S-adenosylmethionine inhibits la ribonucleoprotein domain family member 1 in murine liver and human liver cancer cells.

BACKGROUND AND AIMS: Methionine adenosyltransferase 1A (MAT1A) is responsible for S-adenosylmethionine (SAMe) biosynthesis in the liver. Mice lacking Mat1a have hepatic SAMe depletion and develop NASH and HCC spontaneously. Several kinases are activated in Mat1a knockout (KO) mice livers. However, characterizing the phospho-proteome and determining whether they contribute to liver pathology remain open for study. Our study aimed to provide this knowledge. APPROACH AND RESULTS: We performed phospho-proteomics in Mat1a KO mice livers with and without SAMe treatment to identify SAMe-dependent changes that may contribute to liver pathology. Our studies used Mat1a KO mice at different ages treated with and without SAMe, cell lines, in vitro translation and kinase assays, and human liver specimens. We found that the most striking change was hyperphosphorylation and increased content of La-related protein 1 (LARP1), which, in the unphosphorylated form, negatively regulates translation of 5'-terminal oligopyrimidine (TOP)-containing mRNAs. Consistently, multiple TOP proteins are induced in KO livers. Translation of TOP mRNAs ribosomal protein S3 and ribosomal protein L18 was enhanced by LARP1 overexpression in liver cancer cells. We identified LARP1-T449 as a SAMe-sensitive phospho-site of cyclin-dependent kinase 2 (CDK2). Knocking down CDK2 lowered LARP1 phosphorylation and prevented LARP1-overexpression-mediated increase in translation. LARP1-T449 phosphorylation induced global translation, cell growth, migration, invasion, and expression of oncogenic TOP-ribosomal proteins in HCC cells. LARP1 expression is increased in human NASH and HCC. CONCLUSIONS: Our results reveal a SAMe-sensitive mechanism of LARP1 phosphorylation that may be involved in the progression of NASH to HCC.

Functional splicing network reveals extensive regulatory potential of the core spliceosomal machinery.

Pre-mRNA splicing relies on the poorly understood dynamic interplay between >150 protein components of the spliceosome. The steps at which splicing can be regulated remain largely unknown. We systematically analyzed the effect of knocking down the components of the splicing machinery on alternative splicing events relevant for cell proliferation and apoptosis and used this information to reconstruct a network of functional interactions. The network accurately captures known physical and functional associations and identifies new ones, revealing remarkable regulatory potential of core spliceosomal components, related to the order and duration of their recruitment during spliceosome assembly. In contrast with standard models of regulation at early steps of splice site recognition, factors involved in catalytic activation of the spliceosome display regulatory properties. The network also sheds light on the antagonism between hnRNP C and U2AF, and on targets of antitumor drugs, and can be widely used to identify mechanisms of splicing regulation.

TRIM21 improves apatinib treatment in gastric cancer through suppressing EZH1 stability.

Gastric cancer (GC) is a common tumor with high metastatic rate worldwide. Promoting chemosensitivity is effective for improving therapeutic outcome and survival rate for GC patients. Tripartite motif-containing 21 (TRIM21), a member of TRIM-containing proteins, plays crucial roles in regulating numerous cellular events involved in tumor progression. However, it's regulatory effects on GC growth and drug sensitivity are still unclear. In the present study, we identified that TRIM21 expression was remarkably decreased in human GC tissues compared with the adjacent normal ones, and its down-regulation was closely linked to higher recurrence and lower overall survival rate among GC patients. We then found that apatinib (APA)-reduced GC cell proliferation was significantly abolished by TRIM21 knockdown; however, promoting TRIM21 expression further improved the sensitivity of GC cells to APA treatment, as proved by the remarkably decreased cell viability and colony formation. Furthermore, TRIM21 over-expression dramatically enhanced apoptosis, while its knockdown markedly diminished apoptotic cell death in APA-incubated GC cells. Moreover, stem cell properties of GC cells were also restrained by TRIM21. Our in vivo experiments showed that APA-repressed tumor growth was considerably abolished by TRIM21 knockdown, whereas being further elevated by TRIM21 over-expression. In addition, we showed that TRIM21 markedly decreased enhancer of zeste homolog 1 (EZH1) protein expression levels in GC cells, and importantly, a direct interaction between TRIM21 and EZH1 was verified. Of note, our in vitro studies revealed that EZH1 over-expression remarkably abolished the function of TRIM21 to restrain cell viability and induce apoptosis in APA-incubated GC cells, indicating that EZH1 suppression was necessary for TRIM21 to inhibit GC progression. Together, our findings demonstrated that TRIM21 may be a novel therapeutic target for GC treatment through reducing EZH1 to improve chemosensitivity.

CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea.

CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse organisms, enabling targeted genetic changes to be performed with unprecedented efficiency. Here we report on the first establishment of robust CRISPR/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized Cas9-sgRNA ribonucleoprotein complexes (RNPs) into protoplasts. Editing yields were further improved by development of a novel strategy that combines RNP delivery with cotransformation of transiently stable vectors containing telomeres, which allowed temporary selection and convenient screening for marker-free editing events. We demonstrate that this approach provides superior editing rates compared to existing CRISPR/Cas-based methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae. Genome sequencing of edited strains revealed very few additional mutations and no evidence for RNP-mediated off-targeting. The high performance of telomere vector-mediated editing was demonstrated by random mutagenesis of codon 272 of the sdhB gene, a major determinant of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides by in bulk replacement of the codon 272 with codons encoding all 20 amino acids. All exchanges were found at similar frequencies in the absence of selection but SDHI selection allowed the identification of novel amino acid substitutions which conferred differential resistance levels towards different SDHI fungicides. The increased efficiency and easy handling of RNP-based cotransformation is expected to accelerate molecular research in B. cinerea and other fungi.

Tripartite-motif protein 21 knockdown extenuates LPS-triggered neurotoxicity by inhibiting microglial M1 polarization via suppressing NF-κB-mediated NLRP3 inflammasome activation.

Tripartite motif-containing 21 (TRIM21) has been confirmed to mediate the production of inflammatory mediators via NF-κB signaling. However, the function of TRIM21 in microglia-mediated neuroinflammation remains unclear. This study aimed to explore the effect of TRIM21 on LPS-activated BV2 microglia and its underlying mechanism. BV2 cells exposed to lipopolysaccharide (LPS) were used to simulated neuroinflammation in vitro. Loss-of-function and gain-of-function of TRIM21 in BV2 cells were used to assess the effect of TRIM21 on LPS-induced neuroinflammation. BV2 microglia and HT22 cells co-culture system were used to investigate whether TRIM21 regulated neuronal inflammation-mediated neuronal death. TRIM21 knockdown triggered the polarization of BV2 cells from M1 to M2 phenotype. Knockdown of TRIM21 reduced the secretion of TNF-α, IL-6, and IL-1ß, while increased the content of IL-4 in LPS-treated cells. Knockdown of TRIM21 inhibited the expression of p65 and the binding activity of NF-κB-DNA. Additionally, TRIM21 siRNA eliminated the increase in NLRP3 and cleaved caspase-1 proteins expression and caspase-1 activity induced by LPS. TRIM21 knockdown could resist cytotoxicity induced by activated microglia, including increasing the viability of co-cultured HT22 cells and reducing the emancipation of LDH. Moreover, the increased apoptosis and caspase-3 activity of HT22 neurons induced by activated BV2 cells were blocked by TRIM21 siRNA. Blocking of NF-κB abolished the effect of TRIM21 in promoting the expression of M1 phenotype marker genes. Similarly, the blockade of NF-κB pathway eliminated the promotion of TRIM21 on neurotoxicity induced by neuroinflammation. TRIM21 knockdown suppressed the M1 phenotype polarization of microglia and neuroinflammation-mediated neuronal damage via NF-κB/NLRP3 inflammasome pathway, which suggested that TRIM21 might be a potential therapeutic target for the therapy of central nervous system diseases.

TRIM21 - A potential novel therapeutic target in cancer.

Tripartite motif-containing protein 21 (TRIM21) is well known to be involved in innate immunity, systemic lupus erythematosus and Sjögren's syndrome. In addition, TRIM21 involvement in cancer proliferation has been observed. However, the clinical significance of TRIM21 and its role in cancer cell proliferation and suppression remains elusive. Here we discuss the effects of TRIM21 on major cancer promoting proteins such as NF-κB, STAT3, BCL2, p53, p27 and Snail, comparing its signaling pathways under normal conditions and in the presence of a variety of carcinogenesis effectors (oncogenic, genotoxic and UV irradiation). Depending on the cancer type and the carcinogenesis effector, TRIM21 may enhance cancer proliferation, or alternatively it may increase the ubiquitination of many cancer-triggering proteins, resulting in their proteasomal degradation. This indicates the importance of TRIM21 in cancer proliferation and/or apoptosis and suggests its potential as a novel cancer therapeutic target.

Role of the RNA-binding protein La in cancer pathobiology.

RNA-binding proteins are important regulators of RNA metabolism and are of critical importance in all steps of the gene expression cascade. The role of aberrantly expressed RBPs in human disease is an exciting research field and the potential application of RBPs as a therapeutic target or a diagnostic marker represents a fast-growing area of research.Aberrant overexpression of the human RNA-binding protein La has been found in various cancer entities including lung, cervical, head and neck, and chronic myelogenous leukaemia. Cancer-associated La protein supports tumour-promoting processes such as proliferation, mobility, invasiveness and tumour growth. Moreover, the La protein maintains the survival of cancer cells by supporting an anti-apoptotic state that may cause resistance to chemotherapeutic therapy.The human La protein represents a multifunctional post-translationally modified RNA-binding protein with RNA chaperone activity that promotes processing of non-coding precursor RNAs but also stimulates the translation of selective messenger RNAs encoding tumour-promoting and anti-apoptotic factors. In our model, La facilitates the expression of those factors and helps cancer cells to cope with cellular stress. In contrast to oncogenes, able to initiate tumorigenesis, we postulate that the aberrantly elevated expression of the human La protein contributes to the non-oncogenic addiction of cancer cells. In this review, we summarize the current understanding about the implications of the RNA-binding protein La in cancer progression and therapeutic resistance. The concept of exploiting the RBP La as a cancer drug target will be discussed.

Ubiquitination of IGF2BP3 by E3 ligase MKRN2 regulates the proliferation and migration of human neuroblastoma SHSY5Y cells.

Neuroblastoma (NB) is a paediatric tumour that shows great biomolecule and clinical heterogeneity, and patients with NB often develop various neurological complications. Currently, the disease is mainly treated by surgery and still lacks specific therapeutic drugs; therefore, targets are urgently needed. Makorin ring finger protein 2 (MKRN2) is an E3 ligase whose effects on neuroblastoma have not been illustrated. shRNAs for MKRN2 have been designed, and MKRN2-knockdown human neuroblastoma SHSY5Y cells were established. MKRN2 knockdown promotes the proliferation and migration of SHSY5Y cells. Because MKRN2 is an E3 ligase, we performed a series of experiments, and Insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3) was identified as a new substrate for MKRN2. IGF2BP3 is an RNA-binding protein that regulates the stability of many mRNAs, including CD44 and PDPN, and our study demonstrated that MKRN2 regulates the expression of CD44 and PDPN in an IGF2BP3-dependent manner. These results suggest that MKRN2 might be a potential therapeutic target for neuroblastoma.

TRIM21 Mitigates Human Lung Microvascular Endothelial Cells' Inflammatory Responses to LPS.

Endothelial cell (EC) inflammation is regarded as an important pathogenic feature of many inflammatory diseases, including acute lung injury and sepsis. An increase in EC inflammation results in neutrophil infiltration from the blood to the site of inflammation, further promoting EC permeability. The ubiquitin E3 ligase TRIM21 has been implicated in human disorders; however, the roles of TRIM21 in endothelial dysfunction and acute lung injury have not been reported. Here, we reveal an antiinflammatory property of TRIM21 in a mouse model of acute lung injury and human lung microvascular ECs. Overexpression of TRIM21 by lentiviral vector infection effectively dampened LPS-induced neutrophil infiltration, cytokine release, and edema in mice. TRIM21 inhibited human lung microvascular endothelial cell inflammatory responses as evidenced by attenuation of the NF-κB pathway, release of IL-8, expression of intercellular adhesion molecules, and adhesion of monocytes to ECs. Furthermore, we demonstrated that TRIM21 was predominantly degraded by an increase in its monoubiquitination and lysosomal degradation after inflammatory stimuli. Thus, inhibition of vascular endothelial inflammation by TRIM21 provides a novel therapeutic target to lessen pulmonary inflammation.

Heterogeneous nuclear ribonucleoprotein K inhibits heat shock-induced transcriptional activity of heat shock factor 1.

When cells are exposed to heat shock and various other stresses, heat shock factor 1 (HSF1) is activated, and the heat shock response (HSR) is elicited. To better understand the molecular regulation of the HSR, we used 2D-PAGE-based proteome analysis to screen for heat shock-induced post-translationally modified cellular proteins. Our analysis revealed that two protein spots typically present on 2D-PAGE gels and containing heterogeneous nuclear ribonucleoprotein K (hnRNP K) with trioxidized Cys132 disappeared after the heat shock treatment and reappeared during recovery, but the total amount of hnRNP K protein remained unchanged. We next tested whether hnRNP K plays a role in HSR by regulating HSF1 and found that hnRNP K inhibits HSF1 activity, resulting in reduced expression of hsp70 and hsp27 mRNAs. hnRNP K also reduced binding affinity of HSF1 to the heat shock element by directly interacting with HSF1 but did not affect HSF1 phosphorylation-dependent activation or nuclear localization. hnRNP K lost its ability to induce these effects when its Cys132 was substituted with Ser, Asp, or Glu. These findings suggest that hnRNP K inhibits transcriptional activity of HSF1 by inhibiting its binding to heat shock element and that the oxidation status of Cys132 in hnRNP K is critical for this inhibition.

Naf1 Regulates HIV-1 Latency by Suppressing Viral Promoter-Driven Gene Expression in Primary CD4+ T Cells.

HIV-1 latency is characterized by reversible silencing of viral transcription driven by the long terminal repeat (LTR) promoter of HIV-1. Cellular and viral factors regulating LTR activity contribute to HIV-1 latency, and certain repressive cellular factors modulate viral transcription silencing. Nef-associated factor 1 (Naf1) is a host nucleocytoplasmic shuttling protein that regulates multiple cellular signaling pathways and HIV-1 production. We recently reported that nuclear Naf1 promoted nuclear export of unspliced HIV-1 gag mRNA, leading to increased Gag production. Here we demonstrate new functions of Naf1 in regulating HIV-1 persistence. We found that Naf1 contributes to the maintenance of HIV-1 latency by inhibiting LTR-driven HIV-1 gene transcription in a nuclear factor kappa B-dependent manner. Interestingly, Naf1 knockdown significantly enhanced viral reactivation in both latently HIV-1-infected Jurkat T cells and primary central memory CD4+ T cells. Furthermore, Naf1 knockdown in resting CD4+ T cells from HIV-1-infected individuals treated with antiretroviral therapy significantly increased viral reactivation upon T-cell activation, suggesting an important role of Naf1 in modulating HIV-1 latency in vivo Our findings provide new insights for a better understanding of HIV-1 latency and suggest that inhibition of Naf1 activity to activate latently HIV-1-infected cells may be a potential therapeutic strategy. IMPORTANCE: HIV-1 latency is characterized mainly by a reversible silencing of LTR promoter-driven transcription of an integrated provirus. Cellular and viral proteins regulating LTR activity contribute to the modulation of HIV-1 latency. In this study, we found that the host protein Naf1 inhibited HIV-1 LTR-driven transcription of HIV genes and contributed to the maintenance of HIV-1 latency. Our findings provide new insights into the effects of host modulation on HIV-1 latency, which may lead to a potential therapeutic strategy for HIV persistence by targeting the Naf1 protein.

Identification of HNRNPK as regulator of hepatitis C virus particle production.

Hepatitis C virus (HCV) is a major cause of chronic liver disease affecting around 130 million people worldwide. While great progress has been made to define the principle steps of the viral life cycle, detailed knowledge how HCV interacts with its host cells is still limited. To overcome this limitation we conducted a comprehensive whole-virus RNA interference-based screen and identified 40 host dependency and 16 host restriction factors involved in HCV entry/replication or assembly/release. Of these factors, heterogeneous nuclear ribonucleoprotein K (HNRNPK) was found to suppress HCV particle production without affecting viral RNA replication. This suppression of virus production was specific to HCV, independent from assembly competence and genotype, and not found with the related Dengue virus. By using a knock-down rescue approach we identified the domains within HNRNPK required for suppression of HCV particle production. Importantly, HNRNPK was found to interact specifically with HCV RNA and this interaction was impaired by mutations that also reduced the ability to suppress HCV particle production. Finally, we found that in HCV-infected cells, subcellular distribution of HNRNPK was altered; the protein was recruited to sites in close proximity of lipid droplets and colocalized with core protein as well as HCV plus-strand RNA, which was not the case with HNRNPK variants unable to suppress HCV virion formation. These results suggest that HNRNPK might determine efficiency of HCV particle production by limiting the availability of viral RNA for incorporation into virions. This study adds a new function to HNRNPK that acts as central hub in the replication cycle of multiple other viruses.

MicroRNA-33a disturbs influenza A virus replication by targeting ARCN1 and inhibiting viral ribonucleoprotein activity.

In order to explore the roles of microRNA(s) [miRNA(s)] in the influenza A virus life cycle, we compared the miRNA profiles of 293T and HeLa cell lines, as influenza A virus can replicate efficiently in 293T cells but only poorly in HeLa cells. We analysed differentially expressed miRNAs and identified five, including miR-33a, that could disturb influenza A virus replication significantly. Using TargetScan analysis, we found that ARCN1 could be a potential target of miR-33a. To confirm whether miR-33a could truly target ARCN1, we generated a luciferase reporter for the ARCN1 3' untranslated region (UTR) and performed a luciferase assay. The data indicated that miR-33a could suppress the luciferase activity of the reporter for the ARCN1 3' UTR but not a reporter in which the predicted miR-33a targeting sites on ARCN1 3' UTR were mutated. We performed immunoblotting to confirm that miR-33a could downregulate the protein level of ARCN1. Consistently, the level of ARCN1 protein in HeLa cells was significantly lower than that in 293T cells. We also demonstrated that ectopic expression of ARCN1 could partially rescue the inhibitory effect of miR-33a on virus replication. Furthermore, we demonstrated that miR-33a could impede virus replication at the stage of virus internalization, which was similar to the pattern for knockdown of ARCN1, indicating that miR-33a inhibits influenza virus infection by suppressing ARCN1 expression. In addition, we found that miR-33a could also weaken the viral ribonucleoprotein activity in an ARCN1-independent manner. In conclusion, we found that miR-33a is a novel inhibitory factor for influenza A virus replication.

Human Papillomavirus 16 Oncoprotein Expression Is Controlled by the Cellular Splicing Factor SRSF2 (SC35).

UNLABELLED: High-risk human papillomaviruses (HR-HPV) cause anogenital cancers, including cervical cancer, and head and neck cancers. Human papillomavirus 16 (HPV16) is the most prevalent HR-HPV. HPV oncogenesis is driven by two viral oncoproteins, E6 and E7, which are expressed through alternative splicing of a polycistronic RNA to yield four major splice isoforms (E6 full length, E6*I, E6*II, E6*X). The production of multiple mRNA isoforms from a single gene is controlled by serine/arginine-rich splicing factors (SRSFs), and HPV16 infection induces overexpression of a subset of these, SRSFs 1, 2, and 3. In this study, we examined whether these proteins could control HPV16 oncoprotein expression. Small interfering RNA (siRNA) depletion experiments revealed that SRSF1 did not affect oncoprotein RNA levels. While SRSF3 knockdown caused some reduction in E6E7 expression, depletion of SRSF2 resulted in a significant loss of E6E7 RNAs, resulting in reduced levels of the E6-regulated p53 proteins and E7 oncoprotein itself. SRSF2 contributed to the tumor phenotype of HPV16-positive cervical cancer cells, as its depletion resulted in decreased cell proliferation, reduced colony formation, and increased apoptosis. SRSF2 did not affect transcription from the P97 promoter that controls viral oncoprotein expression. Rather, RNA decay experiments showed that SRSF2 is required to maintain stability of E6E7 mRNAs. These data show that SRSF2 is a key regulator of HPV16 oncoprotein expression and cervical tumor maintenance. IMPORTANCE: Expression of the HPV16 oncoproteins E7 and E6 drives HPV-associated tumor formation. Although increased transcription may yield increased levels of E6E7 mRNAs, it is known that the RNAs can have increased stability upon integration into the host genome. SR splicing factors (SRSFs) control splicing but can also control other events in the RNA life cycle, including RNA stability. Previously, we demonstrated increased levels of SRSFs 1, 2, and 3 during cervical tumor progression. Now we show that SRSF2 is required for expression of E6E7 mRNAs in cervical tumor but not nontumor cells and may act by inhibiting their decay. SRSF2 depletion in W12 tumor cells resulted in increased apoptosis, decreased proliferation, and decreased colony formation, suggesting that SRSF2 has oncogenic functions in cervical tumor progression. SRSF function can be targeted by known drugs that inhibit SRSF phosphorylation, suggesting a possible new avenue in abrogating HPV oncoprotein activity.

Sororin pre-mRNA splicing is required for proper sister chromatid cohesion in human cells.

Sister chromatid cohesion, which depends on cohesin, is essential for the faithful segregation of replicated chromosomes. Here, we report that splicing complex Prp19 is essential for cohesion in both G2 and mitosis, and consequently for the proper progression of the cell through mitosis. Inactivation of splicing factors SF3a120 and U2AF65 induces similar cohesion defects to Prp19 complex inactivation. Our data indicate that these splicing factors are all required for the accumulation of cohesion factor Sororin, by facilitating the proper splicing of its pre-mRNA. Finally, we show that ectopic expression of Sororin corrects defective cohesion caused by Prp19 complex inactivation. We propose that the Prp19 complex and the splicing machinery contribute to the establishment of cohesion by promoting Sororin accumulation during S phase, and are, therefore, essential to the maintenance of genome stability.

Loss of hnRNP K impairs synaptic plasticity in hippocampal neurons.

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA-binding protein implicated in RNA metabolism. Here, we investigated the role of hnRNP K in synapse function. We demonstrated that hnRNP K regulates dendritic spine density and long-term potentiation (LTP) in cultured hippocampal neurons from embryonic rats. LTP requires the extracellular signal-regulated kinase (ERK)1/2-mediated phosphorylation and cytoplasmic accumulation of hnRNP K. Moreover, hnRNP K knockdown prevents ERK cascade activation and GluA1-S845 phosphorylation and surface delivery, which are essential steps for LTP. These findings establish hnRNP K as a new critical regulator of synaptic transmission and plasticity in hippocampal neurons.

Preclinical and clinical efficacy of XPO1/CRM1 inhibition by the karyopherin inhibitor KPT-330 in Ph+ leukemias.

As tyrosine kinase inhibitors (TKIs) fail to induce long-term response in blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL), novel therapies targeting leukemia-dysregulated pathways are necessary. Exportin-1 (XPO1), also known as chromosome maintenance protein 1, regulates cell growth and differentiation by controlling the nucleocytoplasmic trafficking of proteins and RNAs, some of which are aberrantly modulated in BCR-ABL1(+) leukemias. Using CD34(+) progenitors from CML, B-ALL, and healthy individuals, we found that XPO1 expression was markedly increased, mostly in a TKI-sensitive manner, in CML-BC and Ph(+) B-ALL. Notably, XPO1 was also elevated in Ph(-) B-ALL. Moreover, the clinically relevant XPO1 inhibitor KPT-330 strongly triggered apoptosis and impaired the clonogenic potential of leukemic, but not normal, CD34(+) progenitors, and increased survival of BCR-ABL1(+) mice, 50% of which remained alive and, mostly, became BCR-ABL1 negative. Moreover, KPT-330 compassionate use in a patient with TKI-resistant CML undergoing disease progression significantly reduced white blood cell count, blast cells, splenomegaly, lactate dehydrogenase levels, and bone pain. Mechanistically, KPT-330 altered the subcellular localization of leukemia-regulated factors including RNA-binding heterogeneous nuclear ribonucleoprotein A1 and the oncogene SET, thereby inducing reactivation of protein phosphatase 2A tumor suppressor and inhibition of BCR-ABL1 in CML-BC cells. Because XPO1 is important for leukemic cell survival, KPT-330 may represent an alternative therapy for TKI-refractory Ph(+) leukemias.

Diagnosis and risk stratification in patients with anti-RNP autoimmunity.

INTRODUCTION: Anti-RNP autoantibodies occur either in mixed connective tissue disease (MCTD) (with a frequently favorable prognosis), or in systemic lupus erythematosus (SLE) cases with aggressive major organ disease. It is uncertain how to assess for the risk of severe disease in anti-RNP + patients. METHODS: Following institutional review board-approved protocols, clinical data and blood were collected from patients with known or suspected anti-RNP autoimmunity and normal controls in a cohort study. Samples were screened for parameters of immune activation. Groups were compared based on clinical diagnoses, disease classification criteria, disease activity and specific end-organ clinical manifestations. RESULTS: Ninety-seven per cent of patients satisfying Alarcon-Segovia MCTD criteria also met Systemic Lupus International Collaborating Clinic (SLICC) SLE criteria, while 47% of the anti-RNP + SLE patients also met MCTD criteria. Among SLICC SLE patients, MCTD criteria were associated with reduced rates of renal disease (odds ratio (OR) 4.3, 95% confidence interval (CI) 1.3-14.0), increased rates of Raynaud's phenomenon (OR 3.5, 95% CI 1.3-9.5) and increased serum B-cell maturation antigen, transmembrane activator and CAML interactor and TNFα levels. Circulating immune markers and markers of type I interferon activation were not effective at distinguishing clinical subgroups. CONCLUSIONS: Among anti-RNP patients, the question of MCTD versus SLE is not either/or: most MCTD patients also have lupus. MCTD classification criteria (but not a broad set of immune markers) distinguish a subset of SLE patients at reduced risk for renal disease.

Ro60 requires Y3 RNA for cell surface exposure and inflammation associated with cardiac manifestations of neonatal lupus.

Cardiac neonatal lupus (NL) is presumed to arise from maternal autoantibody targeting an intracellular ribonucleoprotein, Ro60, which binds noncoding Y RNA and only becomes accessible to autoantibodies during apoptosis. Despite the importance of Ro60 trafficking in the development of cardiac NL, the mechanism underlying cell surface exposure is unknown. To evaluate the influence of Y RNA on the subcellular location of Ro60 during apoptosis and activation of macrophages, stable Ro60 knockout murine fibroblasts expressing wild-type or mutated FLAG-Ro60 were assessed. FLAG3-Ro60(K170A R174A) binds Y RNA, whereas FLAG3-Ro60(H187S) does not bind Y RNA; fibroblasts expressing these constructs showed equivalent intracellular expression of Ro60. In contrast, apoptotic fibroblasts containing FLAG3-Ro60(K170A R174A) were bound by anti-Ro60, whereas FLAG3-Ro60(H187S) was not surface expressed. RNA interference of mY3 RNA in wild-type fibroblasts inhibited surface translocation of Ro60 during apoptosis, whereas depletion of mY1 RNA did not affect Ro60 exposure. Furthermore, Ro60 was not exposed following overexpression of mY1 in the mY3-depleted fibroblasts. In an in vitro model of anti-Ro60-mediated injury, Y RNA was shown to be an obligate factor for TLR-dependent activation of macrophages challenged with anti-Ro60-opsonized apoptotic fibroblasts. Murine Y3 RNA is a necessary factor to support the surface translocation of Ro60, which is pivotal to the formation of immune complexes on apoptotic cells and a TLR-dependent proinflammatory cascade. Accordingly, the Y3 RNA moiety of the Ro60 ribonucleoprotein imparts a critical role in the pathogenicity of maternal anti-Ro60 autoantibodies.

The N-terminal fragment of PA subunit of the influenza A virus effectively inhibits ribonucleoprotein (RNP) activity via suppression of its RNP expression.

The influenza RNP, which is formed from PB1, PB2, PA, NP subunits, and vRNA, is autonomously replicated and transcribed in the infected cell. The simplest method to inhibit RNP activity is to impair the formation of the RNP. Thereupon we confirmed whether the peptides/fragments mimicking one of RNP components can interfere with their formation. During the process of this inhibitory study we found interesting suppression of protein expression of the RNP components by the N-terminal fragment of PA subunit. Especially, we found two residues (D108 and K134) on the fragment that were critical for the suppression. Furthermore, we determined the combination of three amino acids (P28, M86 and E100) on the fragment that are important for the strong suppression, and identified the minimum essential region (residues from 1 to 188) of the PA subunit that allowed its suppression. Our findings indicate that the N-terminal fragment of PA subunit may become one of candidates for an effective inhibitor of influenza RNP activity.