SSA/Ro60 antibodies” and “anti‐SSA/Ro52 antibodies” (9/21, 43%). Notably, six centers (29%) considered either positive anti‐Ro60 or anti‐Ro52 antibodies as positive anti‐SSA antibodies, all of which adopted the latter reporting system. Conclusion Significant variabilities existed among anti‐SSA/Ro assays. Nearly 30% of centers misinterpreted the definition of positive anti‐SSA antibodies, which may be attributed to the confusing reporting systems of line immunoassay. Therefore, we advocate standardization of the nomenclature of anti‐SSA/Ro antibodies, changing the “anti‐SSA/Ro52” label in favor of the “anti‐Ro52” antibodies for a clear designation., Significant variabilities existed among anti‐SSA/Ro assays, with LIA as the most common used method. Nearly one‐third of the centers misinterpreted the definition of positive anti‐SSA antibodies, which may be attributed to the confusing reporting systems of LIA. We advocate changing the “anti‐SSA/Ro52” label in favor of “anti‐Ro52 antibodies” for a clear designation

Background Pineal cyst is a benign lesion commonly occurring in people of any age. Until now, the underlying molecular alterations have not been explored. Methods We performed whole exome sequencing of 93 germline samples and 21 pineal cyst tissue samples to illustrate its genetic architecture and somatic mutations. The dominant and recessive inheritance modes were considered, and a probability was calculated to evaluate the significance of variant overrepresentation. Results By analyzing pineal cyst as a Mendelian disease with a dominant inheritance pattern, we identified 42,325 rare germline variants, and NM_001004711.1:c.476A>G was highly enriched (FDRT, was overrepresented (FDR, Whole exome sequencing of 93 germline samples and 21 pineal cyst tissue samples was performed to study the molecular mechanism of pineal cysts. A list of overrepresented germline genetic variants and somatic mutations were identified for future validation. ​

Protein arginine methyltransferase 1 (PRMT1) stimulates erythroid differentiation, but the signaling events upstream are yet to be identified. Ca2+ plays crucial roles during erythroid differentiation. Here, we show that Ca2+ enhances methylation during induced erythroid differentiation and that Ca2+ directly upregulates the catalytic activity of recombinant PRMT1 by increasing Vmax toward the substrate heterogeneous nuclear ribonucleoprotein A2. We demonstrate that PRMT1 is essential and responsible for the effect of Ca2+ on differentiation. Depletion of Ca2+ suppresses PRMT1-mediated activation of p38α and p38α-stimulated differentiation. Furthermore, Ca2+ stimulates methylation of p38α by PRMT1. This study uncovers a novel regulatory mechanism for PRMT1 by Ca2+ and identifies the PRMT1/p38α axis as an intracellular mediator of Ca2+ signaling during erythroid differentiation.

Backgrounds To explore the expression and functions of the tripartite motif-containing protein 21 (TRIM21) in oral lichen planus(OLP) lesions. Methods Paraffin sections of buccal mucosa samples from 15 cases of reticular oral lichen planus (OLP) patients and 10 healthy controls were used for immunohistochemistry to determine expression and distribution of TRIM21. Buccal mucosae from 11 OLP patients and seven healthy controls were analyzed by qPCR to quantify its gene expression. Peripheral blood mononuclear cells and CD3+ cells from four pairs of age- and sex-matched OLP patients and healthy controls were isolated for immunocytochemistry and culture. Following lentivirus-mediated overexpression of TRIM21 gene in CD3+ cells, CCK-8 was applied to evaluate cell proliferation. Cytokines including IL-2, IL-4, IL-5, IL-6, IL-10, TNF-α, and IFN-γ in the supernatants were measured by the cytometric bead array and verified by ELISA. Results A larger number of TRIM21-positive cells infiltrating the lamina propria were observed in OLP lesions by immunohistochemistry than those of healthy controls. Significantly higher transcription of TRIM21 was revealed by qPCR. TRIM21 overexpression in CD3+ cells significantly enhanced the proliferation and IL-6 secretion in CD3+ cells from 12 to 72 hours. Conclusion Overexpressed TRIM21 in OLP may be a primary proinflammatory molecule rather than a secondary and inducible regulatory factor in immunopathogenesis of OLP.

Cartilage hair hypoplasia (CHH), anauxetic dysplasia 1, and anauxetic dysplasia 2 are rare metaphyseal dysplasias caused by biallelic pathogenic variants in RMRP and POP1, which encode the components of RNAse-MRP endoribonuclease complex (RMRP) in ribosomal biogenesis pathway. Nucleolus and neural progenitor protein (NEPRO), encoded by NEPRO (C3orf17), is known to interact with multiple protein subunits of RMRP. We ascertained a 6-year-old girl with skeletal dysplasia and some features of CHH. RMRP and POP1 did not harbor any causative variant in the proband. Parents-child trio exomes revealed a candidate biallelic variant, c.435G>C, p.(Leu145Phe) in NEPRO. Two families with four affected individuals with skeletal dysplasia and a homozygous missense variant, c.280C>T, p.(Arg94Cys) in NEPRO, were identified from literature and their published phenotype was compared in detail to the phenotype of the child we described. All the five affected individuals have severe short stature, brachydactyly, skin laxity, joint hypermobility, and joint dislocations. They also have short metacarpals, broad middle phalanges, and metaphyseal irregularities. Protein modeling and stability prediction showed that the mutant protein has decreased stability. Both the reported variants are in the same domain of the protein. Our report delineates the clinical and radiological characteristics of an emerging ribosomopathy caused by biallelic variants in NEPRO.

Apoptotic chromatin condensation inducer in the nucleus (Acinus) is an RNA-binding protein that has a functional role in inducing apoptotic chromatin condensation and regulating messenger RNA (mRNA) processing. Acinus interacts with the spliceosomal machinery and is a member of the ASAP (apoptosis and splicing-associated protein complex) as well as the EJC (exon junction complex), which gets deposited onto mRNA during splicing. In this study, we have used in vivo splicing assays to characterize the function of Acinus in pre-mRNA splicing more closely. We show that full-length Acinus-S', an isoform of Acinus, does not have a role in modulating splice site selection in human immunodeficiency virus 1 minigene reporter system. In contrast, we observed that the tethering of arginine/serine (RS) and RNPS1-SAP18-binding (RSB) domains of Acinus could regulate the selection of alternative splice sites, thereby revealing the potential of Acinus in stimulating alternative splicing. Altogether, our data suggest that the RS and RSB domains play a critical role in regulating splicing activity via selection of distinct splice sites during pre-mRNA splicing.

The autoantigen La protein is an important component of telomerase and a predominantly nuclear phosphoprotein. As a telomerase subunit, La protein associates with the telomerase ribonucleoprotein and influences telomere length. In the reverse transcription, La protein stimulates enzymatic activity and increases repeated addition processivity of telomerase. As nuclear phosphoprotein, La protein binds the 3' poly(U)-rich elements of nascent RNA polymerase III transcripts to facilitate its correct folding and maturation. In this work, we identified a La protein homolog (TbLa) from Trypanosoma brucei (T. brucei). We revealed that TbLa interacts with ribosome-associated protein P34/P37, 40S ribosomal protein SA, and 60S ribosomal subunit L5 in T. brucei. In the interactions between TbLa protein and (P34/P37)/L5/SA, RNA recognition motif (RRM) domain of TbLa was indicated to make the major contribution to the processes. We determined the solution structure of TbLa RRM domain. NMR chemical shift perturbations revealed that the positively charged RNA-binding pocket of TbLa RRM domain is responsible for its interaction with ribosomal and ribosome-associated proteins P37/L5/SA. Furthermore, depletion of TbLa affected the maturation process of 5S rRNA and ribosomal assembly, suggesting TbLa protein might play a significant role in the ribosomal biogenesis pathway in T. brucei. Taken together, our results provide a novel insight and structural basis for better understanding the roles of TbLa and RRM domain in ribosomal biogenesis in T. brucei. DATABASE: Structural data are available in the PDB under the accession number 5ZUH.

Prothymosin‐α (PTMA) is a small, acidic protein that is usually transported into the nucleus and involves many cellular and immunological functions. Previous studies demonstrated that aberrant location of PTMA expression exists in human bladder cancer, but the role of PTMA protein expression remains elusive. In this study, we created ectopic nuclear or cytoplasmic PTMA expression in human bladder cancer cells by infecting lentiviruses carrying wild type or deleted nuclear localization signal of the PTMA gene. The in vivo tumorigenesis assay showed PTMA protein with deleted nuclear localization signal promotes J82 xenograft tumor growth in mice and shortens their survival more so than the wild type. Chromatin immunoprecipitation showed that wild‐type PTMA protein binds to the PTEN promoter and enhances phosphatase and tensin homolog (PTEN) expression. Through immunoblot proteomics and in vivo ubiquitination studies, PTMA protein can bind with tripartite motif‐containing protein 21 (TRIM21) and block its ubiquitination. Also, TRIM21 can downregulate both forms of PTMA protein. In human bladder tumors, loss of nuclear PTMA expression was an unfavorable prognostic indicator for shorter disease‐free survival (hazard ratio, 1.54; P = 0.009). Our data support that nuclear PTMA protein serves as a tumor suppressor in bladder cancer through upregulating PTEN and orchestrating TRIM21 for the regulation of Nrf2 signaling.

Ibrutinib is a small molecule drug that targets Bruton's tyrosine kinase in B‐cell malignancies and is highly efficient at killing mantle cell lymphoma and chronic lymphocytic leukemia. However, the anti‐cancer activity of ibrutinib against solid tumors, such as non‐small cell lung cancer (NSCLC), remains low. To improve the cytotoxicity of ibrutinib towards lung cancer, we synthesized a series of ibrutinib derivatives, of which Ibr‐7 exhibited superior anti‐cancer activity to ibrutinib, especially against epithelial growth factor receptor (EGFR) wild‐type NSCLC cell lines. Ibr‐7 was observed to dramatically suppress the mammalian target of Rapamycin complex 1 (mTORC1)/S6 signaling pathway, which is only slightly affected by ibrutinib, thus accounting for the superior anti‐cancer activity of Ibr‐7 towards NSCLC. Ibr‐7 was shown to overcome the elevation of Mcl‐1 caused by ABT‐199 mono‐treatment, and thus exhibited a significant synergistic effect when combined with ABT‐199. In conclusion, we used a molecular substitution method to generate a novel ibrutinib derivative, termed Ibr‐7, which exhibits enhanced anti‐cancer activity against NSCLC cells as compared with the parental compound.

Stress granules (SGs) are ribonucleoprotein (RNP) assemblies that form in eukaryotic cells as a result of limited translation in response to stress. SGs form during viral infection and are thought to promote the antiviral response because many viruses encode inhibitors of SG assembly. However, the antiviral endoribonuclease RNase L also alters SG formation, whereby only small punctate SG-like bodies that we term RNase L-dependent bodies (RLBs) form during RNase L activation. How RLBs relate to SGs and their mode of biogenesis is unknown. Herein, using immunofluorescence, live-cell imaging, and MS-based analyses, we demonstrate that RLBs represent a unique RNP granule with a protein and RNA composition distinct from that of SGs in response to dsRNA lipofection in human cells. We found that RLBs are also generated independently of SGs and the canonical dsRNA-induced SG biogenesis pathway, because RLBs did not require protein kinase R, phosphorylation of eukaryotic translation initiation factor 2 subunit 1 (eIF2α), the SG assembly G3BP paralogs, or release of mRNAs from ribosomes via translation elongation. Unlike the transient interactions between SGs and P-bodies, RLBs and P-bodies extensively and stably interacted. However, despite both RLBs and P-bodies exhibiting liquid-like properties, they remained distinct condensates. Taken together, these observations reveal that RNase L promotes the formation of a unique RNP complex that may have roles during the RNase L-mediated antiviral response.

Objective We aimed to evaluate the correlation of circular RNA La‐related RNA‐binding protein 4 (circ‐LARP4) with tumor characteristics and prognosis, and its effect on chemosensitivity in breast cancer. Methods Circ‐LARP4 from tumor and adjacent tissues of 283 female breast cancer patients underwent resection was detected by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR). Tumor features, disease‐free survival (DFS), and overall survival (OS) were recorded. In vitro, circ‐LARP4 in human normal mammary epithelial cells (HMEC) and breast cancer cell lines was detected by RT‐qPCR. MCF‐7 and MDA‐MB‐231 cells were transfected with circ‐LARP4 overexpression plasmid (as OE‐Circ group) and control overexpression plasmid (as OE‐Control group). Relative cell viability under different concentrations of doxorubicin was measured. Results Circ‐LARP4 was decreased in tumor tissues than adjacent tissues (P

A large number of plant RNA viruses circulate between plants and insects. For RNA viruses, host alternations may impose a differential selective pressure on viral populations and induce variations in viral genomes. Here, we report the variations in the 3′‐terminal regions of the multiple‐segment RNA virus Rice stripe virus (RSV) that were discovered through de novo assembly of the genome using RNA sequencing data from infected host plants and vector insects. The newly assembled RSV genome contained 16‐ and 15‐nt extensions at the 3′‐termini of two genome segments compared with the published reference RSV genome. Our study demonstrated that these extensional sequences were consistently observed in two RSV isolates belonging to distinct genetic subtypes in RSV‐infected rice, wheat and tobacco. Moreover, the de novo assembled genome of Southern rice black‐streaked dwarf virus also contained 3′‐terminal extensions in five RNA segments compared with the reference genome. Time course experiments confirmed that the 3′‐terminal extensions of RSV were enriched in the vector insects, were gradually eliminated in the host plant and potentially affected viral replication. These findings indicate that variations in the 3′‐termini of viral genomes may be different adaptive strategies for plant RNA viruses in insects and plants.

Summary Despite the great achievements in genome editing, accurately detecting mutations induced by sequence‐specific nucleases is still a challenge in plants, especially in polyploidy plants. An efficient detection method is particularly vital when the mutation frequency is low or when a large population needs to be screened. Here, we applied purified CRISPR ribonucleoprotein complexes to cleave PCR products for genome‐edited mutation detection in hexaploid wheat and diploid rice. We show that this mutation detection method is more sensitive than Sanger sequencing and more applicable than PCR/RE method without the requirement for restriction enzyme site. We also demonstrate that this detection method is especially useful for genome editing in wheat, because target sites are often surrounded by single nucleotide polymorphisms. Using this screening method, we were also able to detect foreign DNA‐free tagw2 mutations induced by purified TALEN protein. Finally, we show that partial base editing mutations can also be detected using high‐fidelity SpCas9 variants or FnCpf1. The PCR/RNP method is low‐cost and widely applicable for rapid detection of genome‐edited mutation in plants.

Localization of mRNAs depends on specific RNA-binding proteins (RBPs) and critically contributes not only to cell polarization but also to basal cell function. The yeast RBP Khd1p binds to several hundred mRNAs, the majority of which encodes secreted or membrane proteins. We demonstrate that a subfraction of Khd1p associates with artificial liposomes and endoplasmic reticulum (ER), and that Khd1p endomembrane association is partially dependent on its binding to RNA. ER targeting of at least two mRNAs, MID2 and SLG1/WSC1, requires KHD1 but is independent of their translation. Together, our results suggest interdependence of Khd1p and mRNA for their targeting to the ER and presents additional evidence for signal sequence-independent, RBP-mediated mRNA targeting.

The practical application of biosensors can be determined by evaluating the sensing ability of fluorophore-modified derivatives of a receptor with appropriate recognition characteristics for target molecules. One of the key determinants for successfully obtaining a useful biosensor is wide variation in the fluorophores attached to a given receptor. Thus, using a larger fluorophore-modified receptor library provides a higher probability of obtaining a practically useful biosensor. However, no effective method has yet been developed for constructing such a diverse library of fluorophore-modified receptors. Herein, we report a method for constructing fluorophore-modified receptors by using a chemical library of synthetic fluorophores with a thiol-reactive group. This library was converted into a library of fluorophore-modified adenosine-binding ribonucleopeptide (RNP) receptors by introducing the fluorophores to the Rev peptide of the RNP complex by alkylation of the thiol group. This method enabled the construction of 263 fluorophore-modified ATP-binding RNP receptors and allowed the selection of suitable receptor-based fluorescent sensors that target ATP.

Local axonal protein synthesis plays a crucial role in the formation and function of neuronal circuits. Understanding the role of this mechanism in specific circuits requires identifying the protein composition and mRNA cargos of the ribonucleoprotein particles (RNPs) that form the substrate for axonal translation. FXGs (Fragile X granules) are axonal RNPs present in a stereotyped subset of mature axons in the intact brain that contain one or more of the Fragile X related (FXR) proteins (FMRP, FXR2P, and FXR1P) along with mRNA and ribosomes. Here we performed a systematic survey of the FXR protein composition and mRNA association of FXGs in the brain. We have identified four FXG types that can be categorized based on their FXR protein complement. All FXGs contain FXR2P, with FMRP and/or FXR1P present in circuit-selective subsets. Individual neuronal cell types predominantly express a single FXG type, with FMRP-containing FXGs the most prevalent in forebrain neurons. All FXG types associate with ribosomes and mRNA, but the specific mRNA cargos are a function of FXG type, brain region and neuron class. Transcripts for β-catenin and its regulator APC associate with a subset of forebrain FXGs. Moreover, both these transcripts can colocalize within individual FXGs, suggesting that the axonal translation of functionally related proteins may be coordinately regulated with high spatiotemporal resolution. Cell type-dependent expression of specific RNP types with distinct mRNA cargos, such as FXGs, presents a potential mechanism for regulating local translation and its output in a circuit-dependent manner.

Adenosine Deaminases acting on RNA (ADARs) are proteins that catalyze widespread A-to-I editing within RNA sequences. We recently reported that ADAR2 edits and stabilizes nuclear-retained Cat2 transcribed nuclear RNA (Ctn RNA). Here, we report that ADAR1 coordinates with ADAR2 to regulate editing and stability of Ctn RNA. We observe an RNA-dependent interaction between ADAR1 and ADAR2. Furthermore, ADAR1 negatively regulates interaction of Ctn RNA with RNA-destabilizing proteins. We also show that breast cancer (BC) cells display elevated ADAR1 but not ADAR2 levels, compared to non-tumorigenic cells. Additionally, BC patients with elevated levels of ADAR1 show low survival. Our findings provide insights into overlapping substrate preferences of ADARs and potential involvement of ADAR1 in breast cancer.

Stress granules are cytoplasmic structures that form in response to a variety of cellular stresses. They contain mRNAs and many proteins including numerous types of RNA-binding proteins, and have been studied in connection to major cellular events such as protein synthesis as well as disease. Despite the well-known fact that stress granules encapsulate mRNPs (mRNA-protein complexes), much of the research has naturally focused on the protein components of stress granules. The specific details of mRNP entry into and exit from stress granules and the functional reasons for these dynamics are not fully understood. Here, we review studies that have concentrated on the aspects of mRNP accumulation in stress granules and produced quantitative data concerning mRNP/stress granule interactions.

Intracellular ribonucleoprotein (RNP) granules are membrane-less droplet organelles that are thought to regulate posttranscriptional gene expression. While liquid-liquid phase separation may drive RNP granule assembly, the mechanisms underlying their supramolecular dynamics and internal organization remain poorly understood. Herein, we demonstrate that RNA, a primary component of RNP granules, can modulate the phase behavior of RNPs by controlling both droplet assembly and dissolution in vitro. Monotonically increasing the RNA concentration initially leads to droplet assembly by complex coacervation and subsequently triggers an RNP charge inversion, which promotes disassembly. This RNA-mediated reentrant phase transition can drive the formation of dynamic droplet substructures (vacuoles) with tunable lifetimes. We propose that active cellular processes that can create an influx of RNA into RNP granules, such as transcription, can spatiotemporally control the organization and dynamics of such liquid-like organelles.

Objective Sjogren's syndrome (SS) is a systemic autoimmune disease that primarily affects lacrimal and salivary glands. We previously reported that FliC derived from Escherichia coli could induce autoimmune pancreatitis-like lesions. From these results, we speculated that FliC could also induce SS-like exocrinopathy. In this study, we investigated the effects of chronic exposure to FliC on lacrimal and salivary glands and the possibility that it might lead to an autoimmune response. Methods C57BL/6 mice were repeatedly injected with FliC and histological changes, serum levels of cytokine/chemokines and autoantibodies were evaluated at different time points after the final injection. The presence of sialadenitis was diagnosed by histological methods. Results In FliC-treated groups, 57% of subjects developed inflammatory cell infiltrates around ducts in mandibular salivary glands, but not lacrimal glands. In addition, serum levels of total IgG, IgG1, and IgG2a were significantly higher in FliC-treated groups. Intriguingly, serum anti-SSA/Ro levels were also significantly higher in FliC-treated groups. Cytokine analysis revealed that serum levels of IL-1β, IL-12p70, IL-13, IFN-γ, IL-15, and IL-23 seemed to be higher in FliC-treated mice. Conclusions Our data suggest that FliC-treated mice develop an SS-like phenotype. Our model may elucidate the relationship between commensal bacteria and SS.

Summary Chloroplast RNA metabolism depends on a multitude of nuclear-encoded RNA-binding proteins (RBPs). Most known chloroplast RBPs address specific RNA targets and RNA-processing functions. However, members of the small chloroplast ribonucleoprotein family (cpRNPs) play a global role in processing and stabilizing chloroplast RNAs. Here, we show that the cpRNP CP33A localizes to a distinct sub-chloroplastic domain and is essential for chloroplast development. The loss of CP33A yields albino seedlings that exhibit aberrant leaf development and can only survive in the presence of an external carbon source. Genome-wide RNA association studies demonstrate that CP33A associates with all chloroplast mRNAs. For a given transcript, quantification of CP33A-bound versus free RNAs demonstrates that CP33A associates with the majority of most mRNAs analyzed. Our results further show that CP33A is required for the accumulation of a number of tested mRNAs, and is particularly relevant for unspliced and unprocessed precursor mRNAs. Finally, CP33A fails to associate with polysomes or to strongly co-precipitate with ribosomal RNA, suggesting that it defines a ribodomain that is separate from the chloroplast translation machinery. Collectively, these findings suggest that CP33A contributes to globally essential RNA processes in the chloroplasts of higher plants.

Summary Phloem sap contains a large number of macromolecules, including proteins and RNAs from different classes. Proteome analyses of phloem samples from different plant species under denaturing conditions identified hundreds of proteins potentially involved in diverse processes. Surprisingly, these studies also found a significant number of ribosomal and proteasomal proteins. This led to the suggestion that active ribosome and proteasome complexes might be present in the phloem, challenging the paradigm that protein synthesis and turnover are absent from the enucleate sieve elements of angiosperms. However, the existence of such complexes has as yet not been demonstrated.In this study we used three‐dimensional gel electrophoresis to separate several protein complexes from native phloem sap from Brassica napus. Matrix‐assisted laser desorption ionization‐time of flight MS analyses identified more than 100 proteins in the three major protein‐containing complexes.All three complexes contained proteins belonging to different ribosomal fragments and blue native northern blot confirmed the existence of ribonucleoprotein complexes. In addition, one complex contained proteasome components and further functional analyses confirmed activity of a proteasomal degradation pathway and showed a large number of ubiquitinated phloem proteins.Our results suggest specialized roles for ubiquitin modification and proteasome‐mediated degradation in the phloem.

RNA-binding protein with serine-rich domain 1 (RNPS1) is a component of pre-splicing and post-splicing multiprotein complexes, which activates constitutive and alternative splicing. RNPS1 participates in the formation of the spliceosome and activates the pre-mRNA splicing process. In the present study, we found that ubiquitin-specific protease 4 (USP4) is a binding partner of RNPS1. Although RNPS1 is polyubiquitinated by both K48- and K63-linkages, USP4 exclusively deubiquitinates K63-linked polyubiquitin chains of RNPS1. We also demonstrate that the catalytic activity of USP4 on ubiquitinated RNPS1 is elevated by squamous cell carcinoma antigen recognized by T cells 3 (Sart3).

Membrane-less organelles, because of their capacity to dynamically, selectively and reversibly concentrate molecules, are very well adapted for local information processing and rapid response to environmental fluctuations. These features are particularly important in the context of neuronal cells, where synapse-specific activation, or localized extracellular cues, induce signaling events restricted to specialized axonal or dendritic subcompartments. Neuronal ribonucleoprotein (RNP) particles, or granules, are nonmembrane bound macromolecular condensates that concentrate specific sets of mRNAs and regulatory proteins, promoting their long-distance transport to axons or dendrites. Neuronal RNP granules also have a dual function in regulating the translation of associated mRNAs: while preventing mRNA translation at rest, they fuel local protein synthesis upon activation. As revealed by recent work, rapid and reversible switches between these two functional modes are triggered by modifications of the networks of interactions underlying RNP granule assembly. Such flexible properties also come with a cost, as neuronal RNP granules are prone to transition into pathological aggregates in response to mutations, aging, or cellular stresses, further emphasizing the need to better understand the mechanistic principles governing their dynamic assembly and regulation in living systems.

The RNA binding proteome (RBPome) was previously investigated using UV crosslinking and purification of poly(A)‐associated proteins. However, most cellular transcripts are not polyadenylated. We therefore developed total RNA‐associated protein purification (TRAPP) based on 254 nm UV crosslinking and purification of all RNA–protein complexes using silica beads. In a variant approach (PAR‐TRAPP), RNAs were labelled with 4‐thiouracil prior to 350 nm crosslinking. PAR‐TRAPP in yeast identified hundreds of RNA binding proteins, strongly enriched for canonical RBPs. In comparison, TRAPP identified many more proteins not expected to bind RNA, and this correlated strongly with protein abundance. Comparing TRAPP in yeast and E. coli showed apparent conservation of RNA binding by metabolic enzymes. Illustrating the value of total RBP purification, we discovered that the glycolytic enzyme enolase interacts with tRNAs. Exploiting PAR‐TRAPP to determine the effects of brief exposure to weak acid stress revealed specific changes in late 60S ribosome biogenesis. Furthermore, we identified the precise sites of crosslinking for hundreds of RNA–peptide conjugates, using iTRAPP, providing insights into potential regulation. We conclude that TRAPP is a widely applicable tool for RBPome characterization.

Pre-mRNA splicing is widely repressed upon heat shock in eukaryotic cells. However, it has been shown that HSP105 pre-mRNA is alternatively spliced in response to heat stress. Using RNAi screening in HeLa cells, we found that RNA-binding proteins hnRNP K and PSF/SFPQ are necessary for the exon 12 exclusion of HSP105 during heat stress. Moreover, exon array analyses showed that a group of genes is alternatively spliced during heat stress in an hnRNP K-dependent manner, whereas hnRNP K is not necessary for the stress-induced alternative splicing of the remaining genes. Among the latter group, we found that SRp38/SRSF10 and SC35/SRSF2 are necessary for the inclusion of exon 13 of TNRC6A during heat stress. Thus, our study clearly showed that several RNA-binding proteins are involved in the splicing regulation in response to heat stress in mammalian cells.

Although RNA are synthesized as single-stranded molecules, most of them are characterized by extensive secondary structures. Two RNA that require distinct folding for biogenesis and function are miRNA and tRNA. While miRNA are processed from hairpin-containing precursors, tRNA are folded into characteristic L-shaped structures. In addition, tRNA and their precursors are a rich source of RNA fragments. Recent findings suggest that their production might be determined by structural characteristics of the tRNA substrates. Importantly, correct folding of pre-tRNA is assisted by the Lupus autoantigen La, an RNA chaperone. In this context, La interacts with pre-tRNA to prevent alternative foldings leading to mis-channeling into the miRNA pathway. Thus, RNA chaperones also function as gatekeepers for correct RNA pathway selection.

HIV replication requires the nuclear export of essential, intron-containing viral RNAs. To facilitate export, HIV encodes the viral accessory protein Rev which binds unspliced and partially spliced viral RNAs and creates a ribonucleoprotein complex that recruits the cellular Chromosome maintenance factor 1 export machinery. Exporting RNAs in this manner bypasses the necessity for complete splicing as a prerequisite for mRNA export, and allows intron-containing RNAs to reach the cytoplasm intact for translation and virus packaging. Recent structural studies have revealed that this entire complex exhibits remarkable plasticity at many levels of organization, including RNA folding, protein-RNA recognition, multimer formation, and host factor recruitment. In this review, we explore each aspect of plasticity from structural, functional, and possible therapeutic viewpoints. WIREs RNA 2016, 7:470-486. doi: 10.1002/wrna.1342 For further resources related to this article, please visit the WIREs website.

Toll-like receptors (TLRs), first identified as pattern recognition receptors, are now recognized to serve as a key interface between innate and adaptive immunity. Systemic Lupus Erythematosus (SLE) is characterized by both continuous and cyclic stimulation of the innate and adaptive immune system by endogenous nucleic acids released from apoptotic or necrotic cells. TLR7 and TLR9 function as innate sensors of viral infection as their ligands are ssRNA and dsDNA, respectively. Recognition of self nucleic acids by endosomal TLRs in B cells and pDCs is thought to be an important step in the pathogenesis of SLE, generating anti-nuclear antibodies and producing type I IFN. In this review, we take a specific look at how TLR7, noncoding RNA, and SSA/Ro60 can contribute to clinical autoimmunity and organ damage in the context of neonatal lupus (NL). Although fifteen times less common than SLE, NL provides a unique opportunity to study two different aspects of autoimmunity: passively acquired tissue injury in a developing fetus and clinical progression of disease in an asymptomatic mother found to have anti-Ro60 autoantibodies only after identification of heart block/rash in a child. Finally, we discuss hydroxychloroquine (HCQ) use by asymptomatic subjects which may forestall the clinical expression of autoimmunity.