© 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– 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) 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 , a GTPase family of responsible for endocytosis in intracellular levels. In both the LDLR and TfR eukaryotic cells, encode the conserved miR-199a and miR-199b internalization processes, 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 -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 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 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 -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 genes using several miRNA target bioinformatic miR-199a/b-5p target genes were assigned to several functional algorithms. Importantly, we identified putative binding sites for annotation clusters and networks as shown in Fig. 1D. Interestingly, miR-199a/b-5p in the 3′UTR of genes involved in vesicle-mediated using software analysis (Panther, http://www. transport and endocytosis. Of note, our present findings indicate that pantherdb.org/) (Thomas et al., 2003), and the protein–protein miR-199a/b-5p regulates the expression of multiple genes interaction database, String (http://string-db.org/) (Szklarczyk et al., participating in clathrin-dependent endocytosis (Cltc, Rab5A, 2011), we observed that the most represented cluster was associated Rab21 and Ldlr) and clathrin-independent endocytosis (Cav-1). with genes involved in cellular transport (Fig. 1D). Among them Furthermore, we demonstrate that miR-199a/b-5p inhibits clathrin- specifically, miR-199a/b-5p was predicted to target a vast network mediated endocytosis through the regulation of CLTC, Rab5A and of genes associated with endocytic functions, including CLTC, Cav- Rab21 expression, affecting the normal function of receptors 1, Rab5A, LDLR and Rab21 (Fig. 1D) (Bucci et al., 1994; Dorsey located in the plasma membrane such as LDLR and TfR. Taken et al., 2007; Doyon et al., 2011; Pellinen et al., 2008; Simpson et al., together, our work shows a new mechanism by which miRNAs 2004; Singh et al., 2003). This intriguing observation led us to regulate intracellular trafficking. In particular, we describe that the investigate the biological role of miR-199a/b-5p in controlling DNM genes along with miR-199 act as a bifunctional locus receptor-mediated endocytosis. encoding the DNM, a GTPase that is a crucial mediator of endocytosis, and miR-199a/b, which also regulates intracellular MiR-199a-5p regulates the expression of endocytosis trafficking, thus adding an unexpected layer of complexity in the mediators regulation of endocytosis. To evaluate the effect of miR-199a/b-5p on CLTC, LDLR, Rab5A and Rab21 expression, we transfected human hepatic Huh7 cells RESULTS with synthetic miR-199a-5p mimics. As seen in Fig. 2A, CLTC, miR-199a/b-5p are potential regulators of transport and LDLR, Rab5A and Rab21 mRNA expression was inhibited in cells vesicle-mediated trafficking processes overexpressing miR-199a-5p compared to cells transfected with a While investigating the genomic location of miRNAs encoded non-targeting control miRNA mimic. Conversely, Huh7 cells in the , we noted the intriguing presence of a transfected with antisense oligonucleotides directed against miR- highly conserved miRNA family, miR-199a/b, embedded 199a-5p (inh-199a-5p) had significantly increased CLTC, LDLR within the intronic sequences of the DNM genes (Fig. 1A). The and Rab5A mRNA expression compared to cells transfected with a miR-199a/b family consists of three members, miR-199a-1, non-targeting control inhibitor (Fig. 2B). Consistent with this, miR-199a-2 and miR-199b, which are transcribed from conserved overexpression of miR-199a-5p markedly reduced CLTC, LDLR, antisense intronic transcripts of the DNM2 locus (human Rab5A and Rab21 protein expression (Fig. 2C, upper panels). 19), DNM3 locus (human chromosome 1) and Moreover, inhibition of endogenous miR-199a-5p increased LDLR, DNM1 locus (human chromosome 9), respectively (Fig. 1A). Rab5A and Rab21 protein in Huh7 cells, suggesting that miR-199a/ Human miR-199a1-5p and miR-199a2-5p have identical mature b-5p plays a physiological role in regulating cellular endocytosis sequences, but the miR-199b-5p mature sequence differs in two (Fig. 2C, lower panels). nucleotides outside of the seed sequence (Fig. 1B). The miR-199a- CLTC, LDLR, Rab5A and Rab21 have one or more predicted 5p mature sequences show higher conservation among vertebrate miR-199a/b-5p-binding sites that are conserved across mammals species than miR-199b-5p (supplementary material Fig. S1A), (Fig. 2D; supplementary material Fig. S1D). To determine whether indicating that miR-199a1 and miR-199a2 are evolutionarily miR-199a-5p specifically targets the 3′UTR of CLTC, LDLR, Journal of Cell Science

3198 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3197-3209 doi:10.1242/jcs.165233

Fig. 1. miR-199 and DNM loci genomic location, human tissue expression and bioinformatic analysis of predicted miR-199a/b target genes. (A) Schematic representation of genomic location of DNM genes and their miR-199a/b intronic family members. Intronic miR-199a2-5p is co-transcribed in a cluster with miR-214. Note that the three members of miR-199a/b are encoded on the opposite strand to the DNM host genes. (B) Sequence alignment between human miR-199 family members. Seed sequences are indicated in boxes. The red color in the miR-199b sequence indicates those nucleotides that have diverged with respect to miR-199a. Stem loop, mature 5p and 3p forms are indicated. (C) Gene expression analyses (qRT-PCR) of miR-199a-5p, DNM2 and DNM3 in different human tissues normalized to snoRD68 for miR-199a-5p and GAPDH, for the DNM2 and DNM3 genes. Data are expressed relative to the amount of miR-199a-5p transcripts expressed in adipose tissue. Results are mean±s.e.m. for three experiments. (D) Gene ontology analysis of the predicted miR-199a/b target genes using Panther software (upper left and bottom panels). A protein–protein interaction analysis scheme of selected predicted miR-199a-5p target genes using String 9.1 software and Navigator 2.2. is shown in the upper right panel.

Rab5A and Rab21, we cloned the entire 3′UTRof the aforementioned mimic (supplementary material Fig. S2A,C, left panels). A similar genes into a luciferase reporter vector and assessed whether miR- effect was observed when we analyzed the expression of Cav-1 199a-5p overexpression could reduce luciferase reporter activity. As by immunofluorescence (supplementary material Fig. S2D). seen in Fig. 2E, miR-199a-5p markedly repressed CLTC, LDLR and Importantly, inhibition of endogenous miR-199a-5p led to an Rab21 3′UTR activity. Surprisingly, Rab5A 3′UTR activity was not increase in Cav-1 expression (supplementary material Fig. S2A,C, influenced by miR-199a-5p overexpression (Fig. 2E), despite its right panels; supplementary material Fig. S2D). We also confirmed inhibitory effect on Rab5A mRNA and protein expression (Fig. 2A, that miR-199a-5p directly targets Cav-1 by assessing the Cav-1 C). As expected, mutation of the miR-199a-5p target sites relieved 3′UTR luciferase activity in cells transfected with miR-199a-5p miR-199a-5p repression of CLTC, LDLR and Rab21 3′UTR activity, mimics (supplementary material Fig. S2B). Taken together, these consistent with a direct interaction of miR-199a-5p with these sites results identify CLTC, LDLR, Rab21 and Cav-1 as direct targets of (Fig. 2E). In addition to these genes, we also identified Cav-1 as a miR-199a-5p. predicted miR-199a-5p target gene. As Cav-1 is expressed at very low levels in hepatic cell lines, we analyzed the role of miR-199a-5p MiR-199a-5p inhibits RME in regulating Cav-1 expression in HeLa cells, which express CLTC, Rab5A and Rab21 are essential components of RME, a significantly higher levels of this protein. The results show that process by which cells internalize molecules (de Hoop et al., 1994; miR-199a-5p overexpression markedly reduced Cav-1 mRNA and Doyon et al., 2011; Simpson et al., 2004). RME is widely used for protein expression compared with cells transfected with control the specific uptake of substances required by the cell, including LDL Journal of Cell Science

3199 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3197-3209 doi:10.1242/jcs.165233

Fig. 2. miR-199a/b regulates LDLR, CLTC, Rab5A and Rab21 expression in Huh7 cells. (A,B) qRT-PCR analysis of LDLR, CLTC, Rab5A and Rab21 expression in Huh7 cells transfected with non-targeting control mimic (CM), miR-199a-5p mimic, or control inhibitor (CI) and miR-199a-5p inhibitor. (C) Western blot analysis of LDLR, CLTC, Rab5A and Rab21 in Huh7 cells transfected with control mimic or miR-199a-5p mimic (upper panels) and control inhibitor or inh- 199a-5p (lower panels). A densitometry analysis is shown in the corresponding histograms; Hsp90 was used as a loading control. (D) Human LDLR, CLTC, Rab21 and Rab5a 3′UTR containing the indicated point mutations (PM) in the miR-199a/b-5p target sites. (E) Luciferase reporter activity in COS7 cells transfected with control mimic or miR-199a-5p mimic and the indicated human 3′UTR containing or not (wild-type, WT) the indicated point mutation in the target miR-199a-5p-binding sites. In A and B, data are expressed as mean±s.e.m. and representative of ≥3 experiments in triplicate. In A–C, data are expressed as mean±s.e.m. and representative of ≥3 experiments performed in duplicate. In E, data are expressed as a percentage of 3′UTR activity of control mimic (±s.e.m.) and are representative of ≥3 experiments performed in triplicate. *P≤0.05. through LDLR (Kang and Folsch, 2011; Mettlen et al., 2010), and transfected with miR-199a-5p at longer time points (2 and 4 h) and in iron, through the TfR (Gan et al., 2002; Tosoni et al., 2005). The cells treated with Dynasore, a widely used DNM inhibitor (Macia LDLR binds to LDL particles and mediates their endocytosis et al., 2006) (Fig. 3C; supplementary material Fig. S3A,C). By together with CLTC, which is necessary for coated vesicle contrast, cells treated with the miR-199a-5p inhibitor (inh-199a-5p) formation, and the Rab5A and Rab21 GTPases, which are had increased DiI–LDL uptake after 2 and 4 h (supplementary involved in regulating vesicle trafficking in the early endosomal material Fig. S3B,C). Treatment of Huh7 cells with U18666A, a compartment (Semerdjieva et al., 2008; Simpson et al., 2004). Our compound that blocks cholesterol trafficking from late endosomes previous results suggest that miR-199a-5p controls LDLR activity and lysosomes to the endoplasmic reticulum (ER), resulting in by directly targeting the LDLR (Fig. 2) but also by regulating its enhanced processing of sterol regulatory binding protein 2 (SREBP2), endocytosis through repression of Rab5A, Rab21 and CLTC and increased expression of LDLR (Liscum and Faust, 1989) was (Fig. 2). Therefore, to functionally assess the role of miR-199a-5p used as a positive control (supplementary material Fig. S3B, in regulating LDLR activity in human hepatic cells, we right panel; supplementary material Fig. S3D). To further confirm overexpressed or inhibited miR-199a-5p and examined the effect of miR-199a-5p in regulating LDLR expression and fluorescence-labeled LDL (DiI–LDL) binding (4°C for 2 h) and activity, we next assessed LDLR–antibody internalization by uptake (37°C for 5, 15 and 30 min) by flow cytometry (Fig. 3A). immunofluorescence. As seen in supplementary material Fig. S4, Transfection of Huh7 cells with miR-199a-5p mimics markedly we found a marked reduction in LDLR internalization as well as a reduced DiI–LDL-specific uptake at different time points. concomitant decrease in CLTC staining in cells transfected with Importantly, the reduction in DiI–LDL internalization at 30 min miR-199a-5p mimics compared to that in control mimic. was significantly greater (34%) than the DiI–LDL binding before Upon internalization, LDL is delivered first to early endosomes incubating cells at 37°C (22%) (Fig. 3B). These results suggest that and then to lysosomes where LDL-derived cholesteryl esters are both DiI–LDL binding and uptake are regulated by miR-199a-5p. As hydrolyzed to unesterified cholesterol (Brown and Goldstein, expected, DiI–LDL uptake was significantly reduced in Huh7 cells 1986). To ascertain whether miR-199a-5p influences intracellular Journal of Cell Science

3200 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3197-3209 doi:10.1242/jcs.165233

Fig. 3. miR-199a/b-5p regulates the LDLR activity. (A) Schematic diagram showing timecourse experiment followed in B. (B) Flow cytometry analysis of DiI–LDL uptake in Huh7 cells transfected with a control mimic (CM) or miR-199a-5p mimic and incubated with 30 μg/ml DiI–LDL for the indicated times at 37°C. Data correspond to the MFI (mean±s.e.m.) of three experiments. a.u.f., arbitrary units of fluorescence. (C) DiI–LDL uptake in Huh7 cells treated or not with Dynasore for 2 h. Data are expressed as a percentage of the control (in the absence of Dynasore). Data correspond to the mean±s.e.m. of three experiments. (D) Representative confocal immunofluorescence images of Huh7 cells transfected as indicated and subjected to 10–30 min of DiI–LDL uptake at 37°C, before being fixed and stained for the early endosome antigen-1 (EEA1), Rab11 and the lysosomal marker CD63, and with TOPRO for the nuclei. Scale bars: 10 μm. (E) Histograms showing colocalization analysis of indicated markers and DiI–LDL in Huh7 cells transfected as indicated, by measuring the Mander’s coefficient. Data are representative of ≥3 experiments. *P≤0.05; #P>0.05. trafficking of LDL-derived cholesterol, we transfected Huh7 cells DiI–LDL for 20 min, we found a little colocalization of DiI–LDL with a control mimic or a miR-199a-5p mimic and assessed DiI– with CD63 in cells transfected with miR-199-5p after 30 min of LDL uptake and subcellular localization by immunofluorescence. incubation with DiI–LDL at 37°C (Fig. 3D,E). We also observed a Interestingly, we observed that overexpression of miR-199a-5p marked reduction in DiI–LDL and CD63 colocalization in cells resulted in accumulation of DiI–LDL particles in the early transfected with miR-199a-5p at 4 h after DiI–LDL incubation endosomal compartment as seen by co-staining with the EEA1 (supplementary material Fig. S3C). As expected, cells treated with marker at early time points (Fig. 3D,E). We also analyzed the U18666A accumulated DiI–LDL in the lysosome compartments, intracellular colocalization of DiI–LDL with Rab11, a recycling whereas Dynasore significantly reduced LDL internalization and endosomal marker, and with the late endosome and lysosomal colocalization with lysosomal CD63 protein (supplementary compartment marker CD63 during the timecourse experiment. material Fig. S3D). Transfection of cells with Inh-199a-5p had no Although miR-199a-5p levels did not influence the colocalization significant effect on the DiI–LDL and CD63 colocalization of Rab11 and DiI–LDL after culturing the cells in the presence of (supplementary material Fig. S3C, right panels). Given that the Journal of Cell Science

3201 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3197-3209 doi:10.1242/jcs.165233

Fig. 4. MiR-199a-5p regulates free cholesterol intracellular localization. Representative immunofluorescence analysis of free cholesterol (Filipin), F-actin and DiI–LDL in Huh7 cells transfected with non-targeting control miRNA (CM), miR-199a-5p mimic or inh-199a-5p and incubated with 30 μg/ml DiI–LDL for 1 h at 37°C. Fluorescence intensity plots are shown in the right panels. PM, plasma membrane. White arrows indicate the region of the cell subjected to image analysis. Scale bars: 10 μm. cholesterol uptake in the cells is mediated mainly by the Importantly, we found that miR-199a-5p overexpression in LDLR– internalization of LDL through LDLR, we next assessed the GFP-positive cells impaired surface internalization of LDLR upon intracellular location of cholesterol using Filipin, a dye that stains engagement with antibodies and incubation at 37°C (Fig. 5B). unesterified cholesterol. Consistent with the diminished LDL These results suggest that the reduction of DiI–LDL uptake is uptake observed in miR-199a-5p-transfected cells, we found a mediated by two mechanisms; direct inhibition of LDLR expression striking accumulation of free cholesterol at the plasma membrane and the repression of numerous components of the endocytic compared with that in cells treated with control mimic (Fig. 4, machinery. middle panel and intensity plot). Inhibition of endogenous miR- Given the direct effect of miR-199a-5p on LDLR expression and 199a-5p expression showed a similar free cholesterol distribution to activity, we wondered whether or not other receptor-mediated that in Huh7 cells transfected with control mimic (Fig. 4). Taken processes would be affected by miR-199a-5p. Therefore we next together, these results suggest that cells overexpressing miR-199a- assessed the effect of miR-199a-5p on TfR endocytosis. TfR 5p have a trafficking defect that causes missorting of LDL particles regulates the import of the transferrin–iron complex through after LDLR internalization. clathrin-mediated endocytosis (Killisch et al., 1992). To this end, Because miR-199a-5p inhibits numerous components of the we transfected human epithelial HeLa cells with miR-199a-5p and endocytic pathway as well as LDLR, the contribution of miR-199a- incubated them with FITC-conjugated transferrin at various time 5p in regulating RME by assessing DiI–LDL uptake could be points. As shown in Fig. 6A, miR-199a-5p significantly inhibited influenced by the significant reduction in LDLR expression transferrin internalization as assessed on a single-cell basis by flow observed in miR-199a-5p-overexpressing cells. To avoid this cytometry. As expected, Dynasore treatment also inhibited caveat, we transfected Huh7 cells with a LDLR–GFP cDNA transferrin internalization (Fig. 6A). This effect was independent construct that lacked the 3′UTR, thereby making it resistant to the of the total and surface TfR expression levels (Fig. 6B,D), inhibitory action of miR-199a-5p, and assessed DiI–LDL cellular suggesting that the observed internalization defect is due to other localization in cells transfected with miR-199a-5p mimics. The endocytic proteins rather than the absence of TfR. To rule out the results show that miR-199a-5p overexpression caused a marked possibility that miR-199a-5p might also be affecting exocytosis, we retention of LDLR–GFP in the plasma membrane as revealed by transfected HeLa cells with miR-199a-5p mimics for 48 h and then actin co-staining, compared to cells transfected with a control mimic treated them with Dynasore. As seen in Fig. 6A, transferrin–FITC (Fig. 5A, intensity plots in right panels). We further analyzed LDLR internalization under these conditions did not change compared to internalization by flow cytometry. To this end, Huh7 cells were cells treated with Dynasore only, suggesting that exocytosis was not transfected with control mimic or miR-199a-5p and LDLR–GFP affected by miR-199a-5p. We next analyzed the intracellular construct and incubated with an anti-LDLR antibody (labeled localization of transferrin–FITC after miR-199a-5p transfection with phycoerythrin) for 2 h at 4°C. Then, cells were incubated at using confocal microscopy. As expected, miR-199a-5p-

37°C for different times to allow LDLR–antibody internalization. overexpressing cells showed a reduced Rab5 staining compared Journal of Cell Science

3202 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3197-3209 doi:10.1242/jcs.165233

Fig. 5. MiR-199a-5p regulates internalization of LDLR. (A) Representative confocal images of DiI–LDL, LDLR–GFP and F-actin expression in Huh7 cells co-transfected with LDLR–GFP and control mimic (CM) or miR-199a-5p mimic and incubated with 30 μg/ml DiI–LDL for 1 h at 37°C. Scale bars: 10 μm (main images); 2.5 µm (magnification images). Fluorescence intensity plots for LDLR–GFP (green), F-actin (blue) and DiI–LDL (red) signals along the arrow in the image are shown in the right panels. PM, plasma membrane. (B) Schematic representation showing the timecourse experiment followed in the right panel. Huh7 cells transfected with LDLR–GFP and control mimic or miR-199a-5p was incubated with anti-LDLR antibodies at 4°C for 2 h, washed and then switched to 37°C to allow internalization for 5 and 10 min. Cells were then stained with anti-phycoerythrin secondary antibodies, washed, fixed with PFA and analyzed by FACS. The graph shows the level of LDLR internalization in gated LDLR–GFP-positive cells as the percentage of phycoerythrin fluorescence. Data are expressed as the geometrical mean (G mean) percentage compared with that for the control mimic at time 0 min (±s.e.m.) and are representative of ≥3 experiments in triplicate. *P≤0.05. with cells transfected with the control mimic (Fig. 6C). Internalized DISCUSSION transferrin–FITC in cells transfected with the control mimic showed Although a large number of studies have shown that several a partial colocalization with EEA1 at 10 min after the start of miRNAs participate in cancer and disease development (Cuk et al., the incubation (Fig. 6C). Most importantly, miR-199a-5p 2013; Pignot et al., 2013; Srivastava et al., 2013), little is known overexpression increased the colocalization of transferrin–FITC about the role of miRNAs in the central cell biology process of with EEA1, suggesting that there is a defect in the endocytic process intracellular trafficking on which other cellular functions depend (Fig. 6C). This effect was not due to changes in EEA1 protein levels on for proper operation. The most salient finding of this study is as measured in cells transfected with miR-199a-5p mimics the identification of miR-199a-5p as an important regulator of (Fig. 6B), suggesting that miR-199a-5p overexpression in cells endocytosis. In particular, our study expands the current triggers malfunctioning of the endosome compartment. Taken understanding of how miRNAs, specifically miR-199a/b-5p, together, these results suggest that miR-199a-5p influences contribute to intracellular trafficking control. Remarkably, the endocytic compartment functioning. miR-199a/b family is encoded within the DNM genes, which are Journal of Cell Science

3203 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3197-3209 doi:10.1242/jcs.165233

Fig. 6. MiR-199a-5p regulates transferrin uptake. (A) Flow cytometry analysis of the transferrin–FITC (Tf-FITC) internalization assay, as described in the Materials and Methods, in HeLa cells transfected with negative control miRNA (CM) or miR-199a-5p mimic and treated as indicated. Data are expressed as the geometrical mean (G mean) of the difference of fluorescence of each time point minus the geometrical mean fluorescence at time 0 min (±s.e.m.) and are representative of ≥3 experiments. (B) Western blot analysis of TfR and EEA1 in HeLa cells transfected with control mimic or miR-199a-5p mimic. Hsp90 was used as a loading control. Quantification of protein fold change is shown in the histogram. (C) Representative confocal images of transferrin–FITC, Rab5 and EEA1 expression in HeLa cells transfected with control mimic or miR-199a-5p mimic, incubated for different times with transferrin–FITC. Histograms at the bottom show a colocalization analysis between EEA1 and transferrin–FITC. Data are representative of ≥3 experiments. *P≤0.05. (D) Left panel, representative confocal images of membrane transferrin–FITC localization in HeLa cells treated as C. Right panel, flow cytometry analysis of surface transferrin–FITC binding in HeLa cells transfected with control mimic and miR-199a-5p. crucial components of the endocytic machinery, suggesting that Our results also demonstrate that miR-199a-5p plays an opposite DNM genes and miR-199a/b form a genomic locus that controls role to DNM in controlling endocytosis. DNM1 is selectively intracellular transport pathways (Fig. 7). Similar to other intronic expressed at very high levels in neurons, where it is crucial for miRNAs, such as miR-33a, miR-33b and miR-208 (Callis et al., synapses to efficiently recycle synaptic vesicles during intense 2009; Rayner et al., 2010), we found that miR-199a/b-5p regulates activity (Baurfend et al., 1995). DNM2 is ubiquitously expressed related physiological processes to those controlled by the host genes and DNM3 is found most prominently in the brain and in the testis in which they are encoded. This finding prompted us to identify and (Cao et al., 1998). For instance, although DNM2 is required for further characterize target genes associated with cellular trafficking. receptor-mediated endocytosis, miR-199a-5p inhibits the Interestingly, we found that a significant number of predicted target expression of numerous genes associated with this process genes for miR-199a/b-5p were associated with cellular transport. including CLTC and GTPases. Our expression profile Journal of Cell Science

3204 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3197-3209 doi:10.1242/jcs.165233

Fig. 7. Proposed model of regulation of RME by DNM and miR-199a/b. Sense strands of the DNM genes are transcribed and translated to synthetize DNM proteins that are involved in endosome trafficking. miR-199a- 5p is transcribed in the nucleus from the antisense strand of introns in the DNM2 and DNM3 gene and regulates receptor-mediated endocytosis and intracellular cholesterol levels by balancing the post-transcriptional levels of genes involved in endocytosis such as LDLR, CLTC, Cav-1, Rab5A and Rab21.

analysis is in agreement with the results obtained in other studies, namely miR-199a-5p and miR-199a-3p, are expressed in human which indicate that miR-199a/b-5p strands are moderately tissues (Shatseva et al., 2011; Shen et al., 2010), and their expressed in several tissues (Gu and Chan, 2012; Sakurai et al., expression originates from the DNM2 and DNM3 introns. 2011). As expected by their opposing roles, the expression of miR- Interestingly, miR-199a-3p, which is highly expressed in some 199 members and their respective DNM host genes were inversely tumor cells, has been reported to target caveolin-2 (Shatseva et al., correlated in most human tissues. This is particularly remarkable in 2011), a key structural protein regulating endocytosis. In addition to the case of the brain, where the expression of DNM1 is markedly miR-199a, the same DNM3 intron also contains miR-214, which is high (Cao et al., 1998) compared with its intronic miRNA, miR- expressed as a cluster together with miR-199a and subsequently 199b-5p, which is expressed at low levels (supplementary material processed to render mature forms. Of note, miR-214 is known to Fig. S2A). Opposing expression levels are also observed in the target phosphatase and tensin homolog (PTEN), which interacts heart, where the expression of miR-199a-5p and miR-199b-5p (da with DNM and regulates receptor recycling (Yang et al., 2008). Costa Martins et al., 2010) is high, in contrast with the moderate More interesting, the mirror miRNA miR-3120, which is fully the expression of DNM2 and almost null expression of DNM1 and complement of miR-214 in the DNM3 intron, is co-expressed with DNM3 (Fig. 1C). This observation suggests that organs that require its host gene mRNA and regulates uncoating of clathrin-coated active trafficking, such as the brain (Faire et al., 1992), express vesicles by targeting Hsc70 and auxilin (Scott et al., 2012). These substantial amounts of DNM and very low levels of miR-199a/b. studies are in agreement with our hypothesis that DNM intronic So far, only a very few studies have shown the participation of miRNAs regulate important aspects of cellular functions that are miRNAs in receptor-mediated internalization or trafficking (Lin similar to those regulated by its host gene. et al., 2014; Serva et al., 2012; Yang et al., 2013). In the case of Aside from using bioinformatic prediction tools, we performed miR-199a/b family, both strands of miR-199a-1 and miR-199a-2, several experiments to dissect the role of miR-199a-5p in regulating Journal of Cell Science

3205 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3197-3209 doi:10.1242/jcs.165233 endocytosis, including the identification of putative regulators by functional response. In summary, our study uncovers an elegant mRNA expression profiling and analysis of protein level changes mechanism by which the miR-199 and DNM genomic locus upon expression and/or inhibition of miR-199a-5p levels together coordinately regulates cellular endocytosis. with a fluorescence confocal microscopy-based assay and rescue of function analysis. In this study, we have characterized a prominent MATERIALS AND METHODS role of miR-199a-5p in regulating endocytosis that mechanistically Materials is facilitated by the direct downregulation of multiple genes Chemicals were obtained from Sigma-Aldrich unless otherwise noted. involved in the endocytic pathway (Fig. 7). As a result, 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) overexpression of miR-199a-5p inhibited clathrin-mediated was purchased from Molecular Probes (Invitrogen). A rabbit polyclonal endocytosis as shown by our in vitro LDL and transferrin antibody against LDLR was obtained from Cayman Chemical and mouse monoclonal antibodies against HSP90, Rab5A, Rab21 and EEA1 were internalization assays. This observation suggests that the ability of purchased from BD Biosciences. The mouse monoclonal antibody against miR-199a-5p to comprehensively control the internalization at LDLR and the goat antibody for TfR were obtained from Santa Cruz different levels (LDLR and CLTC) of essential nutrients, such as Biotechnology. The rabbit polyclonal antibodies to CLTC and Rab11 were cholesterol, present in LDL, and iron. Interestingly, antagonism of purchased from Cell Signaling Technology. Transferrin–FITC and miR-199a-5p enhances LDLR, Rab21 and Rab5A protein secondary fluorescently labeled antibodies were from Molecular Probes expression levels significantly. This observation suggests that (Invitrogen). miRNA mimics and inhibitors were obtained from miR-199a-5p plays a role in regulating constitutive levels of these Dharmacon. The LDLR–GFP plasmid was kindly provided by Peter proteins, increasing LDL uptake and binding in Huh7 cells. In Tontonoz (UCLA, Los Angeles, CA). accordance with this, we observed an increase in the intracellular pool of LDL when inhibiting endogenous expression of miR-199a- Cell culture 5p, suggesting a physiological role in vivo for miR-199a-5p in Human hepatic (Huh7), cervix carcinoma (HeLa) and monkey kidney fibroblast (COS7) cells were obtained from the American Type Tissue controlling internalization pathways in the cell. These results also Collection. Huh7, HeLa and COS7 cells were maintained in Dulbecco’s suggest that antagonism of endogenous miR-199a/b-5p might have modified Eagle’s medium (DMEM) containing 10% fetal bovine serum a potential therapeutic effect for increasing levels of LDLR (FBS) and 2% penicillin-streptomycin in 10 cm2 dishes at 37°C and 5% expression. It is also interesting to note that the DNM proteins CO2. For DiI–LDL uptake and binding experiments, Huh7 cells were encoded by the miR-199a/b-5p host genes associate with CTLC and incubated in DMEM containing 10% lipoprotein-deficient serum (LPDS) mediate the formation of vesicles after endocytosis (Takei et al., supplemented with 30 μg/ml DiI–LDL cholesterol. 1999). Disruption of DNM function results in reduced internalization of receptors (Girard et al., 2011; Gray et al., 2003; Bioinformatic analysis of miRNA target genes Shajahan et al., 2004), such as LDLR. We had hypothesized that Target genes for hsa-miR-199a/b were identified and compared using the miR-199a-5p has a major role in endocytosis, and indeed, our online target prediction algorithm, miRWalk (http://www.umm.uni- rescue of function experiment with ectopic LDLR–GFP did not heidelberg.de/apps/zmf/mirwalk/), which provides target interaction completely restore intracellular levels of DiI–LDL, as expected; information from eight different prediction algorithms. Specifically, the programs miRanda, miRWalk and TargetScan were used. The putative these results are consistent with the finding that many other targets produced by all three of these algorithms for miR-199a were endocytic components are affected when miR-199a-5p levels are uploaded into the gene classification system, PANTHER v8.0 (http://www. elevated. In this regard, a recent study has reported that miR-199a- pantherdb.org) to identify gene targets that were mapped to the transport 5p targets the well-known endocytosis regulator Cav-1 (Lino process (GO 0006810). The functional interactions of these predicted targets Cardenas et al., 2013). We also confirmed that miR-199a-5p for miR-199a/b-5p described in STRING v9.05 (http://string-db.org) were overexpression inhibits Cav-1 expression, but further studies will then combined with the functional annotation groups described in DAVID. assess the contribution of this miRNA to Cav-1 functions. MATLAB and Cytoscape v2.8.3 were used to create the visualization One important question to address further is whether or not networks, as previously described (Huang et al., 2008). STRING changes in miR-199a/b-5p endogenous levels influence interactions with a confidence score of 0.4 or higher were added and endocytosis. The documented observation that in most of cancers highlighted in bold. Smaller annotation clusters and unconnected genes were left out of the visualization due to space constraints. miR-199a-5p expression is downregulated supports the idea that cancer cells can exploit this fact to ensure the intracellular miRNA mimic and inhibitor transfections trafficking necessary for growth, therefore enabling cancer For mimic and inhibitor transfections, Huh7 and HeLa cells were transfected progression (Ramsay et al., 2007; Xu et al., 2012). Intriguingly, with 40 nM miRIDIAN miRNA mimics (miR-199a-5p) or with 60 nM miR-199a-5p overexpression inhibits tumor cell migration without miRIDIAN miRNA inhibitors (Inh-199a-5p) (Dharmacon) using RNAimax affecting cellular proliferation and viability (Cheung et al., 2011; (Invitrogen), or Lipofectamine 2000 (Invitrogen) for co-transfection Duan et al., 2011). To do that, we speculate that miR-199a-5p might experiments with the LDLR–GFP plasmid. All experimental control regulate key endocytic intermediates as described here to fine-tune samples were treated with an equal concentration of a non-targeting control intracellular trafficking routes that are implicated in cancer mimic sequence or inhibitor negative control sequence for use as controls for progression. As discussed previously, miRNAs are known to non-sequence-specific effects in miRNA experiments. Verification of miR- regulate many aspects of cancer cell biology (Iorio and Croce, 2012) 199a-5p overexpression and inhibition was determined using quantitative real-time PCR (qRT-PCR), as described below. and individual miRNAs could be differentially expressed under different stimuli. Because of their different genomic location, future RNA isolation and qRT-PCR experiments will further characterize the specific roles of miR- Total RNA was isolated using TRIzol reagent (Invitrogen) according to the 199a1-5p, miR-199a2-5p and miR-199b-5p in regulating manufacturer’s protocol. For mRNA quantification, cDNA was synthesized endocytosis in this pathological context. In light of our findings, using iScript RT Supermix (Bio-Rad), following the manufacturer’s we can assume that miR-199a/b-5p function in the opposite way to protocol. qRT-PCR analysis was performed in triplicate using iQ SYBR DNM host genes, but additional work is needed to clarify whether green Supermix (BioRad) on an iCycler Real-Time Detection System or not the miR-199 and DNM locus coordinates a unique biological (Eppendorf). The mRNA level was normalized to GAPDH Journal of Cell Science

3206 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3197-3209 doi:10.1242/jcs.165233

(glyceraldehyde-3-phosphate dehydrogenase) as a . The membrane internalization. Cells were then incubated with anti-LDLR human primer sequences used were: GAPDH, 5′-TTGATTTTGGAGGG- monoclonal antibody (C7, Santa Cruz Biotechnology) and 30 µg/ml DiI– ATCTCG-3′ and 5′-CAATGACCCCTTCATTGACC-3′; LDLR, 5′-TGA- LDL for 40 min at 4°C. Following incubation, cells were gently washed TGGGTTCATCTGACCAGT-3′ and 5′-AGTTGGCTGCGTTAATGTG- twice with cold medium and shifted to 37°C to allow for internalization AC-3′; CLTC, 5′-TGAGGCGACTGGGCGGAGTT-3′ and 5′-CCGGGG- of both LDLR–antibody complexes and DiI–LDL for the indicated ACGCAGGAAACTGG-3′; Cav-1, 5′-AGTGCATCAGCCGTGTCTATT- times, and were then acid stripped and fixed with 4% PFA. After 5 min CCA-3′ and 5′-TCTGCAAGTTGATGCGGACATTGC-3′;Rab5A,5′-GG- of 0.2% Triton X-100 permeabilization and 15 min of blocking (PBS GGCTGCTTTTCTAACCCA-3′ and 5′-TTTGCTAGGTCGGCCTTG- with 3% BSA), cells were stained with anti-mouse-IgG conjugated to TT-3′;Rab21,5′-CCTCCGGTGCCTGACGTGGT-3′ and 5′-CAGCC- Alexa Fluor 488 (Molecular Probes) and TO-PRO 3 (Life Technologies) TTCCCCCAGCAGCAC-3′; DNM1, 5′-CACCGTTAGACAGTGCAC- for 1 h at room temperature. After this, cells were washed twice with CA-3′ and 5′-CCCTTGCGGATGACCAGAAT-3′;DNM2,5′-CACAG- 1× PBS and mounted on glass slides with Prolong-Gold (Life CCCCACTCCACAGCG-3′ and 5′-CCTGGGGGAATCCCTGGGGG-3′; Technologies). DNM3, 5′-CCCCCACTCTGGGGCTCCTC-3′ and 5′-GATGGGGGTG- For LDLR–GFP rescue experiments, Huh7 cells were grown on GTCTCCGGCT-3′; and TfR, 5′-GAACTACACCGACCCTCGTG-3′ and coverslips and co-transfected with 1 µg LDLR–GFP plasmid and 40 nM 5′-TGCCACACAGAAGAACCTGC-3′. For miRNA quantification, total of a control mimic or miR-199a-5p mimic. At 48 h post transfection cells RNA was reverse transcribed using the miScript II RT Kit (Qiagen). Primers were incubated with 30 µg/ml DiI–LDL for 1 h at 37°C (uptake). Then, cells specific for human pre-miR-199a1, pre-miR-199a2, pre-miR-199b, miR- were washed twice with 1× PBS, fixed with 4% PFA and blocked (3% BSA 199a-5p and miR-199a-3p (Qiagen) were used and values were normalized in 1× PBS) for 15 min. Following this, cells were washed twice, stained with to SNORD68 (Qiagen) as a housekeeping gene. phalloidin to visualize F-actin and mounted on glass slides with Prolong- Gold (Life Technologies). All images were analyzed using a confocal Western blot analysis microscope (Leica SP5 II) equipped with a 63× Plan Apo Lenses. All gains Cells were lysed in ice-cold buffer containing 50 mM Tris-HCl pH 7.5, for the acquisition of comparable images were maintained at a constant 125 mM NaCl, 1% NP-40, 5.3 mM NaF, 1.5 mM NaP, 1 mM orthovanadate level. Analysis of different images was performed using ImageJ (NIH) and and 1 mg/ml of protease inhibitor cocktail (Roche) and 0.25 mg/ml AEBSF Adobe Photoshop CS5. (Roche). Cell lysates were rotated at 4°C for 1 h before the insoluble material was removed by centrifugation at 12,000 g for 10 min. After normalizing for 3′UTR luciferase reporter assays equal protein concentration, cell lysates were resuspended in SDS sample cDNA fragments corresponding to the entire 3′UTR of human LDLR, buffer before separation by SDS-PAGE. Following overnight transfer of the Cav-1, Rab5A, Rab21 and CLTC were amplified by RT-PCR from total proteins onto nitrocellulose membranes, the membranes were probed with RNA extracted from Huh7 cells with XhoI and NotI linkers. The PCR antibodies against LDLR (1:500), Rab5 (1:1000), CLTC (1:1000), Rab21 product was directionally cloned downstream of the Renilla luciferase open (1:500), EEA-1 (1:1000), TfR (1:1000) or HSP90 (1:1000). Protein bands reading frame in the psiCHECK2™ vector (Promega), which also contains a were visualized using the Odyssey Infrared Imaging System (LI-COR constitutively expressed firefly luciferase gene, which is used to normalize Biotechnology). Densitometry analysis of the gels was carried out using transfections. Point mutations in the seed region of the predicted miR-199a ImageJ software. binding sites within all the above 3′UTR were generated using the Multisite Quikchange kit (Stratagene), according to the manufacturer’s protocol. All LDL receptor and TfR activity assays constructs were confirmed by sequencing. COS7 cells were plated into 12- Human LDL was isolated and labeled with the fluorescent probe DiI as well plates (Costar) and co-transfected with 1 μg of the indicated 3′UTR previously reported (Calvo et al., 1998). Huh7 cells were transfected in 6- or luciferase reporter vectors and miR-199a-5p mimics or control mimics 12-well plates with miRNA mimics and inhibitors in DMEM containing (Dharmacon) using Lipofectamine 2000 (Invitrogen). Luciferase activity 10% LPDS for 48 h. Then, cells were washed once in 1× PBS and incubated was measured using the Dual-Glo luciferase assay system (Promega). in fresh medium containing DiI–LDL (30 µg cholesterol/ml). Non-specific Renilla luciferase activity was normalized to the corresponding firefly uptake was determined in extra wells containing a 50-fold excess of luciferase activity and plotted as a percentage of the control (cells co- unlabeled native LDL (nLDL). Cells were incubated for 5 min to 4 h at 37°C transfected with the corresponding concentration of control mimic). to allow for DiI–LDL uptake. In other instances, cells were incubated Experiments were performed in triplicate wells of a 12-well plate and for 120 min at 4°C to assess anti-LDLR antibody binding. At the end of repeated at least three times. the incubation period, cells were washed, resuspended in 1 ml of PBS and analyzed by flow cytometry (FACScalibur, Becton Dickinson), as Statistics previously described (Suarez et al., 2004). The results are expressed in terms All data are expressed as mean±s.e.m. Statistical differences were of the percentage of specific DiI–LDL uptake after subtracting measured using an unpaired Student’s t-test. A value of P≤0.05 was autofluorescence of cells incubated in the absence of DiI–LDL, which considered statistically significant. Data analysis was performed using was calculated from median fluorescence intensity (MFI) relative to 2 h time GraphPad Prism Software Version 6.03 (GraphPad, San Diego, CA). point. For timecourse experiments, DiI–LDL uptake is represented as MFI *P≤0.05. in arbitrary units. For analysis of transferrin internalization cells were incubated for Acknowledgements 45 min at 37°C in serum-free DMEM. Cells were first incubated on ice We thank Dr Peter Tontonoz for generously providing the LDLR–GFP plasmid. for 20 min followed by addition of 50 µg/ml transferrin–FITC (Sigma) in serum-free medium. After 30 min on ice cells were washed with iced Competing interests The authors declare no competing or financial interests. cold PBS and transferred to a 37°C and 5% CO2 incubator in the presence of unlabeled transferrin for the indicated times. Cells were acid washed to remove surface-bound transferrin and analyzed by FACS or Author contributions fixed in 4% paraformaldehyde (PFA) for 15 min for fluorescence J.F.A. and C.F.-H. conceived and designed the study. J.F.A., A.C.-D. and L.G. microscopy analysis. performed the experiments. Y.S. and C.F.H. assisted with experimental design and data interpretation. J.F.A. and C.F.-H. wrote the manuscript, which was commented on by all authors. Fluorescence microscopy – – For LDLR antibody internalization and DiI LDL uptake assays, Huh7 Funding cells were grown on coverslips and transfected with a miR-199a-5p This work was supported by grants from the National Institutes of Health [grant mimic and a negative control mimic in DMEM containing 10% LPDS. numbers R01HL107953, R01HL106063 to C.F.-H., and 1F31AG043318 to L.G.].

At 48 h post transfection, cells were cooled to 4°C for 20 min to stop Deposited in PMC for release after 12 months. Journal of Cell Science

3207 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3197-3209 doi:10.1242/jcs.165233

Supplementary material Girard, E., Paul, J. L., Fournier, N., Beaune, P., Johannes, L., Lamaze, C. and Supplementary material available online at Védie, B. (2011). The dynamin chemical inhibitor dynasore impairs cholesterol http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.165233/-/DC1 trafficking and sterol-sensitive genes transcription in human HeLa cells and macrophages. PLoS ONE 6, e29042. References Goedeke, L., Vales-Lara, F. M., Fenstermaker, M., Cirera-Salinas, D., Chamorro- Jorganes, A., Ramirez, C. M., Mattison, J. A., de Cabo, R., Suarez, Y. and Ambros, V. (2004). The functions of animal microRNAs. Nature 431, 350-355. Fernandez-Hernando, C. (2013). A regulatory role for microRNA 33* in Bartel, D. P. (2009). MicroRNAs: target recognition and regulatory functions. Cell controlling lipid metabolism gene expression. Mol. Cell. Biol. 33, 2339-2352. 136, 215-233. Gray, N. W., Fourgeaud, L., Huang, B., Chen, J., Cao, H., Oswald, B. J., Hémar, Baurfend, R., David, C., Galli, T., McPherson, P. S., Takei, K. and De Camilli, P. A. and McNiven, M. A. (2003). Dynamin 3 is a component of the postsynapse, (1995). Molecular mechanisms in endocytosis. Cold Spring Harb. where it interacts with mGluR5 and Homer. Curr. Biol. 13, 510-515. Symp. Quant. Biol. 60, 397-404. Gu, S. and Chan, W.-Y. (2012). Flexible and versatile as a chameleon-sophisticated Brown, M. S. and Goldstein, J. L. (1986). A receptor-mediated pathway for functions of microRNA-199a. Int. J. Mol. Sci. 13, 8449-8466. cholesterol homeostasis. Science 232, 34-47. Gu, C., Yaddanapudi, S., Weins, A., Osborn, T., Reiser, J., Pollak, M., Hartwig, J. Bucci, C., Wandinger-Ness, A., Lutcke, A., Chiariello, M., Bruni, C. B. and and Sever, S. (2010). Direct dynamin-actin interactions regulate the actin Zerial, M. (1994). Rab5a is a common component of the apical and basolateral cytoskeleton. EMBO J. 29, 3593-3606. endocytic machinery in polarized epithelial cells. Proc. Natl. Acad. Sci. USA 91, Gu, Z., Noss, E. H., Hsu, V. W. and Brenner, M. B. (2011). Integrins traffic rapidly 5061-5065. via circular dorsal ruffles and macropinocytosis during stimulated cell migration. Bushati, N. and Cohen, S. M. (2007). microRNA functions. Annu. Rev. Cell Dev. J. Cell Biol. 193, 61-70. Biol. 23, 175-205. Harding, C., Heuser, J. and Stahl, P. (1983). Receptor-mediated endocytosis of Callis, T. E., Pandya, K., Seok, H. Y., Tang, R.-H., Tatsuguchi, M., Huang, Z.-P., transferrin and recycling of the transferrin receptor in rat reticulocytes. J. Cell Biol. Chen, J.-F., Deng, Z., Gunn, B., Shumate, J. et al. (2009). MicroRNA-208a is a 97, 329-339. regulator of cardiac hypertrophy and conduction in mice. J. Clin. Invest. 119, Huang, D. W., Sherman, B. T. and Lempicki, R. A. (2008). Systematic and 2772-2786. integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Calvo, D., Gomez-Coronado, D., Suarez, Y., Lasuncion, M. A. and Vega, M. A. Protoc. 4, 44-57. (1998). Human CD36 is a high affinity receptor for the native lipoproteins HDL, Iorio, M. V. and Croce, C. M. (2012). microRNA involvement in human cancer. LDL, and VLDL. J. Lipid Res. 39, 777-788. Carcinogenesis 33, 1126-1133. Cao, H., Garcia, F. and McNiven, M. A. (1998). Differential distribution of dynamin Jones, S. M., Howell, K. E., Henley, J. R., Cao, H. and McNiven, M. A. (1998). Role isoforms in mammalian cells. Mol. Biol. Cell 9, 2595-2609. of dynamin in the formation of transport vesicles from the trans-Golgi network. Cao, H., Thompson, H. M., Krueger, E. W. and McNiven, M. A. (2000). Disruption Science 279, 573-577. of Golgi structure and function in mammalian cells expressing a mutant dynamin. Kang, R. S. and Folsch, H. (2011). ARH cooperates with AP-1B in the exocytosis of J. Cell Sci. 113, 1993-2002. LDLR in polarized epithelial cells. J. Cell Biol. 193, 51-60. Chamorro-Jorganes, A., Araldi, E., Rotllan, N., Cirera-Salinas, D. and Suarez, Y. Killisch, I., Steinlein, P., Romisch, K., Hollinshead, R., Beug, H. and Griffiths, G. (2014). Autoregulation of glypican-1 by intronic microRNA-149 fine tunes the (1992). Characterization of early and late endocytic compartments of the angiogenic response to FGF2 in human endothelial cells. J. Cell Sci. 127, transferrin cycle. Transferrin receptor antibody blocks erythroid differentiation by 1169-1178. trapping the receptor in the early endosome. J. Cell Sci. 103, 211-232. Chendrimada, T. P., Finn, K. J., Ji, X., Baillat, D., Gregory, R. I., Liebhaber, S. A., Lee, Y., Ahn, C., Han, J., Choi, H., Kim, J., Yim, J., Lee, J., Provost, P., Rådmark, Pasquinelli, A. E. and Shiekhattar, R. (2007). MicroRNA silencing through RISC O., Kim, S. et al. (2003). The nuclear RNase III Drosha initiates microRNA recruitment of eIF6. Nature 447, 823-828. processing. Nature 425, 415-419. Cheung, H.-H., Davis, A. J., Lee, T.-L., Pang, A. L., Nagrani, S., Rennert, O. M. Lee, M. Y., Skoura, A., Park, E. J., Landskroner-Eiger, S., Jozsef, L., Luciano, and Chan, W.-Y. (2011). Methylation of an intronic region regulates miR-199a in A. K., Murata, T., Pasula, S., Dong, Y., Bouaouina, M. et al. (2014). Dynamin 2 testicular tumor malignancy. Oncogene 30, 3404-3415. regulation of integrin endocytosis, but not VEGF signaling, is crucial for Cuk, K., Zucknick, M., Madhavan, D., Schott, S., Golatta, M., Heil, J., Marme, F., developmental angiogenesis. Development 141, 1465-1472. Turchinovich, A., Sinn, P., Sohn, C. et al. (2013). Plasma microRNA panel for Lin, D.-H., Yue, P., Zhang, C. and Wang, W.-H. (2014). MicroRNA-194 (miR-194) minimally invasive detection of breast cancer. PLoS ONE 8, e76729. regulates ROMK channel activity by targeting intersectin 1. Am. J. Physiol. Renal da Costa Martins, P. A., Salic, K., Gladka, M. M., Armand, A.-S., Leptidis, S., el Physiol. 306, F53-F60. Azzouzi, H., Hansen, A., Coenen-de Roo, C. J., Bierhuizen, M. F., van der Lino Cardenas, C. L., Henaoui, I. S., Courcot, E., Roderburg, C., Cauffiez, C., Nagel, R. et al. (2010). MicroRNA-199b targets the nuclear kinase Dyrk1a in an Aubert, S., Copin, M.-C., Wallaert, B., Glowacki, F., Dewaeles, E. et al. (2013). auto-amplification loop promoting calcineurin/NFAT signalling. Nat. Cell Biol. 12, miR-199a-5p Is upregulated during fibrogenic response to tissue injury and 1220-1227. mediates TGFbeta-induced lung fibroblast activation by targeting caveolin-1. de Hoop, M. J., Huber, L. A., Stenmark, H., Williamson, E., Zerial, M., Parton, PLoS Genet. 9, e1003291. R. G. and Dotti, C. G. (1994). The involvement of the small GTP-binding protein Liscum, L. and Faust, J. R. (1989). The intracellular transport of low density lipoprotein-derived cholesterol is inhibited in Chinese hamster ovary cells cultured Rab5a in neuronal endocytosis. Neuron 13, 11-22. with 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one. J. Biol. Chem. 264, Dorsey, F. C., Muthusamy, T., Whitt, M. A. and Cox, J. V. (2007). A novel role for a 11796-11806. YXXPhi motif in directing the caveolin-dependent sorting of membrane-spanning Macia, E., Ehrlich, M., Massol, R., Boucrot, E., Brunner, C. and Kirchhausen, T. proteins. J. Cell Sci. 120, 2544-2554. (2006). Dynasore, a cell-permeable inhibitor of dynamin. Dev. Cell 10, 839-850. Doyon, J. B., Zeitler, B., Cheng, J., Cheng, A. T., Cherone, J. M., Santiago, Y., McMahon, H. T. and Boucrot, E. (2011). Molecular mechanism and physiological Lee, A. H., Vo, T. D., Doyon, Y., Miller, J. C. et al. (2011). Rapid and efficient functions of clathrin-mediated endocytosis. Nat. Rev. Mol. Cell Biol. 12, 517-533. clathrin-mediated endocytosis revealed in genome-edited mammalian cells. Nat. Mettlen, M., Loerke, D., Yarar, D., Danuser, G. and Schmid, S. L. (2010). Cargo- Cell Biol. 13, 331-337. and adaptor-specific mechanisms regulate clathrin-mediated endocytosis. J. Cell Duan, Z., Choy, E., Harmon, D., Liu, X., Susa, M., Mankin, H. and Hornicek, F. Biol. 188, 919-933. (2011). MicroRNA-199a-3p is downregulated in human osteosarcoma and Moore, M. S., Mahaffey, D. T., Brodsky, F. M. and Anderson, R. G. (1987). regulates cell proliferation and migration. Mol. Cancer Ther. 10, 1337-1345. Assembly of clathrin-coated pits onto purified plasma membranes. Science 236, Faire, K., Trent, F., Tepper, J. M. and Bonder, E. M. (1992). Analysis of dynamin 558-563. isoforms in mammalian brain: dynamin-1 expression is spatially and temporally Mousley, C. J., Yuan, P., Gaur, N. A., Trettin, K. D., Nile, A. H., Deminoff, S. J., regulated during postnatal development. Proc. Natl. Acad. Sci. USA 89, Dewar, B. J., Wolpert, M., Macdonald, J. M., Herman, P. K. et al. (2012). A 8376-8380. sterol-binding protein integrates endosomal lipid metabolism with TOR signaling Ferguson, S. M. and De Camilli, P. (2012). Dynamin, a membrane-remodelling and nitrogen sensing. Cell 148, 702-715. GTPase. Nat. Rev. Mol. Cell Biol. 13, 75-88. Nielsen, E., Severin, F., Backer, J. M., Hyman, A. A. and Zerial, M. (1999). Rab5 Fernández-Rojo, M. A., Restall, C., Ferguson, C., Martel, N., Martin, S., Bosch, M., regulates motility of early endosomes on microtubules. Nat. Cell Biol. 1, 376-382. Kassan, A., Leong, G. M., Martin, S. D., McGee, S. L. et al. (2012). Caveolin-1 Parachoniak, C. A., Luo, Y., Abella, J. V., Keen, J. H. and Park, M. (2011). GGA3 orchestrates the balance between glucose and lipid-dependent energy functions as a switch to promote Met receptor recycling, essential for sustained metabolism: implications for liver regeneration. Hepatology 55, 1574-1584. ERK and cell migration. Dev. Cell 20, 751-763. Filipowicz, W., Bhattacharyya, S. N. and Sonenberg, N. (2008). Mechanisms of Pellinen, T., Tuomi, S., Arjonen, A., Wolf, M., Edgren, H., Meyer, H., Grosse, R., post-transcriptional regulation by microRNAs: are the answers in sight? Nat. Rev. Kitzing, T., Rantala, J. K., Kallioniemi, O. et al. (2008). Integrin trafficking Genet. 9, 102-114. regulated by Rab21 is necessary for cytokinesis. Dev. Cell 15, 371-385. Gan, Y., McGraw, T. E. and Rodriguez-Boulan, E. (2002). The epithelial-specific Pignot, G., Cizeron-Clairac, G., Vacher, S., Susini, A., Tozlu, S., Vieillefond, A., adaptor AP1B mediates post-endocytic recycling to the basolateral membrane. Zerbib, M., Lidereau, R., Debre, B., Amsellem-Ouazana, D. et al. (2013).

Nat. Cell Biol. 4, 605-609. microRNA expression profile in a large series of bladder tumors: identification of a Journal of Cell Science

3208 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3197-3209 doi:10.1242/jcs.165233

3-miRNA signature associated with aggressiveness of muscle-invasive bladder Simpson, J. C., Griffiths, G., Wessling-Resnick, M., Fransen, J. A. M., Bennett, cancer. Int. J. Cancer 132, 2479-2491. H. and Jones, A. T. (2004). A role for the small GTPase Rab21 in the early Ramsay, A. G., Keppler, M. D., Jazayeri, M., Thomas, G. J., Parsons, M., endocytic pathway. J. Cell Sci. 117, 6297-6311. Violette, S., Weinreb, P., Hart, I. R. and Marshall, J. F. (2007). HS1-associated Singh, R. D., Puri, V., Valiyaveettil, J. T., Marks, D. L., Bittman, R. and Pagano, protein X-1 regulates carcinoma cell migration and invasion via clathrin-mediated R. E. (2003). Selective caveolin-1-dependent endocytosis of glycosphingolipids. endocytosis of integrin alphavbeta6. Cancer Res. 67, 5275-5284. Mol. Biol. Cell 14, 3254-3265. Rayner, K. J., Suarez, Y., Davalos, A., Parathath, S., Fitzgerald, M. L., Tamehiro, Srivastava, A., Goldberger, H., Dimtchev, A., Ramalinga, M., Chijioke, J., N., Fisher, E. A., Moore, K. J. and Fernandez-Hernando, C. (2010). MiR-33 Marian, C., Oermann, E. K., Uhm, S., Kim, J. S., Chen, L. N. et al. (2013). contributes to the regulation of cholesterol homeostasis. Science 328, 1570-1573. MicroRNA profiling in prostate cancer–the diagnostic potential of urinary miR-205 Rodriguez, A., Griffiths-Jones, S., Ashurst, J. L. and Bradley, A. (2004). and miR-214. PLoS ONE 8, e76994. Identification of mammalian microRNA host genes and transcription units. Suarez, Y., Fernandez, C., Gomez-Coronado, D., Ferruelo, A. J., Davalos, A., Genome Res. 14, 1902-1910. Martinez-Botas, J. and Lasuncion, M. A. (2004). Synergistic upregulation of Roux, A., Uyhazi, K., Frost, A. and De Camilli, P. (2006). GTP-dependent twisting low-density lipoprotein receptor activity by tamoxifen and lovastatin. Cardiovasc. of dynamin implicates constriction and tension in membrane fission. Nature 441, Res. 64, 346-355. 528-531. Szklarczyk, D., Franceschini, A., Kuhn, M., Simonovic, M., Roth, A., Minguez, Saini, H. K., Griffiths-Jones, S. and Enright, A. J. (2007). Genomic analysis of P., Doerks, T., Stark, M., Muller, J., Bork, P. et al. (2011). The STRING database human microRNA transcripts. Proc. Natl. Acad. Sci. USA 104, 17719-17724. in 2011: functional interaction networks of proteins, globally integrated and Sakurai, K., Furukawa, C., Haraguchi, T., Inada, K.-I., Shiogama, K., Tagawa, T., scored. Nucleic Acids Res. 39, D561-D568. Fujita, S., Ueno, Y., Ogata, A., Ito, M. et al. (2011). MicroRNAs miR-199a-5p and Takei, K., McPherson, P. S., Schmid, S. L. and De Camilli, P. (1995). Tubular -3p target the Brm subunit of SWI/SNF to generate a double-negative feedback membrane invaginations coated by dynamin rings are induced by GTP-gamma S loop in a variety of human cancers. Cancer Res. 71, 1680-1689. in nerve terminals. Nature 374, 186-190. Scott, H., Howarth, J., Lee, Y. B., Wong, L.-F., Bantounas, I., Phylactou, L., Takei, K., Slepnev, V. I., Haucke, V. and De Camilli, P. (1999). Functional Verkade, P. and Uney, J. B. (2012). MiR-3120 is a mirror microRNA that targets partnership between amphiphysin and dynamin in clathrin-mediated endocytosis. heat shock cognate protein 70 and auxilin messenger RNAs and regulates clathrin Nat. Cell Biol. 1, 33-39. vesicle uncoating. J. Biol. Chem. 287, 14726-14733. Thomas, P. D., Campbell, M. J., Kejariwal, A., Mi, H., Karlak, B., Daverman, R., Semerdjieva, S., Shortt, B., Maxwell, E., Singh, S., Fonarev, P., Hansen, J., Diemer, K., Muruganujan, A. and Narechania, A. (2003). PANTHER: a library of Schiavo, G., Grant, B. D. and Smythe, E. (2008). Coordinated regulation of AP2 protein families and subfamilies indexed by function. Genome Res. 13, uncoating from clathrin-coated vesicles by rab5 and hRME-6. J. Cell Biol. 183, 2129-2141. 499-511. Tosoni, D., Puri, C., Confalonieri, S., Salcini, A. E., De Camilli, P., Tacchetti, C. Serva, A., Knapp, B., Tsai, Y.-T., Claas, C., Lisauskas, T., Matula, P., Harder, N., and Di Fiore, P. P. (2005). TTP specifically regulates the internalization of the Kaderali, L., Rohr, K., Erfle, H. et al. (2012). miR-17–5p regulates endocytic transferrin receptor. Cell 123, 875-888. trafficking through targeting TBC1D2/Armus. PLoS ONE 7, e52555. Urrutia, R., Henley, J. R., Cook, T. and McNiven, M. A. (1997). The : Shajahan, A. N., Timblin, B. K., Sandoval, R., Tiruppathi, C., Malik, A. B. and redundant or distinct functions for an expanding family of related GTPases? Proc. Minshall, R. D. (2004). Role of Src-induced dynamin-2 phosphorylation in Natl. Acad. Sci. USA 94, 377-384. -mediated endocytosis in endothelial cells. J. Biol. Chem. 279, van Rooij, E., Quiat, D., Johnson, B. A., Sutherland, L. B., Qi, X., Richardson, 20392-20400. J. A., Kelm, R. J., Jr and Olson, E. N. (2009). A family of microRNAs encoded by Shatseva, T., Lee, D. Y., Deng, Z. and Yang, B. B. (2011). MicroRNA miR-199a-3p myosin genes governs myosin expression and muscle performance. Dev. Cell 17, regulates cell proliferation and survival by targeting caveolin-2. J. Cell Sci. 124, 662-673. 2826-2836. Xu, N., Zhang, J., Shen, C., Luo, Y., Xia, L., Xue, F. and Xia, Q. (2012). Cisplatin- Shen, Q., Cicinnati, V. R., Zhang, X., Iacob, S., Weber, F., Sotiropoulos, G. C., induced downregulation of miR-199a-5p increases drug resistance by activating Radtke, A., Lu, M., Paul, A., Gerken, G. et al. (2010). Role of microRNA-199a-5p autophagy in HCC cell. Biochem. Biophys. Res. Commun. 423, 826-831. and discoidin domain receptor 1 in human hepatocellular carcinoma invasion. Yang, H., Kong, W., He, L., Zhao, J.-J., O’Donnell, J. D., Wang, J., Wenham, Mol. Cancer 9, 227. R. M., Coppola, D., Kruk, P. A., Nicosia, S. V. et al. (2008). MicroRNA Siegel, G., Obernosterer, G., Fiore, R., Oehmen, M., Bicker, S., Christensen, M., expression profiling in human ovarian cancer: miR-214 induces cell survival and Khudayberdiev, S., Leuschner, P. F., Busch, C. J. L., Kane, C. et al. (2009). A cisplatin resistance by targeting PTEN. Cancer Res. 68, 425-433. functional screen implicates microRNA-138-dependent regulation of the Yang, S., Liu, X., Li, X., Sun, S., Sun, F., Fan, B. and Zhao, S. (2013). MicroRNA- depalmitoylation enzyme APT1 in dendritic spine morphogenesis. Nat. Cell 124 reduces caveolar density by targeting caveolin-1 in porcine kidney epithelial Biol. 11, 705-716. PK15 cells. Mol. Cell. Biochem. 384, 213-219. Journal of Cell Science

3209