TENT2, TUT4, and TUT7 selectively regulate miRNA sequence and abundance.

TENTs generate miRNA isoforms by 3' tailing. However, little is known about how tailing regulates miRNA function. Here, we generate isogenic HEK293T cell lines in which TENT2, TUT4 and TUT7 are knocked out individually or in combination. Together with rescue experiments, we characterize TENT-specific effects by deep sequencing, Northern blot and in vitro assays. We find that 3' tailing is not random but highly specific. In addition to its known adenylation, TENT2 contributes to guanylation and uridylation on mature miRNAs. TUT4 uridylates most miRNAs whereas TUT7 is dispensable. Removing adenylation has a marginal impact on miRNA levels. By contrast, abolishing uridylation leads to dysregulation of a set of miRNAs. Besides let-7, miR-181b and miR-222 are negatively regulated by TUT4/7 via distinct mechanisms while the miR-888 cluster is upregulated specifically by TUT7. Our results uncover the selective actions of TENTs in generating 3' isomiRs and pave the way to investigate their functions.

The Flavonoid Naringenin Alleviates Collagen-Induced Arthritis through Curbing the Migration and Polarization of CD4+ T Lymphocyte Driven by Regulating Mitochondrial Fission.

Rheumatoid arthritis (RA) is a progressive autoimmune disease. Due to local infiltration and damage to the joints, activated CD4+ T cells play a crucial role in the progression of RA. However, the exact regulatory mechanisms are perplexing, which makes the effective management of RA frustrating. This study aimed to investigate the effect of mitochondria fission on the polarization and migration of CD4+ T cells as well as the regulatory mechanism of NAR, so as to provide enlightenment on therapeutic targets and novel strategies for the treatment of RA. In this study, a collagen-induced arthritis (CIA) model was established, and rats were randomly given saline or naringenin (NAR, 10 mg/kg, 20 mg/kg, 50 mg/kg, i.p.) once a day, before being euthanized on the 42nd day of primary immunization. The pain-like behavior, articular index scores, account of synovial-infiltrated CD4+ T cells, and inflammatory factors were investigated in each group. In vitro, spleen CD4+ T lymphocytes were derived from each group. In addition, mitochondrial division inhibitor 1 (Mdivi-1) or NAR was added to the cell medium containing C-X-C motif chemokine ligand 12 (CXCL12) in order to induce CD4+ T lymphocytes, respectively. The polarization capacity of CD4+ T cells was evaluated through the immunofluorescence intensity of the F-actin and myosin light chain phosphorylated at Ser19 (pMLC S19), and the mitochondrial distribution was determined by co-localization analysis of the translocase of outer mitochondrial membrane 20 (TOM20, the mitochondrial marker) and intercellular adhesion molecule 1 (ICAM1, the uropod marker). The mitochondrial fission was investigated by detecting dynamin-related protein 1 (Drp1) and mitochondrial fission protein 1 (Fis1) using Western blot and immunofluorescence. This study revealed that high-dose NAR (50 mg/kg, i.p.) alleviated pain-like behavior and articular index scores, reduced the serum level of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α), and accounted for CD4+ T lymphocytes that infiltrated into the synovial membrane of the CIA group. Meanwhile, NAR (50 mg/kg, i.p.) suppressed the polarization of spleen CD4+ T lymphocytes, reduced the redistribution of mitochondria in the uropod, and inhibited the expression of Drp1 and Fis1 in the CIA model. Furthermore, the in vitro experiments confirmed that NAR reduced mitochondrial fission, which in turn inhibited the CXCL12-induced polarization and migration of CD4+ T lymphocytes. Our results demonstrated that the flavonoid NAR was a promising drug for the treatment of RA, which could effectively interfere with mitochondrial fission, thus inhibiting the polarization and migration of CD4+ T cells in the synovial membrane.

AGO-bound mature miRNAs are oligouridylated by TUTs and subsequently degraded by DIS3L2.

MicroRNAs (miRNAs) associated with Argonaute proteins (AGOs) regulate gene expression in mammals. miRNA 3' ends are subject to frequent sequence modifications, which have been proposed to affect miRNA stability. However, the underlying mechanism is not well understood. Here, by genetic and biochemical studies as well as deep sequencing analyses, we find that AGO mutations disrupting miRNA 3' binding are sufficient to trigger extensive miRNA 3' modifications in HEK293T cells and in cancer patients. Comparing these modifications in TUT4, TUT7 and DIS3L2 knockout cells, we find that TUT7 is more robust than TUT4 in oligouridylating mature miRNAs, which in turn leads to their degradation by the DIS3L2 exonuclease. Our findings indicate a decay machinery removing AGO-associated miRNAs with an exposed 3' end. A set of endogenous miRNAs including miR-7, miR-222 and miR-769 are targeted by this machinery presumably due to target-directed miRNA degradation.

3' Uridylation Confers miRNAs with Non-canonical Target Repertoires.

Many microRNAs (miRNAs) exist alongside abundant miRNA isoforms (isomiRs), most of which arise from post-maturation sequence modifications such as 3' uridylation. However, the ways in which these sequence modifications affect miRNA function remain poorly understood. Here, using human miR-27a in cell lines as a model, we discovered that a nonfunctional target site unable to base-pair extensively with the miRNA seed sequence can regain function when an upstream adenosine is able to base-pair with a post-transcriptionally added uridine in the miR-27a tail. This tail-U-mediated repression (TUMR) is abolished in cells lacking the uridylation enzymes TUT4 and TUT7, indicating that uridylation alters miRNA function by modulating target recognition. We identified a set of non-canonical targets in human cells that are specifically regulated by uridylated miR-27a. We provide evidence that TUMR expands the targets of other endogenous miRNAs. Our study reveals a function of uridylated isomiRs in regulating non-canonical miRNA targets.

Structural Differences between Pri-miRNA Paralogs Promote Alternative Drosha Cleavage and Expand Target Repertoires.

MicroRNA (miRNA) processing begins with Drosha cleavage, the fidelity of which is critical for downstream processing and mature miRNA target specificity. To understand how pri-miRNA sequence and structure influence Drosha cleavage, we studied the maturation of three pri-miR-9 paralogs, which encode the same mature miRNA but differ in the surrounding scaffold. We show that pri-miR-9-1 has a unique Drosha cleavage profile due to its distorted and flexible stem structure. Cleavage of pri-miR-9-1, but not pri-miR-9-2 or pri-miR-9-3, generates an alternative miR-9 with a shifted seed sequence that expands the scope of its target RNAs. Analyses of low-grade glioma patient samples indicate that the alternative-miR-9 has a potential role in tumor progression. Furthermore, we provide evidence that distortion of pri-miRNA stems induced by asymmetric internal loops correlates with Drosha cleavage at non-canonical sites. Our studies reveal that pri-miRNA paralogs can have distinct functions via differential Drosha processing.

Effect of Lang-Chuang-Ding Decoction () on DNA Methylation of CD70 Gene Promoter in Peripheral Blood Mononuclear Cells of Female Patients with Systemic Lupus Erythematosus.

OBJECTIVE: To investigate the effect of Lang-chuang-ding Decoction (, LCD) on the expression of DNA methylation of CD70 gene promoter in peripheral blood mononuclear cells (PBMCs) of females with systemic lupus erythematosus (SLE). METHODS: PBMCs isolated from female patients with SLE or healthy donors were cultured and treated with LCD medicated serum or normal serum for 24 or 48 h. The mRNA expressions of CD70 gene in PBMCs were detected by reverse transcription polymerase chain reaction (PCR); the DNA methylation of the CD70 gene promoter region was detected by methylation-specific PCR. RESULTS: After treated with medicated serum for 48 h, the mRNA expression levels of CD70 in PBMCs of SLE patients were signifificantly higher than those of healthy donors (P<0.05); the DNA methylation levels of CD70 promoter region in PBMCs of SLE patients treated with medicated serum for 48 h were signifificantly higher than those treated with fetal bovine serum (P<0.01). CONCLUSION: LCD could inhibit CD70 gene expression in PBMCs of SLE patients by promoting the DNA methylation of CD70 gene promoter.

Progressive antigenic drift and phylogeny of human influenza A(H3N2) virus over five consecutive seasons (2009-2013) in Hangzhou, China.

Vaccine efficacy (VE) can be affected by progressive antigenic drift or any new reassortment of influenza viruses. To effectively track the evolution of human influenza A(H3N2) virus circulating in Hangzhou, China, a total of 65 clinical specimens were selected randomly from outpatients infected by A(H3N2) viruses during the study period from November 2009 to December 2013. The results of reduced VE and antigenic drift of the correspondent epitopes (C-D-E to A-B) suggest that the current vaccine provides suboptimal protection against the A(H3N2) strains circulating recently. Phylogenetic analysis of the entire HA and NA sequences demonstrated that these two genes underwent independent evolutionary pathways during recent seasons. The H3-based phylogenetic tree showed that a special strain A/Hangzhou/A289/2012 fell in a cluster among viruses with reduced VE predominantly circulating in 2013. Our findings underscore a possible early warning for the circulation of A(H3N2) variants with antigenic drift during the previous seasons.

Molecular evolution of HA gene of the influenza A H1N1 pdm09 strain during the consecutive seasons 2009-2011 in Hangzhou, China: several immune-escape variants without positively selected sites.

BACKGROUND: Even under immune pressure, the highly active influenza A H1N1 pdm09 variants emerged again in December 2010. Did the variability lead to poor vaccine effectiveness? OBJECTIVES: To study the genetic distance and antigenic drift of the influenza A H1N1 pdm09 strains based on the sequence analysis of HA virus gene segments during consecutive seasons 2009-2011 in Hangzhou, China. STUDY DESIGN: 39 Clinical samples from influenza-like-illness patients with culture-confirmed influenza A H1N1 pdm09 infections were collected over seasons in routine influenza surveillance. The HA gene was amplified and sequenced. A perspective analysis of genetic distance, antigenic drift and positively selected sites were conducted. RESULTS: Analyses of human influenza A H1N1 pdm09 strains isolated in Hangzhou revealed that during the seasons 2009-2011, the antigenic drift had occurred, away from the northern hemisphere 2010/2011 influenza vaccine strain A/California/07/2009. The 2010/2011 viruses cluster in two main branches with a significant genetic distance, characterized by either S202T and S468N, or K180T/I, V216A, P288S, I312V and I389F. Interestingly, the epitopes corresponding to the immune-escape characteristic have altered much, but none of the amino acid substitutions in 2010/2011 variants were positively selected. CONCLUSIONS: The results of genetic surveillance in this study might account for frequent outbreaks of the influenza A H1N1 pdm09 strains since December 2010 and the disappearance after three months circulation. It facilitates early detection of antigenic sites for the virus to escape immunological restraint in 2010/2011 season. Continuous monitoring of antigenic changes is recommended.

[Study on low-temperature alkaline lipase from Bohaisea-9145].

Lipase-overproducing marine yeast Bohaisea-9145 isolated from the Bohai sea region was identified as psychrotrophlic Yarrowia lipolytica. The optimum composition of culture medium was ground soybean 4%, rice bran 4%, peanut power 4%, crude peanut oil 0.5%, MgSO4 0.05%, KH2PPO4 0.2%. The optimum temperature and pH were 26 +/- 1 degrees C pH5.0, the time of fermentation cycle was 23h. The lipase activity was improved to 258.67 U/mL through optimization, four-fold more than that of initial strain. The lipase displayed maximum activity at pH 8.5, 35 degrees C and was stable within the range of pH 4.0 - 9.0 at low temperature. It had high thermosensitivity, good compatibility with familiar metal ions and chemical reagents. It was antioxidant and had strong resistance to high salinity. As a novel marine low-temperature lipase, it has promising prospect in many fields, especially as an additive of detergent.