Mechanism of translational regulation by miR-2 from sites in the 5¢ untranslated region or the open reading frame. Rna

European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
RNA (Impact Factor: 4.94). 10/2010; 16(12):2493-502. DOI: 10.1261/rna.2384610
Source: PubMed


MicroRNAs (miRs) commonly regulate translation from target mRNA 3' untranslated regions (UTRs). While effective miR-binding sites have also been identified in 5' untranslated regions (UTRs) or open reading frames (ORFs), the mechanism(s) of miR-mediated regulation from these sites has not been defined. Here, we systematically investigate how the position of miR-binding sites influences translational regulation and characterize their mechanistic basis. We show that specific translational regulation is elicited in vitro and in vivo not only from the 3'UTR, but equally effectively from six Drosophila miR-2-binding sites in the 5'UTR or the ORF. In all cases, miR-2 triggers mRNA deadenylation and inhibits translation initiation in a cap-dependent fashion. In contrast, single or dual miR-2-binding sites in the 5'UTR or the ORF yield rather inefficient or no regulation. This work represents the first demonstration that 5'UTR and ORF miR-binding sites can function mechanistically similarly to the intensively investigated 3'UTR sites. Using single or dual binding sites, it also reveals a biological rationale for the high prevalence of miR regulatory sites in the 3'UTR.

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Available from: Francesca Moretti, Jan 24, 2014
    • "A single strand of this mature miRNA is loaded into the miRNA-induced silencing complex (miRISC) which allows the miRNA to base-pair with a specifi c target mRNA. Pairing of a miRNA usually occurs within the 3′UTR of the mRNA, although 5′UTRs and open reading frame pairing has also been reported (McIver et al. 2012b ;Moretti et al. 2010). miRNA nucleotides 2–8 of the 5′ end of the RNA commonly form the 'seed region' of the sequence where perfect pairing takes place and which isessential for effi cient target binding (Fig. 6.2). "
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    ABSTRACT: Testicular germ and somatic cells express many classes of small ncRNAs, including Dicer-independent PIWI-interacting RNAs, Dicer-dependent miRNAs, and endogenous small interfering RNA. Several studies have identified ncRNAs that are highly, exclusively, or preferentially expressed in the testis and epididymis in specific germ and somatic cell types. Temporal and spatial expression of proteins is a key requirement of successful spermatogenesis and large-scale gene transcription occurs in two key stages, just prior to transcriptional quiescence in meiosis and then during spermiogenesis just prior to nuclear silencing in elongating spermatids. More than 60 % of these transcripts are then stockpiled for subsequent translation. In this capacity ncRNAs may act to interpret and transduce cellular signals to either maintain the undifferentiated stem cell population and/or drive cell differentiation during spermatogenesis and epididymal maturation. The assignation of specific roles to the majority of ncRNA species implicated as having a role in spermatogenesis and epididymal function will underpin fundamental understanding of normal and disease states in humans such as infertility and the development of germ cell tumours.
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    • "The potential of miRNA regulation of mRNAs through binding sites that occur in 5 ′ UTRs was demonstrated early on (Lytle et al. 2007; Moretti et al. 2010). However, only a few validated examples of naturally occurring 5 ′ UTR miRNA targets exist in the literature to date (Jopling et al. 2005; Lytle et al. 2007; Orom et al. 2008; Lee et al. 2009; Grey et al. 2010; Vora et al. 2013). "
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    ABSTRACT: MicroRNAs (miRNAs) are short noncoding RNAs that regulate the expression of their targets in a sequence-dependent manner. For protein-coding transcripts, miRNAs regulate expression levels through binding sites in either the 3' untranslated region (3' UTR) or the amino acid coding sequence (CDS) of the targeted messenger RNA (mRNA). Currently, for the 5' untranslated region (5' UTR) of mRNAs, very few naturally occurring examples exist whereby the targeting miRNA down-regulates the expression of the corresponding mRNA in a seed-dependent manner. Here we describe and characterize two miR-103a-3p target sites in the 5' UTR of GPRC5A, a gene that acts as a tumor suppressor in some cancer contexts and as an ongocene in other cancer contexts. In particular, we show that the interaction of miR-103a-3p with each of these two 5' UTR targets reduces the expression levels of both GPRC5A mRNA and GPRC5A protein in one normal epithelial and two pancreatic cancer cell lines. By ectopically expressing "sponges" that contain instances of the wild-type 5' UTR targets we also show that we can reduce miR-103a-3p levels and increase GPRC5A mRNA and protein levels. These findings provide some first knowledge on the post-transcriptional regulation of this tumor suppressor/oncogene and present additional evidence for the participation of 5' UTRs in miRNA driven post-transcriptional regulatory control.
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    • "The mechanism by which a miRNA can diminish protein expression is unclear, but several proposals are there from different experimental evidences. miRNAs can interfere with translation process at the stage of initiation (Figure 2) or elongation (Figure 3), or target mRNA may be affected by isolating it from ribosomal machinery [8] [9] [10]. "
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