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1.
Nucleic Acids Res ; 44(6): 2873-87, 2016 Apr 07.
Article in English | MEDLINE | ID: mdl-26809675

ABSTRACT

The mechanism by which micro (mi)RNAs control their target gene expression is now well understood. It is however less clear how the level of miRNAs themselves is regulated. Under specific conditions, abundant and highly complementary target RNA can trigger miRNA degradation by a mechanism involving nucleotide addition and exonucleolytic degradation. One such mechanism has been previously observed to occur naturally during viral infection. To date, the molecular details of this phenomenon are not known. We report here that both the degree of complementarity and the ratio of miRNA/target abundance are crucial for the efficient decay of the small RNA. Using a proteomic approach based on the transfection of biotinylated antimiRNA oligonucleotides, we set to identify the factors involved in target-mediated miRNA degradation. Among the retrieved proteins, we identified members of the RNA-induced silencing complex, but also RNA modifying and degradation enzymes. We further validate and characterize the importance of one of these, the Perlman Syndrome 3'-5' exonuclease DIS3L2. We show that this protein interacts with Argonaute 2 and functionally validate its role in target-directed miRNA degradation both by artificial targets and in the context of mouse cytomegalovirus infection.


Subject(s)
Exoribonucleases/genetics , MicroRNAs/genetics , Nucleotidyltransferases/genetics , RNA Stability , RNA, Messenger/genetics , Animals , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Base Sequence , Biotinylation , Cell Line, Tumor , Cytomegalovirus/genetics , Cytomegalovirus Infections/genetics , Cytomegalovirus Infections/virology , Exoribonucleases/metabolism , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Hepatocytes/cytology , Hepatocytes/metabolism , Humans , Mice , MicroRNAs/antagonists & inhibitors , MicroRNAs/metabolism , Molecular Sequence Data , Nucleotidyltransferases/metabolism , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/metabolism , Polynucleotide Adenylyltransferase , RNA, Messenger/metabolism , mRNA Cleavage and Polyadenylation Factors/genetics , mRNA Cleavage and Polyadenylation Factors/metabolism
2.
PLoS One ; 9(10): e111266, 2014.
Article in English | MEDLINE | ID: mdl-25360821

ABSTRACT

We evaluated micro (mi) RNA-mediated regulation of BAFF expression in fibroblasts using two concomitant models: (i) synovial fibroblasts (FLS) isolated from healthy controls (N) or Rheumatoid Arthritis (RA) patients; (ii) human dermal fibroblasts (HDF) isolated from healthy controls (N) or Systemic Sclerosis (SSc) patients. Using RT-qPCR and ELISA, we first showed that SScHDF synthesized and released BAFF in response to Poly(I:C) or IFN-γ treatment, as previously observed in RAFLS, whereas NHDF released BAFF preferentially in response to IFN-γ. Next, we demonstrated that miR-30a-3p expression was down regulated in RAFLS and SScHDF stimulated with Poly(I:C) or IFN-γ. Moreover, we demonstrated that transfecting miR-30a-3p mimic in Poly(I:C)- and IFN-γ-activated RAFLS and SScHDF showed a strong decrease on BAFF synthesis and release and thus B cells survival in our model. Interestingly, FLS and HDF isolated from healthy subjects express higher levels of miR-30a-3p and lower levels of BAFF than RAFLS and SScHDF. Transfection of miR-30a-3p antisense in Poly(I:C)- and IFN-γ-activated NFLS and NHDF upregulated BAFF secretion, confirming that this microRNA is a basal repressors of BAFF expression in cells from healthy donors. Our data suggest a critical role of miR-30a-3p in the regulation of BAFF expression, which could have a major impact in the regulation of the autoimmune responses occurring in RA and SSc.


Subject(s)
Arthritis, Rheumatoid/genetics , Arthritis, Rheumatoid/pathology , B-Cell Activating Factor/biosynthesis , Fibroblasts/metabolism , MicroRNAs/genetics , Scleroderma, Systemic/genetics , Scleroderma, Systemic/pathology , 3' Untranslated Regions/genetics , Arthritis, Rheumatoid/immunology , B-Cell Activating Factor/genetics , B-Lymphocytes/cytology , B-Lymphocytes/drug effects , Base Sequence , Cell Survival/drug effects , Down-Regulation/drug effects , Down-Regulation/genetics , Female , Fibroblasts/drug effects , Humans , Interferon-gamma/pharmacology , Male , Middle Aged , Poly I-C/pharmacology , Scleroderma, Systemic/immunology
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