ABSTRACT
MicroRNA (miRNA) genes produce three major RNA products; primary (pri-), precursor (pre-), and mature miRNAs. Each product includes sequences complementary to cognate targets, thus they all can in principle interact with the targets. In a recent study we showed that pri-miRNAs play a direct role in target recognition and repression in the absence of functional mature miRNAs. Here we examined the functional contribution of pri-miRNAs in target regulation when full-length functional miRNAs are present. We found that pri-let-7 loop nucleotides control the production of the 5' end of mature miRNAs and modulate the activity of the miRNA gene. This insight enabled us to modulate biogenesis of functional mature miRNAs and dissect the causal relationships between mature miRNA biogenesis and target repression. We demonstrate that both pri- and mature miRNAs can contribute to target repression and that their contributions can be distinguished by the differences between the pri- and mature miRNAs' sensitivity to bind to the first seed nucleotide. Our results demonstrate that the regulatory information encoded in the pri-/pre-miRNA loop nucleotides controls the activities of pri-miRNAs and mature let-7 by influencing pri-miRNA and target complex formation and the fidelity of mature miRNA seed generation.
Subject(s)
MicroRNAs/genetics , Nucleotides/genetics , RNA Precursors/genetics , RNA Processing, Post-Transcriptional , Animals , Base Sequence , Blotting, Northern , Caenorhabditis elegans/genetics , Cell Line , Gene Expression Regulation , Humans , MicroRNAs/chemistry , MicroRNAs/metabolism , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , Nucleotides/metabolism , RNA Precursors/chemistry , RNA Precursors/metabolismABSTRACT
Major RNA products of a microRNA (miRNA) gene--the long primary transcript (pri-miRNA), the â¼70-nucleotide (nt) precursor miRNA (pre-miRNA), and the â¼21-nt mature miRNA--all contain the same sequence required for target gene recognition. Thus, it is intrinsically difficult to discern the contribution of individual RNA species or to rule out a function of miRNA precursor species in target repression. Here, we describe a novel approach to dissect the functional contribution of pri-miRNA without compromising important cellular pathways. We show that pri-let-7 has a direct function in target repression in the absence of properly processed mature let-7. Moreover, we show that loop nucleotides provide regulatory controls of the activity of pri-let-7 by modulating interactions between pri-let-7 and target RNAs in vitro and in vivo. Finally, we show that human let-7a-3 pri-miRNA can directly interact with target mRNAs. These findings illustrate that the regulatory information encoded in structured pri-miRNAs may be translated into function through direct interactions with target mRNAs.
Subject(s)
Gene Expression Regulation/physiology , MicroRNAs/physiology , RNA, Messenger/metabolism , Animals , Base Sequence , Blotting, Northern , Caenorhabditis elegans , Cell Line , Gene Expression Regulation/genetics , Humans , Mice , MicroRNAs/biosynthesis , MicroRNAs/metabolism , Molecular Sequence Data , Mutation/genetics , Polymerase Chain ReactionABSTRACT
Recognition of immunoglobulin G (IgG) by surface receptors for the Fc domain of immunoglobulin G (Fcgamma), FcgammaRs, can trigger both humoral and cellular immune responses. Two human cytomegalovirus (HCMV)-encoded type I transmembrane receptors with Fcgamma-binding properties (vFcgammaRs), gp34 and gp68, have been identified on the surface of HCMV-infected cells and are assumed to confer protection against IgG-mediated immunity. Here we show that Fcgamma recognition by both vFcgammaRs occurs independently of N-linked glycosylation of Fcgamma, in contrast with the properties of host FcgammaRs. To gain further insight into the interaction with Fcgamma, truncation mutants of the vFcgammaR gp68 ectodomain were probed for Fcgamma binding, resulting in localization of the Fcgamma binding site on gp68 to residues 71 to 289, a region including an immunoglobulin-like domain. Gel filtration and biosensor binding experiments revealed that, unlike host FcgammaRs but similar to the herpes simplex virus type 1 (HSV-1) Fc receptor gE-gI, gp68 binds to the C(H)2-C(H)3 interdomain interface of the Fcgamma dimer with a nanomolar affinity and a 2:1 stoichiometry. Unlike gE-gI, which binds Fcgamma at the slightly basic pH of the extracellular milieu but not at the acidic pH of endosomes, the gp68/Fcgamma complex is stable at pH values from 5.6 to pH 8.1. These data indicate that the mechanistic details of Fc binding by HCMV gp68 differ from those of host FcgammaRs and from that of HSV-1 gE-gI, suggesting distinct functional and recognition properties.
