Nup358 binds to AGO proteins through its SUMO ‐interacting motifs and promotes the association of target mRNA with miRISC
2016; Springer Nature; Volume: 18; Issue: 2 Linguagem: Inglês
10.15252/embr.201642386
ISSN1469-3178
AutoresManas Ranjan Sahoo, Swati Gaikwad, Deepak Khuperkar, Maitreyi Ashok, Mary Helen, Santosh K. Yadav, Aditi Singh, Indrasen Magre, Prachi Deshmukh, Supriya Dhanvijay, Pabitra K. Sahoo, Yogendra Ramtirtha, M. S. Madhusudhan, P. Gayathri, Vasudevan Seshadri, Jomon Joseph,
Tópico(s)interferon and immune responses
ResumoArticle30 December 2016free access Transparent process Nup358 binds to AGO proteins through its SUMO-interacting motifs and promotes the association of target mRNA with miRISC Manas Ranjan Sahoo Manas Ranjan Sahoo National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Swati Gaikwad Swati Gaikwad National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Deepak Khuperkar Deepak Khuperkar National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Maitreyi Ashok Maitreyi Ashok National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Mary Helen Mary Helen National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Santosh Kumar Yadav Santosh Kumar Yadav National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Aditi Singh Aditi Singh National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Indrasen Magre Indrasen Magre National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Prachi Deshmukh Prachi Deshmukh National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Supriya Dhanvijay Supriya Dhanvijay National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Pabitra Kumar Sahoo Pabitra Kumar Sahoo National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Yogendra Ramtirtha Yogendra Ramtirtha Division of Biology, Indian Institute of Science Education and Research, Pune, India Search for more papers by this author Mallur Srivatsan Madhusudhan Mallur Srivatsan Madhusudhan Division of Biology, Indian Institute of Science Education and Research, Pune, India Search for more papers by this author Pananghat Gayathri Pananghat Gayathri Division of Biology, Indian Institute of Science Education and Research, Pune, India Search for more papers by this author Vasudevan Seshadri Vasudevan Seshadri National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Jomon Joseph Corresponding Author Jomon Joseph [email protected] orcid.org/0000-0002-3042-6286 National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Manas Ranjan Sahoo Manas Ranjan Sahoo National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Swati Gaikwad Swati Gaikwad National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Deepak Khuperkar Deepak Khuperkar National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Maitreyi Ashok Maitreyi Ashok National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Mary Helen Mary Helen National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Santosh Kumar Yadav Santosh Kumar Yadav National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Aditi Singh Aditi Singh National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Indrasen Magre Indrasen Magre National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Prachi Deshmukh Prachi Deshmukh National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Supriya Dhanvijay Supriya Dhanvijay National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Pabitra Kumar Sahoo Pabitra Kumar Sahoo National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Yogendra Ramtirtha Yogendra Ramtirtha Division of Biology, Indian Institute of Science Education and Research, Pune, India Search for more papers by this author Mallur Srivatsan Madhusudhan Mallur Srivatsan Madhusudhan Division of Biology, Indian Institute of Science Education and Research, Pune, India Search for more papers by this author Pananghat Gayathri Pananghat Gayathri Division of Biology, Indian Institute of Science Education and Research, Pune, India Search for more papers by this author Vasudevan Seshadri Vasudevan Seshadri National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Jomon Joseph Corresponding Author Jomon Joseph [email protected] orcid.org/0000-0002-3042-6286 National Centre for Cell Science, S.P. Pune University Campus, Pune, India Search for more papers by this author Author Information Manas Ranjan Sahoo1,‡, Swati Gaikwad1,‡, Deepak Khuperkar1, Maitreyi Ashok1, Mary Helen1, Santosh Kumar Yadav1, Aditi Singh1, Indrasen Magre1, Prachi Deshmukh1, Supriya Dhanvijay1, Pabitra Kumar Sahoo1, Yogendra Ramtirtha2, Mallur Srivatsan Madhusudhan2, Pananghat Gayathri2, Vasudevan Seshadri1 and Jomon Joseph *,1 1National Centre for Cell Science, S.P. Pune University Campus, Pune, India 2Division of Biology, Indian Institute of Science Education and Research, Pune, India ‡These authors contributed equally to this work *Corresponding author. Tel: +91 20 25708084; E-mail: [email protected] EMBO Reports (2017)18:241-263https://doi.org/10.15252/embr.201642386 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract MicroRNA (miRNA)-guided mRNA repression, mediated by the miRNA-induced silencing complex (miRISC), is an important component of post-transcriptional gene silencing. However, how miRISC identifies the target mRNA in vivo is not well understood. Here, we show that the nucleoporin Nup358 plays an important role in this process. Nup358 localizes to the nuclear pore complex and to the cytoplasmic annulate lamellae (AL), and these structures dynamically associate with two mRNP granules: processing bodies (P bodies) and stress granules (SGs). Nup358 depletion disrupts P bodies and concomitantly impairs the miRNA pathway. Furthermore, Nup358 interacts with AGO and GW182 proteins and promotes the association of target mRNA with miRISC. A well-characterized SUMO-interacting motif (SIM) in Nup358 is sufficient for Nup358 to directly bind to AGO proteins. Moreover, AGO and PIWI proteins interact with SIMs derived from other SUMO-binding proteins. Our study indicates that Nup358–AGO interaction is important for miRNA-mediated gene silencing and identifies SIM as a new interacting motif for the AGO family of proteins. The findings also support a model wherein the coupling of miRISC with the target mRNA could occur at AL, specialized domains within the ER, and at the nuclear envelope. Synopsis The nucleoporin Nup358 promotes the association of target mRNA with miRISC possibly at specialized ER domains and at the nuclear envelope. The study also identifies SIM as a new interacting motif for AGO family proteins. Nup358 depletion disrupts P body formation and impairs the coupling of target mRNA with miRISC. Nup358 interacts with AGO proteins through its SUMO-interacting motifs (SIMs), and SIM is identified as a new binding motif for AGO family proteins. The association of target mRNA with miRISC possibly occurs at Nup358-positive structures on the ER called annulate lamellae and at the nuclear envelope. Introduction Regulation of gene expression at the translational level is shown to be involved in diverse cellular processes and has emerged as an area of intense investigation. Small non-coding RNAs, particularly microRNAs (miRNAs), appear to significantly contribute to this layer of regulation. miRNAs, which are of ~22 nucleotides length, suppress translation of mRNAs that possess partial or complete sequence complementarity, mostly at the 3′-untranslated region (UTR) 1. Predictions based on sequence analysis have indicated that miRNAs could target over 50% of human protein-coding genes 2. The genes encoding miRNAs are generally transcribed by RNA polymerase II to produce primary miRNAs (pri-miRNAs), which are processed into precursor miRNAs (pre-miRNAs) by the microprocessor complex containing Drosha and DGCR8 in the nucleus 3. The pre-miRNA, in complex with exportin-5 and RanGTP, is exported through the nuclear pore complex (NPC) into the cytoplasm, where it is further processed by Dicer into double-stranded miRNA. One of the strands stably associates with Argonaute (AGO) protein to generate a functional miRISC. Humans have four AGO isoforms: AGO1–AGO4 4. A glycine–tryptophan (GW)-rich protein, GW182 (also called TNRC6), interacts directly with AGO proteins and is essential for miRISC-mediated translational repression and/or degradation of target mRNAs through recruitment of deadenylation and decapping complexes. The suppression and/or degradation of target mRNAs is believed to occur in the cytoplasmic foci termed as "processing bodies (P bodies)" 56. As downstream effectors, GW182 family of proteins directly bind to AGO proteins through conserved GW/WG sequence. This motif is also present in other AGO-interacting proteins and is referred to as "AGO hook" 78. Argonaute proteins have a highly conserved role in RNA silencing 4. AGO family is divided into two clades based on their functions: AGO and PIWI subfamilies. As described earlier, AGO subfamily proteins are present ubiquitously and are involved in small interfering RNA (siRNA)-mediated cleavage of mRNA or miRNA-mediated suppression of mRNA translation 4. However, the members of PIWI subclade are mostly present in germ cells and are involved in silencing transposons, maintenance of genome integrity, and gametogenesis 9. Although the subcellular location where the loading of miRNAs to AGO proteins (miRISC formation) and association of miRISC with the target mRNAs occur is not well understood, recent studies have indicated a role for endoplasmic reticulum (ER) in these processes. It was shown that Arabidopsis AGO1 associates peripherally with ER, and miRISC could inhibit the translation of target mRNAs on the ER 10. Another study indicated that rough ER could be the site for miRNA and siRNA loading to AGO proteins and translational regulation of target mRNAs 11. A central question that is yet unresolved is how miRISC identifies the target mRNAs in vivo. Although a sorting mechanism could be envisaged that couples the RNAs exported from the nucleus with the miRISC, there is no available evidence for the existence of such machinery. The nuclear envelope (NE) that encircles the nucleus is an extension of ER and is made up of a double-layered membrane. Nuclear pore complexes (NPCs) form the molecular gates on the NE, through which the transport of macromolecules between the nucleus and the cytoplasm occurs 12. The protein components of NPCs are termed as nucleoporins (Nups), and each mammalian NPC contains around 30 different nucleoporins in multiple copies 13. The spatial distribution of individual nucleoporins within the NPC structure could vary 14. Although the nucleoporins are fundamentally expected to be involved in the regulation of nucleo-cytoplasmic transport, several of them are shown to have multiple other functions 1516. Apart from the localization to NPCs on the NE, some nucleoporins also accumulate in annulate lamellae (AL), which are stacked ER membrane-containing pore-like structures 171819. These AL pore complexes show gross structural similarities to that of NPCs at electron microscopy level 1719. Although AL structures have been extensively analyzed in male and female gametes, other proliferating non-germ cells also possess varying quantities of AL 1719. The functional role for these structures in any cellular processes is unclear. Previous studies have implicated AL as the storehouse of excess nucleoporins to be supplied as and when the cell requires, for example, to meet the increasing demand for nucleoporins in the assembly of new NPCs during initial zygotic cell divisions. However, there is experimental evidence arguing against such a function 20. Consistent with being a part of the endoplasmic reticulum, electron microscopic studies also have suggested AL to often have RNA and ribosomes in their close vicinity 1719. Previous studies have shown that AL associate with MEX-3-positive RNP granules in the arrested Caenorhabditis elegans oocytes and that several nucleoporins play a role in the complete assembly of these RNP granules 21. However, whether AL associate with other mRNP granules and play a role in their functions is not known. Nup358 is a nucleoporin that localizes to the cytoplasmic side of the NPC and has been implicated in several functions 22232425262728293031. Depletion of Nup358 does not appear to grossly affect transport of macromolecules across the NE, although some studies suggest a role for this nucleoporin in specific receptor- and cargo-dependent transport 3233343536. Nup358 has been identified as a small ubiquitin-like modifier (SUMO) E3 ligase 28 and is shown to mediate in vivo SUMOylation of substrates such as topoisomerase II 37, borealin 38, and Ran 39. SUMO is a small protein that gets covalently conjugated to target proteins through specific lysine residues and modulates their function 4041. SUMO pathway is shown to be involved in multiple cellular processes 42. In humans, there are four SUMO isoforms: SUMO1–4. In addition to the covalent interaction, SUMO associates with other proteins through directly binding to specific SUMO-interacting motif (SIM), which is characterized by a conserved set of hydrophobic amino acids 4041. Multiple SIMs have been identified in many SUMO-interacting proteins and functionally validated 43. The presence of a stretch of negatively charged amino acids adjacent to the N- or C-terminus of the hydrophobic sequence (SIM) is shown to contribute to the strength, orientation, and paralog specificity of SUMO binding 42. SUMO conjugation to the substrate lysine requires concerted action of SUMO-specific E1 (Aos1/Uba2 heterodimer), E2 (Ubc9), and multiple E3 ligases 42. RanGTPase-activating protein (RanGAP) is the first SUMO substrate identified 444546. SUMO gets attached to lysine 524 of human RanGAP, which targets it to the NPC through binding to Nup358. Structural and functional analyses showed that SUMO-RanGAP interacts with Nup358 through a region having internal repeats (IR) harboring two SIMs 4748. Nup358-IR also possesses the SUMO E3 ligase activity 28. Each of the two repeats, IR1 and IR2, has a SIM-binding and a Ubc9-binding domain 4950. However, studies have shown that IR1 (SIM1) is involved in SUMO~RanGAP1 interaction, which is stabilized by Ubc9 as it directly binds to IR1, RanGAP1, and SUMO 4751. In vitro studies have illustrated that SUMO-RanGAP and Ubc9 form a stable complex with IR1, and not with IR2 515253. Although no conclusive evidence exists, it is believed that SUMO-dependent binding of RanGAP1 to Nup358 would enhance RanGAP's ability to activate the hydrolysis of GTP on Ran in the export complex 5455. Endogenously, bulk of RanGAP is SUMO-modified and has been shown to associate with Nup358 throughout the cell cycle 2556. Here, we show that Nup358-positive AL structures dynamically associate with cytoplasmic mRNPs such as P bodies and stress granules (SGs). Furthermore, our study reveals interaction between Nup358 and components of miRISC, AGO, and GW182. The results suggest an unanticipated function for this nucleoporin in miRNA-mediated gene silencing by aiding in the coupling of miRISC with target mRNAs. The results also indicate a possible role for AL in the miRISC–mRNA coupling process. Interestingly, characterization of Nup358–AGO interaction led to identification of SIM as a new conserved interaction motif for AGO family of proteins. Our data also suggest that Nup358–AGO interaction is essential for miRNA-mediated suppression of mRNA translation. Results Nup358-positive AL structures and NE associate with SGs and P bodies Localization of endogenous Nup358 in HeLa cells using a specific antibody showed that, in addition to NE, this nucleoporin is present in cytoplasmic punctate structures along with RanGAP1, a known interacting partner of Nup358 (Fig 1A) 4546. To validate whether the cytoplasmic Nup358-positive structures represented AL, we immunostained for other nucleoporins and found these entities to contain Nup214 (Fig 1A) and Nup62 (Fig EV1A), but not Nup153 (Fig EV1A). Moreover, these Nup358-positive structures were associated with ER, particularly marking distinct domains within the ER (Fig EV1B). Co-localization with a set of nucleoporins and association with ER indicated that Nup358-positive cytoplasmic structures represented the previously characterized AL 5758. Exogenously expressed GFP-tagged Nup358 (GFP-Nup358) also accumulated in AL, as confirmed by its co-localization with AL-specific nucleoporins such as Nup214 and Nup62 (Fig EV1C). Figure 1. Nup358-positive AL structures associate with P bodies and SGs, and Nup358 depletion leads to impairment of P body assembly Confocal microscopic image showing HeLa cells immunostained for Nup358 (green, upper panel) or Nup214 (green, lower panel) and RanGAP1 (red) using specific antibodies. DNA was stained with Hoechst 33342 (blue). Scale bars, 10 μm. COS-7 cells were transfected with GFP-Nup358 and one of the cells was subjected to live imaging using confocal microscopy, and the frames at the indicated time points have been provided. Arrows denoted by different colors indicate individual AL structures. Note that one of the AL structures (marked by yellow arrow) originated from the NE (dotted line) and fused with another AL structure (marked by red arrow), which later fused with a different AL structure (marked by blue arrow). N, nucleus; C, cytoplasm. Scale bar, 5 μm. Maximum-intensity projection confocal image of a sodium arsenite-treated HeLa cell immunostained for endogenous Nup358 (green), P bodies (red, Dcp1a as a marker), and SGs (blue, eIF3η as a marker) using specific antibodies. Scale bar, 10 μm. The histograms represent fluorescence intensity profile along the dotted arrows. Adjacent graph represents quantitative data showing percentage of P bodies (top) or SGs (bottom) associated with nuclear envelope (NE), Nup358-positive AL, with each other or unassociated with any of the other mentioned structures (others). Data are presented as mean ± SD (n = 3). HeLa cells were transfected with Nup358 siRNA (siNup358) or Nup214 siRNA (siNup214). Cells were fixed and stained for endogenous Nup358 (green, upper panel) or Nup214 (green, lower panel) and endogenous P body marker (Dcp1a, red). Graph represents the quantitative data as mean ± SD. The data were obtained from three independent experiments, and in each experiment, 100 cells were counted from different fields for the presence of intact microscopically distinct P bodies and expressed as percentage. Western blots (WB) indicate the extent of Nup358 depletion and the level of Dcp1a. Arrow indicates Nup358/Nup214 depleted cell and arrow head shows non-depleted cell. Scale bar, 10 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Endogenous and overexpressed Nup358-positive cytoplasmic puncta represent AL HeLa cells were immunostained for Nup62 (red, upper panel) or Nup153 (red, lower panel) along with Nup358 (green) using specific antibodies. DNA was stained with Hoechst 33342 (blue, merge). Percentages of Nup358-positive cytoplasmic puncta co-localizing with Nup62 (upper panel) and Nup358 with Nup153 (lower panel) are indicated. Scale bars, 10 μm. HeLa cells were fixed and stained for endogenous Nup358 (green) and PDI (red, as an ER marker). Scale bar, 10 μm. COS-7 cells were transfected with GFP-Nup358 (green) and stained for endogenous RanGAP1, Nup214, Nup62, or Nup153 (red) using specific antibodies. DNA is shown in blue (merge). Percentages of GFP-Nup358-positive cytoplasmic puncta co-localizing with respective proteins are indicated. Scale bar, 10 μm. COS-7 cells expressing GFP-Nup358 (green) and RFP-Dcp1 (red) were fixed and analyzed for their relative distribution within the cytoplasm. Often GFP-Nup358-positive AL structures are in close proximity/associated with P bodies (RFP-Dcp1a). The region marked by square is magnified (inset). Dotted line indicates nuclear periphery. Graph represents quantitative data showing percent of P bodies (RFP-Dcp1a) associated with nuclear envelope (NE), GFP-Nup358-positive AL, or unassociated with either of the structures (free). Scale bar, 10 μm. Data are presented as mean ± SD (n = 3). COS-7 cells expressing GFP-Nup358 (green) and RFP-G3BP1 (red) were fixed and analyzed by fluorescence microscopy. Often GFP-Nup358-positive AL structures are in close proximity/associated with SGs induced by RFP-G3BP1 expression. The region marked by square is magnified (inset). Dotted line indicates nuclear periphery. Graph represents quantitative data showing percent of SGs (RFP-G3BP1) associated with nuclear envelope (NE), GFP-Nup358-positive AL, or unassociated with either of the structures (free). Scale bar, 10 μm. Data are presented as mean ± SD (n = 3). HeLa cells were transfected with control siRNA (siControl) or siRNA against Nup358 (siNup358). Cells were treated with 0.5 mM sodium arsenite for 30 min prior to fixation and stained for endogenous Nup358 (green) and eIF3η (red) as a SG marker using specific antibodies. DNA was stained with Hoechst 33342 (merge, blue). Arrows indicate a Nup358-depleted cell. Scale bars, 10 μm. HeLa cells were transfected with control siRNA (siControl) or Nup214 siRNA (siNup214). Cells were treated as indicated in (A) and were fixed and stained for endogenous Nup358 (green) and a SG marker (eIF3η, red). DNA was stained with Hoechst 33342 (merge, blue). Arrows indicate a Nup214-depleted cell. Scale bars, 10 μm. Download figure Download PowerPoint The nature and origin of AL have been unclear, and to monitor these, we analyzed the dynamics of GFP-Nup358-labeled AL using live cell imaging (Movie EV1). We observed that AL were highly dynamic and often underwent homotypic fusion with neighboring AL structures. Interestingly, we noticed that some AL structures were budding off from the NE and fusing with the pre-existing cytoplasmic AL (Fig 1B and Movie EV2). These results suggested that cytoplasmic AL could originate from NE and are extensively dynamic entities. Further, we sought to investigate the distribution of AL in relation to other cytoplasmic structures. Interestingly, we found that two cytoplasmic messenger ribonucleoprotein (mRNP) granules, namely SGs and P bodies, were often associated with or present juxtaposed to AL (Fig 1C). We subjected HeLa cells to oxidative stress through sodium arsenite treatment to induce SGs 59 and immunostained for endogenous Nup358, eIF3η (SG marker), and Dcp1a (P body marker). P bodies and SGs were found often juxtaposed to each other in the cytoplasm as previously reported 59. Interestingly, we observed that many individual Nup358-positive AL structures were present adjacent to SGs or P bodies, and in some cases, all three structures appeared to physically associate with each other (Fig 1C). Quantitative analysis suggested that ~20% of P bodies were associated with either Nup358-positive AL or SGs, whereas ~50% of them were found to be associated with neither AL nor SGs, and ~6% were associated with the NE (Fig 1C). Similarly, ~20% of SGs were associated with either Nup358-positive AL or P bodies, whereas ~50% of them were found to be associated with neither AL nor P bodies, and ~10% were associated with the NE (Fig 1C). In unstressed cells, ~16% of endogenous P bodies associated with Nup358-positive AL structures. The physical association was much more striking when GFP-Nup358 was exogenously expressed along with RFP-Dcp1a (P body marker) or RFP-G3BP1 (SG marker) (Fig EV1D and E). Under this condition, ~47% of P bodies associated with Nup358-positive AL, whereas < 10% associated with the NE (Fig EV1D). Similarly, ~58% of SGs were present juxtaposed to Nup358-positive AL and ~10% were associated with NE (Fig EV1E). Live cell imaging, interestingly, indicated a dynamic interplay between the two mRNP granules and Nup358-positive AL/NE (Movies EV3 and EV4). Depletion of Nup358 disrupts P body formation The dynamic association of Nup358-positive AL/NE with SGs and P bodies prompted us to investigate whether any functional link exists between these entities. Toward this, we tested whether siRNA-mediated Nup358 depletion caused any effect on the assembly of mRNP granules. Removal of Nup358 did not have any gross effect on SG assembly (assessed by SG-specific marker, eIF3η) as compared to control cells (Fig EV1F). Neither did depletion of Nup214, another nucleoporin present on the cytoplasmic face of NPC and AL, show any effect on SG assembly (Fig EV1G). Interestingly, knockdown of Nup358, but not Nup214, led to dramatic impairment of P body assembly as assessed by Dcp1a staining (Fig 1D). However, the levels of Dcp1a were comparable between control and Nup358 knockdown cells (Fig 1D). These results demonstrated a specific requirement for Nup358 in P body formation, and possibly in its function. Nup358 is required for miRNA-mediated translation suppression Previous studies have shown that mRNAs suppressed by miRISC localize to P bodies 56, and disturbances in miRNA pathway lead to disruption of microscopically visible distinct P body structures 60. We sought to find out whether Nup358 depletion affected the miRNA pathway. As let-7a is one of the abundant miRNAs expressed in HeLa cells, a Renilla luciferase (RL) reporter construct that expresses RL mRNAs containing three imperfect let-7a binding sites in its 3′-UTR (RL-3xBulge) was used to monitor let-7a-mediated translation suppression in HeLa cells 61. Compared to control Renilla luciferase (RL-control) mRNAs that did not have any let-7a binding site, RL-3xBulge generally showed ~60% suppression (Fig 2B). We measured the RL activity in cells depleted of Dicer, Nup214, or Nup358 (Fig 2A) and found that similar to Dicer knockdown, Nup358 depletion caused significant reversal of miRNA-mediated suppression (Fig 2B). Cells with Nup214 knockdown, however, showed no significant change in the reporter activity as compared to control siRNA-treated cells (Fig 2B). These results indicated a specific role for Nup358 in miRNA function. Figure 2. Nup358 is required for miRNA function Western analysis of HeLa cells, treated with control (siControl), Dicer (siDicer), Nup214 (siNup214), or Nup358 (siNup358) siRNA, for assessing the extent of protein depletion using indicated antibodies. Vinculin was used as loading control. HeLa cells were initially transfected with indicated siRNAs, followed by Renilla luciferase (RL) reporter constructs: RL-control (no let-7a binding site in the 3′-UTR) or RL-3xBulge (3 imperfect let-7a binding sites in the 3′-UTR). Firefly luciferase (FL) was co-transfected to serve as internal control. RL/FL luminescence ratio was calculated. Data are presented as mean ± SD (n = 3), P-values were calculated using Student's t-test. HEK293T cells were transfected with indicated siRNAs, followed by pCMV-FL-miR30 (P) reporter with either pSUPER-control or pSUPER-miR30 constructs. RL was co-transfected as internal control. FL/RL luminescence ratio was calculated. Data are presented as mean ± SD (n = 3), P-values were calculated using Student's t-test. HeLa cells were initially transfected with control (siControl), Dicer (siDicer), or Nup358 (siNup358)-specific siRNAs, followed by FL constructs containing wild-type (HMGA2-wt) or mutant (HMGA2-mut) HMGA2 3′-UTR, along with RL as internal control. Graph was plotted using data from three independent experiments. FL/RL luminescence ratio was calculated. Data are presented as mean ± SD (n = 3), P-values were calculated using Student's t-test. Download figure Download PowerPoint To test the generality of Nup358 function in miRNA pathway, we utilized another miRNA reporter system in HEK293T cells, involving firefly luciferase (FL) that contains eight miR-30a perfect binding sites (sequence with complete complementarity to miR-30a) in the 3′-UTR 62. Nup358 depletion also significantly impaired miR-30a activity (Fig 2C). Additionally, Nup358 was required for let-7-mediated suppression of FL mRNAs engineered to contain the 3′-UTR of HMGA2 that harbors multiple functional let-7 binding sites (Fig 2D) 63. Moreover, knockdown of Nup358 using three independent siRNAs targeted to different regions of Nup358 gene led to significant de-repression of RL-3xBulge reporter mRNA (Fig EV2A). When different concentrations of siRNAs were used to deplete Nup358 to varying levels, the de-repression occurred in a dose-dependent manner (Fig EV2B). Ectopic expression of GFP-Nup358 in HeLa cells enhanced miR-30-mediated suppression of FL-reporter mRNA as compared to GFP-control (Fig EV2C). Moreover, exogenous expression of Nup358 also rescued the de-repression caused by Nup358 knockdown (Fig EV2D). Taken together, these results suggested that Nup358 play
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