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The Mir-199–Dynamin Regulatory Axis Controls Receptor-Mediated Endocytosis Juan F

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The Mir-199–Dynamin Regulatory Axis Controls Receptor-Mediated Endocytosis Juan F © 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 3197-3209 doi:10.1242/jcs.165233 RESEARCH ARTICLE The miR-199–dynamin regulatory axis controls receptor-mediated endocytosis Juan F. Aranda1,2, Alberto Canfrán-Duque1,2, Leigh Goedeke1,2, Yajaira Suárez1,2 and Carlos Fernández-Hernando1,2,* ABSTRACT mechanism for the selective uptake of essential nutrients such as Small non-coding RNAs (microRNAs) are important regulators of low-density lipoprotein (LDL), through the LDL receptor (LDLR) gene expression that modulate many physiological processes; (Brown and Goldstein, 1986), or iron, through transferrin receptor however, their role in regulating intracellular transport remains (TfR) (Harding et al., 1983). Thus, factors that affect RME have a largely unknown. Intriguingly, we found that the dynamin (DNM) direct effect on these receptors, and, in the case of LDLR, to regulate genes, a GTPase family of proteins responsible for endocytosis in intracellular cholesterol levels. In both the LDLR and TfR eukaryotic cells, encode the conserved miR-199a and miR-199b internalization processes, clathrin plays a key role during the family of miRNAs within their intronic sequences. Here, we formation of coated vesicles (Moore et al., 1987). Once vesicles are demonstrate that miR-199a and miR-199b regulate endocytic internalized, their passage through a broad endosomal compartment transport by controlling the expression of important mediators of system is required; first they are rapidly transported into early endocytosis such as clathrin heavy chain (CLTC), Rab5A, low- endosomes, where Rab5A is a key regulator (Nielsen et al., 1999), density lipoprotein receptor (LDLR) and caveolin-1 (Cav-1). and subsequently to late endosomes and lysosomes. Whichever Importantly, miR-199a-5p and miR-199b-5p overexpression route of entry is used, a crucial step in endocytosis and intracellular markedly inhibits CLTC, Rab5A, LDLR and Cav-1 expression, thus transport is the formation of endocytic vesicles, which specifically preventing receptor-mediated endocytosis in human cell lines (Huh7 requires the participation of the dynamin (DNM) GTPase family to and HeLa). Of note, miR-199a-5p inhibition increases target gene promote their scission and fission from the plasma membrane expression and receptor-mediated endocytosis. Taken together, our (Ferguson and De Camilli, 2012; Jones et al., 1998; Roux et al., work identifies a new mechanism by which microRNAs regulate 2006; Takei et al., 1995). DNM proteins are also involved in other intracellular trafficking. In particular, we demonstrate that the DNM, membrane remodeling processes, such as fission of clathrin-coated miR-199a-5p and miR-199b-5p genes act as a bifunctional locus that vesicles from the trans-Golgi network (TGN) (Cao et al., 2000) and regulates endocytosis, thus adding an unexpected layer of complexity membrane ruffling through the interaction with actin nucleators (Gu in the regulation of intracellular trafficking. et al., 2010). In mammals, the DNM gene family is encoded by three separate genes: DNM1, DNM2 and DNM3. Although all three DNM KEY WORDS: miRNA, miR-199, Endocytosis, LDLR genes share a high degree of sequence similarity, thus resulting in proteins containing similar domains, they differ in their tissue- INTRODUCTION specific expression (Urrutia et al., 1997). Endocytosis is an essential process in cell physiology by which It is presently accepted, that small non-coding RNAs, known as eukaryotic cells take up macromolecules and particles from the microRNAs (miRNAs), are key regulators of several cellular surrounding medium. Many physiological processes, including cell processes (Bushati and Cohen, 2007). This regulatory control is migration, angiogenesis, metabolism and development, depend on carried out through repression of gene expression at the post- proper functioning of endocytosis and thus cells have developed transcriptional level by base pairing with complementary regions multiple mechanisms to ensure proper intracellular trafficking and mainly within the 3′ untranslated regions (3′UTRs) of target endocytosis (Fernández-Rojo et al., 2012; Gu et al., 2011; Lee et al., mRNAs, thus promoting mRNA degradation, translational 2014; Mousley et al., 2012; Parachoniak et al., 2011). There are repression, or both (Ambros, 2004; Bartel, 2009; Filipowicz et al., multiple pathways of endocytosis into cells, including, clathrin- 2008). Most miRNAs are first transcribed into long transcripts of dependent, caveolin-dependent and clathrin- and caveolin- primary miRNAs (pri-miRNAs) that are then processed independent internalization. In all of them, the material to be sequentially in the nucleus by Drosha and DGCR8 to generate internalized is surrounded by an area of plasma membrane, which pre-miRNAs, and by Dicer in the cytoplasm to generate the mature then buds off inside the cell to form a vesicle containing the ingested ∼22 nt miRNA duplex (Lee et al., 2003) that, after strand selection, material that is delivered to intracellular organelles and cytosol mediates the targeting activity when incorporated into the RISC (McMahon and Boucrot, 2011). The best-characterized form of this complex (Chendrimada et al., 2007). There is mounting evidence process is receptor-mediated endocytosis (RME), which provides a that suggests that both strands, the guide or ‘5p’ strand as well as the miRNA* (also known as the passenger or ‘3p’ strand) have important regulatory activity (Chamorro-Jorganes et al., 2014; 1Integrative Cell Signaling and Neurobiology of Metabolism Program, Section of Comparative Medicine, Yale University School of Medicine, New Haven, CT 06510, Goedeke et al., 2013). Approximately half of the miRNA genes can USA. 2Vascular Biology and Therapeutics Program, Yale University School of be found in intergenic regions, whereas the intragenic miRNAs are Medicine, New Haven, CT 06510, USA. predominantly located inside introns and usually oriented on the *Author for correspondence ([email protected]) same DNA strand of the host gene (Saini et al., 2007). Intergenic miRNA genes have their own promoter region and, thus, their Received 30 October 2014; Accepted 2 July 2015 expression is regulated by the same molecular mechanisms that Journal of Cell Science 3197 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3197-3209 doi:10.1242/jcs.165233 control the expression of protein-coding genes. By contrast, same- conserved. Given this seed sequence conservation, we focused strand intronic miRNAs are co-transcribed with their host gene our study on miR-199a-5p. (Rodriguez et al., 2004) and then processed to finally become Mammalian miRNAs are present in the genome either as mature functional miRNAs. A number of studies have shown that independent transcriptional units or embedded within the introns intronic miRNAs localized in the same orientation as their host of protein-coding genes. To determine whether the expression of genes usually cooperate with them to regulate similar cellular the miR-199a/b family members and DNM genes are co-regulated, functions (Rayner et al., 2010; van Rooij et al., 2009). However, we measured their expression in different human tissues. As seen in exceptions to this common scheme of co-transcription have been Fig. 1C and supplementary material Fig. S1B, we observed that the reported. In fact, ∼26% of intronic miRNAs are antisense orientated mature miR-199a-5p (miR-199a1-5p and miR-199a2-5p), miR- and transcribed independently of their host genes (Rodriguez et al., 199b-5p and their respective precursors (pre-miR-199a-1, pre-miR- 2004; Siegel et al., 2009). Despite this, intronic miRNA can support 199a-2 and pre-miR-199b) (supplementary material Fig. S1C) were the function of its host gene by silencing genes that are functionally widely expressed in most tissues. Remarkably, compared with other antagonistic to the host or act synergistically with the host by tissues, mature miR-199a-5p was expressed at very low levels in the coordinating the expression of genes with related functions. brain, which expresses high levels of DNM3 (Fig. 1C). Similarly, Interestingly, the miR-199a and miR-199b (hereafter denoted the expression of miR-199b-5p in the brain is markedly reduced miR-199a/b) family members are encoded within introns of the compared with other tissues (supplementary material Fig. S1B). DNM genes in the opposite orientation to the host gene. The miR- Interestingly, miR-199b-5p levels were inversely correlated with 199a/b family is composed of three members, miR-199a1, miR- DNM1 expression (supplementary material Fig. S1B), suggesting 199a2 and miR-199b located within the DNM2, DNM3 and DNM1 that miR-199b-5p is regulated independently of its host gene. genes, respectively. MiR-199a/b gene sequences exhibit high We next sought to ascertain the potential function of miR-199a/b- conservation across species and share the same seed sequence, 5p. To this end, we employed a combination of bioinformatic thereby potentially targeting the same group of genes. Interestingly, algorithms [Targetscan (http://www.targetscan.org) and miRanda predicted target genes for miR-199a/b-5p (guide) strands are (http://www.microrna.org)] that predict miRNA targets largely broadly conserved among species compared to the miR-199a/b-3p based on the ability of the miRNA sequence to undergo specific (passenger) strand. Therefore, here, we investigated potential miR- base-pairing within the putative 3′UTR target. The predicted 199a/b-5p target
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