ABSTRACT
In this issue of Molecular Cell, Denzler et al. (2014) report a quantitative study of microRNA function in adult mouse liver, suggesting that the natural abundance of miRNAs and their binding sites generally excludes the previously proposed regulation of miRNAs by competitive endogenous RNAs (ceRNAs).
Subject(s)
MicroRNAs/genetics , RNA Interference , Animals , MaleABSTRACT
Understanding the function of individual microRNA (miRNA) species in mice would require the production of hundreds of loss-of-function strains. To accelerate analysis of miRNA biology in mammals, we combined recombinant adeno-associated virus (rAAV) vectors with miRNA 'tough decoys' (TuDs) to inhibit specific miRNAs. Intravenous injection of rAAV9 expressing anti-miR-122 or anti-let-7 TuDs depleted the corresponding miRNA and increased its mRNA targets. rAAV producing anti-miR-122 TuD but not anti-let-7 TuD reduced serum cholesterol by >30% for 25 weeks in wild-type mice. High-throughput sequencing of liver miRNAs from the treated mice confirmed that the targeted miRNAs were depleted and revealed that TuDs induced miRNA tailing and trimming in vivo. rAAV-mediated miRNA inhibition thus provides a simple way to study miRNA function in adult mammals and a potential therapy for dyslipidemia and other diseases caused by miRNA deregulation.
Subject(s)
Dependovirus/genetics , Genetic Vectors/genetics , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , Animals , Base Sequence , Binding Sites , Cell Line , Cholesterol/metabolism , Liver/metabolism , Mice , Mice, Inbred C57BL , MicroRNAs/metabolism , Molecular Sequence Data , RNA, Antisense/genetics , RNA, Antisense/metabolism , Recombinant Proteins/geneticsABSTRACT
Small RNAs loaded into Argonaute proteins direct silencing of complementary target mRNAs. It has been proposed that multiple, imperfectly complementary small interfering RNAs or microRNAs, when bound to the 3' untranslated region of a target mRNA, function cooperatively to silence target expression. We report that, in cultured human HeLa cells and mouse embryonic fibroblasts, Argonaute1 (Ago1), Ago3, and Ago4 act cooperatively to silence both perfectly and partially complementary target RNAs bearing multiple small RNA-binding sites. Our data suggest that for Ago1, Ago3, and Ago4, multiple, adjacent small RNA-binding sites facilitate cooperative interactions that stabilize Argonaute binding. In contrast, small RNAs bound to Ago2 and pairing perfectly to an mRNA target act independently to silence expression. Noncooperative silencing by Ago2 does not require the endoribonuclease activity of the protein: A mutant Ago2 that cannot cleave its mRNA target also silences noncooperatively. We propose that Ago2 binds its targets by a mechanism fundamentally distinct from that used by the three other mammalian Argonaute proteins.
Subject(s)
Eukaryotic Initiation Factors/metabolism , RNA Interference , RNA, Small Interfering/genetics , Animals , Base Sequence , Cells, Cultured , Eukaryotic Initiation Factors/deficiency , Humans , Mice , Protein BindingABSTRACT
In this study we describe the identification and characterization of a novel cytosolic protein of the guanine exchange factor (GEF) family. The human cDNA corresponds to predicted human protein FLJ00128/FLJ10357 located on chromosome 14q11.2. The deduced protein sequence contains in its C-terminus a RhoGEF domain followed by a pleckstrin domain. Its N-terminus, central region and RhoGEF/pleckstrin domain are homologous to the recently identified zebrafish Quattro protein, which is involved in morphogenetic movements mediated by the actin cytoskeleton. Based on the homology of our protein's RhoGEF domain to the RhoGEF domains of Trio, Duo and Duet and its homology with Quattro, we named it Solo. The Solo mRNA is ubiquitously expressed but enriched in brain, its expression peaks perinatally and it undergoes extensive alternative splicing. In both myoblasts and neuroblastoma cells, the Solo protein is concentrated around the nucleus.
Subject(s)
Carrier Proteins/genetics , Gene Expression Profiling , Guanine Nucleotide Exchange Factors/genetics , Amino Acid Sequence , Animals , Blood Proteins/genetics , Blood Proteins/metabolism , Blotting, Northern , Blotting, Western , Brain/embryology , Brain/growth & development , Brain/metabolism , Carrier Proteins/metabolism , Cell Line , Cell Line, Tumor , Cloning, Molecular , Cytoplasm/metabolism , DNA, Complementary/chemistry , DNA, Complementary/genetics , Exons , Guanine Nucleotide Exchange Factors/metabolism , Humans , Immunohistochemistry , Immunoprecipitation , Introns , Male , Mice , Molecular Sequence Data , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Reverse Transcriptase Polymerase Chain Reaction , Rho Guanine Nucleotide Exchange Factors , Saccharomyces cerevisiae/genetics , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Two-Hybrid System Techniques , tau Proteins/genetics , tau Proteins/metabolismABSTRACT
Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. The N-terminal domain of the protein interacts with the axonal membrane, and is modulated by differential inclusion of exons 2 and 3. These two tau exons are alternatively spliced cassettes, in which exon 3 never appears independently of exon 2. Previous work with tau minigene constructs indicated that exon 3 is intrinsically suboptimal and its primary regulator is a weak branch point. In this study, we confirm the role of the weak branch point in the regulation of exon 3 but also show that the exon is additionally regulated by a combination of exonic enhancers and silencers. Furthermore, we demonstrate that known splicing regulators affect the ratio of exon 3 isoforms, Lastly, we tentatively pinpoint the site of action of several splicing factors which regulate tau exon 3.
