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Imp interacts with Lin28 to regulate adult stem cell proliferation in the Drosophila intestine [1]

['Perinthottathil Sreejith', 'Department Of Biomedical Genetics', 'University Of Rochester Medical Center', 'Rochester', 'New York', 'United States Of America', 'School Of Biological Sciences', 'Technology', 'Chonnam National University', 'Gwangju']

Date: 2022-11 

Stem cells are essential for the development and long-term maintenance of tissues and organisms. Preserving tissue homeostasis requires exquisite control of all aspects of stem cell function: cell potency, proliferation, fate decision and differentiation. RNA binding proteins (RBPs) are essential components of the regulatory network that control gene expression in stem cells to maintain self-renewal and long-term homeostasis in adult tissues. While the function of many RBPs may have been characterized in various stem cell populations, how these interact and are organized in genetic networks remains largely elusive. In this report, we show that the conserved RNA binding protein IGF2 mRNA binding protein (Imp) is expressed in intestinal stem cells (ISCs) and progenitors in the adult Drosophila midgut. We demonstrate that Imp is required cell autonomously to maintain stem cell proliferative activity under normal epithelial turnover and in response to tissue damage. Mechanistically, we show that Imp cooperates and directly interacts with Lin28, another highly conserved RBP, to regulate ISC proliferation. We found that both proteins bind to and control the InR mRNA, a critical regulator of ISC self-renewal. Altogether, our data suggests that Imp and Lin28 are part of a larger gene regulatory network controlling gene expression in ISCs and required to maintain epithelial homeostasis.

Stem cells are essential to maintain healthy organs. However, dysregulation of their function is a potential major driver of diseases, including cancer and neurodegeneration, and significantly contributes to the aging process. For these reasons, numerous mechanisms control the ability of stem cells to divide and give rise to functional daughter cells. In this study, we used the Drosophila fruitfly as a genetically amenable experimental model to characterize the function of a conserved protein, the IGF2 mRNA binding protein, in the regulation of adult intestinal stem cells. We found that it is essential for stem cell proliferation under normal conditions and in response to tissue damage. We also report that it interacts with another known regulator, Lin28. Importantly, these two factors largely control stem cell biology and development in mammals, including humans, and are often dysregulated in cancer. This suggests that our work is shedding new light on the conserved mechanisms that maintain long-term stem cell function across organisms.

Funding: This work was supported by the grant R01GM108712 from the National Institutes of Health https://www.nigms.nih.gov/ to BB and the grant NRF-2021R1A2C1010334 from the National Research Foundation of Korea https://www.nrf.re.kr/eng/main/ to CK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Sreejith et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, we report that Imp is expressed in Drosophila adult intestinal progenitors, and that it is required and sufficient cell-autonomously to promote proliferation of ISCs. We found that Imp and Lin28 genetically cooperate to control ISC proliferation and that these two proteins can physically interact. We propose a model in which Imp and Lin28 act non-redundantly to regulate the stability of the InR mRNA in to control adult intestinal proliferative homeostasis under normal conditions and in response to tissue damage.

In addition to IMP and Lin28, several post-transcriptional regulators of gene expression controlling intestinal tissue homeostasis have been identified in ISCs. For example, while Lin28 drives symmetric cell division by enhanced insulin signaling, independently of let-7, FMRP (Fragile X Mental Retardation protein), another RBP, acts to oppose Lin28-driven proliferation [ 24 , 26 ]. Similarly, Tis11, an Adenine-Uridine Riche Element (ARE) binding protein promote RNA destabilization to restore proliferative homeostasis after tissue repair [ 27 ]. Lastly, the role of microRNAs in the ISC lineage is starting to be explored; for example, expression of miR-8 and miR-305 controls cell differentiation and self-renewal respectively by affecting the expression of specific components of the conserved signaling pathways that regulate these processes [ 28 , 29 ]. Altogether, these observations suggest that post-transcriptional regulators of genes expression form a regulatory network that largely remains to be explored in ISCs.

Our work takes advantage of Drosophila as a genetic model, where both Imp and Lin28 proteins are highly conserved. While there are three IMP (IMP1, 2, 3) orthologues, and two Lin28 (Lin28A, B) orthologues in mammals, the fly genome encodes only one Imp and one Lin28 gene. In recent studies, we and others identified the role of both these RBPs in the regulation of several adult fly stem cell populations. In the testis, Lin28 is expressed in the niche cells and is required for the self-renewal of testis germline stem cells indirectly via regulating self-renewal factor Unpaired (upd), independently of the microRNA let-7 [ 22 ]. In the same cells, Imp also binds to and control the stability of the Upd messenger [ 23 ], suggesting that Imp and Lin28 may cooperate to control the function of the stem cell niche. To investigate the function of Imp in stem cells themselves, we turned to adult intestinal stem cells (ISC). The adult midgut is maintained by multipotent ISCs that can give rise to progenitors committed to the absorptive enterocyte (ECs) fate, the Enteroblasts (EBs), as well as progenitors committed to the secretory enteroendocrine cell fate (pre-EEs and EEs). In the intestinal epithelium, Lin28 is highly enriched in intestinal stem cells and is required for adult stem cell expansion by regulating the mRNA encoding for the insulin receptor (InR) [ 24 ]. However, while the expression of IMP in these cells has been suggested [ 25 ], its function in ISCs remain to be thoroughly investigated.

RBPs, including IMPs and Lin28s, have been extensively studied in developing tissues, adult stem cells or in tumor cells. However, beside the individual function of RBPs, increasing evidence suggest that the interaction between RBPs is critical in defining the fate of RNA and gene expression [ 10 , 11 ]. Investigating how RBPs cooperate and/or compete to regulate the post-transcriptional gene network thus becomes crucial to understand the establishment and maintenance of different cellular phenotypes, like stem cell pluripotency and self-renewal. Recently, several studies have identified genetic and biochemical interactions between IMPs and Lin28s in various stem and progenitor populations [ 12 – 14 ]. However, many questions remain regarding the role of this potential interaction in adult tissue stem cells, or in cancer cells where higher expression of these proteins have been associated with disease state [ 15 – 21 ].

Self-renewal is essential for normal and pathological stem cell function and relies on complex gene regulatory networks. Thus, understanding the molecular mechanisms of gene regulation in stem and progenitor cells is critical for designing strategies to reprogram somatic cells, promote tissue regeneration, treat degenerative diseases, or understand the tumorigenesis process. RNA binding proteins (RBPs) have emerged as key regulators of cell pluripotency and self-renewal [ 1 , 2 ]. Particularly, IGF2 mRNA binding proteins (IGF2BPs / IMPs) and Lin28 are two families of RBPs that have been identified as critical regulators of gene regulation in stem cells and progenitors, controlling many essential aspects of their biology during development and in adult tissues [ 3 , 4 ]. On one hand, IMPs are a group of highly conserved RBPs that regulate mRNA targets by promoting their stability either by preventing mRNA degradation, by localizing mRNA to cytoplasmic granules, or by blocking siRNA/miRNA mediated silencing [ 3 ]. As a result, IMPs are essential to maintain stem cell identity and function in many biological contexts [ 3 , 5 ]. On the other hand, Lin28, a highly conserved RBP, was identified early as one of the critical factors sufficient to reprogram human somatic cells into pluripotent stem cells and regulates metabolic genes in these cells [ 6 , 7 ]. Mechanistically, Lin28 are best characterized for its regulation of microRNA let-7 biogenesis but, more recently, Lin28s have been shown to associate with mRNA-ribonucleoprotein (mRNP) complexes and acts as regulators of mRNA stability and translation by binding to many transcripts in embryonic stem cells (ESCs) and other stem cells [ 8 , 9 ].

Results

Imp expression is highly enriched in adult intestinal progenitors Based on the functional interaction between IMPs and LIN28s in many stem cell populations, on the function of both Imp and Lin28 in the fly testis niche, on the expression of Lin28 in ISCs, and a recent report [25], we hypothesized that Imp could play a role in controlling ISC function. First, to investigate the expression pattern of Imp in the adult intestinal epithelium, young adult midguts were stained with an antibody directed against the endogenous Imp protein. We found significant Imp expression in all progenitor cells (ISC and EBs), as well as weak expression in EEs (Fig 1). This was first demonstrated by colocalization of Imp protein with Delta, a well-established ISC marker, as well as low signal in prospero-positive EEs (Fig 1A). Also, we show that Imp is expressed in all escargot-positive cells, using the esg-Gal4>UAS-GFP;tubulin-Gal80ts (esgGFPts) driver line (Fig 1B). We knocked-down the expression of Imp in esg-positive cells, using the temperature-inducible esgGFPts driver and a dsRNA construct directed against Imp (UAS-ImpRNAi); the loss of Imp signal in this condition confirmed the specificity of our staining and the efficacy of the RNAi line (Fig 1B). To support the anti-Imp antibody staining, we next used the protein trap line GFP-Imp [30]. We found the expression of the Imp fusion protein in all intestinal progenitors, as shown by the co-expression with the ISC- and EB- specific esg-LacZ reporter (Fig 1C) and colocalization with the anti-Imp staining (Fig 1D). Altogether these data indicate that Imp is enriched in ISCs and EBs in the midgut epithelium, suggesting it may play an essential regulatory function in ISCs. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Imp expression in adult intestinal stem cell progenitors. (A) Confocal images of 7-day old wild type intestinal epithelium stained with antibodies specific to Imp and Delta (Dl, ISC marker) and Prospero (Pros, enteroendocrine cell marker). (B) Confocal images of 5-day old adult guts (esg-GFPts) stained with antibody specific to Imp shows the expression of Imp in Stem cell progenitors where GFP is expressed in ISCs and EBs. Adult specific knockdown of Imp using Imp RNAi (esg-GFPts>ImpRNAi) leads to loss of Imp staining in the stem cell progenitors compared to controls (esgGFPts>+). (C) Confocal images of the intestine of 7-day old trans-heterozygote for GFP-Imp-Trap and progenitor specific Lac-Z reporter (GFP-Imp;esg-LacZ) stained with antibody specific to GFP and β-galactosidase show the expression of Imp in all LacZ-positive ISCs and EBs. (D) Confocal images of 7-day old GFP-Imp gut co-stained for delta, Prospero, Imp and GFP antibody confirms the expression of Imp in ISCs and EBs. In all panels, scale bar: 10μm. https://doi.org/10.1371/journal.pgen.1010385.g001

Imp expression is required and sufficient cell-autonomously for ISC proliferation Based on its expression in ISCs and EBs, we asked whether, like Lin28, Imp is required for ISC proliferation. Since Imp mutants are homozygous lethal [31], we used MARCM (Mosaic analysis with a repressible cell marker; [32]) clonal analysis to induce and trace Imp7 homozygous mutant ISC lineages in heterozygous adults. Intestines were analyzed at different time points after clonal induction (days after heat shock) to assess the number of labelled cells per clones. In control intestines, we observed a persistent increase in the size of marked clones reporting the proliferative activity of individual ISCs (Fig 2A). However, Imp7 homozygous mutant clones remained constantly small, mostly composed of 1 to 2 Dl-positive Sox21a-positive ISCs and very few Sox21a EBs [33], even 30 days after clonal induction (Fig 2A). This demonstrates that Imp is required cell autonomously for ISC proliferation but dispensable for their survival under normal conditions. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Imp is required cell autonomously in Intestinal Stem cells. (A) MARCM clonal analysis of control and Imp homozygous null ISCs shows that Imp is essential for ISC proliferation. Clones are labeled by GFP expression (green), Sox21a identifies ISCs and EBs, Delta (Dl) specifically stains ISCs and Prospero (Pros) highlights enteroendocrine cells. Insert ‘a’ presents a higher magnification image of an Imp mutant clone, illustrating the presence Dl-positive and Sox21a-positive cells in these very small GFP+ cell clusters. Box plot represents the distribution of number of cells per clone in an age-dependent manner. Imp mutant clones fail to grow, even after 30 days of clonal induction (days after heat shock, AHS). Student’s t-test **** p-value = < 0.0001; ns = 0.426. (B) Imp is required specifically in ISCs for cell proliferation. Cell specific knockdown of Imp in ISCs, but not in EBs, abolishes DSS- induced cell division. Proliferation is measured by the number of phospho-Histone H3 (pH3) positive cells per gut 48-hour exposure to DSS, in control animals or when Imp is knocked-down in ISCs (ISC-YFPts), ISCs+EBs (esgGFPts) or EBs (GBE-GFPts). (C, D) Over-expression of Imp in ISCs (ISC-YFPts) or ISCs+EBs (esgGFPts) is sufficient to promote cell proliferation, as shown by the expansion of the esg-positive and Dl-positive cells (C) and the increased number in pH3-positive mitotic cells (arrowheads, D). Delta (Dl) specifically stains for ISCs and Prospero (Pros) stains for EEs. DNA is stained by Hoechst. In B and D, each data point represents the number of pH3-positive cells in a gut; n>10 guts per genetic and treatment conditions. Student’s t-test **** p-value = < 0.0001; * p = <0.05; ns p = 0.3562. In panels A and C, scale bar: 10μm. https://doi.org/10.1371/journal.pgen.1010385.g002 While the intestinal epithelium remains relatively quiescent at homeostasis, ISC proliferation can be increased in response to various external stimuli, such as exposure to toxicants or bacterial infection [34–36]. The chemical stressor dextran sulfate sodium (DSS) induces a rapid and robust ISC response [34]. Therefore, we tested the role of Imp in intestinal progenitors in this stress paradigm. We combined UAS-ImpRNAi with cell type specific drivers to conditionally knockdown Imp in ISCs and/or EBs and ISC proliferation by feeding these transgene-expressing flies 4% DSS or sucrose (control). Proliferation in the intestinal epithelium of these animals was measured by counting the number of cells positive for mitotic marker phospho-histoneH3 (pH3). We found that loss of Imp specifically in ISCs, not in EBs, lead to blockage of proliferation: DSS-induced proliferation is inhibited when ImpRNAi is driven by the esgGFPts (ISCs+EBs; esgGal4>UAS-GFP,tub-Gal80ts) or the ISC-YFPts (ISCs only; esGal4>UAS-YFP,tub-Gal80ts,GBE-Su(H)Gal80), but not when we used the GBE-GFPts driver (EBs only; GBE-Su(H)Gal4>UAS-GFP,tub-Gal80ts) (Figs 2B and S1A). Next, we exposed esgGFPts>UAS-ImpRNAi to the ROS-producing compound paraquat (PQ), to promote stem cell division [37], and observed a similar block of ISC proliferation in this oxidative stress paradigm (S1B Fig). This confirmed that, like under homeostatic conditions, Imp is required cell-autonomously in ISCs to promote cell division after tissue damage. This supports the notion that it acts downstream of the stress-responsive transcription factor ets21c in these cells [25]. Conversely, we found that long-term over-expression of Imp using the esgGFPts or ISC-YFPts drivers results in very high numbers of mitotic cells in the intestine, demonstrating that Imp is sufficient to promote ISC proliferation cell-autonomously (Fig 2C). Altogether, our data establish that Imp is a critical regulator of ISC proliferation during homeostatic tissue turnover and in response to tissue challenge.

Imp and Lin28 cooperate to control ISC proliferation In the testis, Imp and Lin28 are part of a regulatory network that control the expression of the self-renewal factor Upd in hub cells. Thus, we genetically interrogated the relationship between Imp and Lin28 in ISCs. First, we asked whether increased levels of Imp and Lin28 can cooperate to promote ISC proliferation. As opposed to Imp long-term over-expression (Fig 2C), two-day overexpression of each factor individually, using the esgGFPts driver, results in a significant but limited increase in proliferation (Fig 3A). However, when combined, the co-overexpression of Imp and Lin28 synergistically increases ISC proliferation, as seen by high levels of pH3+ cells, 2 days after transgenes induction, and the large expansion in the number of esg>GFP+ cells, 5 days after induction. This results in flies with increased sensitivity to DSS treatment, as shown by their significantly reduced survival under these culture conditions (S2A Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 3. Imp and Lin28 cooperate to regulate ISC proliferation. (A) Co-expression of Lin28 and IMP leads to synergistically increased ISC proliferation. Box plot representing the quantification of the number of pH3 positive cells per gut that shows that ISCs/EBs specific co-expression of Imp and Lin28 leads to high number of mitotic cells 2 days after transgene induction. Representative images are shown to illustrate the expansion of esg-positive cells 5 days post induction of both transgenes. GFP is expressed in ISCs and EBs, Delta stains for ISCs and Prospero (Pros) stains for EEs. DNA is stained by Hoechst. Student’s t-test **** p-value = <0.0001; ** p = 0.0036. (B) Double knockdown of Imp and Lin28 results in a long-term loss of progenitors in adult guts, 7 days after transgene activation. Plot presenting the comparison of the percentage of esg-positive in the posterior midgut (measured by the ratio of GFP positive cells vs the total number of cells, counted by DNA-positive nuclei) showing a reduced number of progenitors when either Imp or lin28 are knocked down, which is exacerbated when both Imp and Lin28 are knocked down. Representative confocal images are shown to illustrate the changes in the number of GFP-positive cells. (C) Combined loss of Imp and Lin28 reduces adult female lifespan. Adult specific knockdown of Imp and Lin28 in esg-positive cells leads to accelerated death when flies are reared at 29°C. Kaplan-Meier survival curves of three populations of 25 flies are shown for each genetic condition. In A and B, scale bar: 10μm. https://doi.org/10.1371/journal.pgen.1010385.g003 Next, we asked whether the combined loss-of-function of Imp and Lin28 may affect ISC and tissue homeostasis. We used the esgGFPts driver to express UAS-ImpRNAi+UAS-Lin28RNAi in ISC/EBs starting in young adult flies. Like ImpRNAi expression alone, this manipulation completely blocks DSS-induced ISC proliferation (S1A Fig). However, this combination also significantly decreases the number of esg-positive cells in the intestine, compared to wild-type animals or flies expressing only one of the RNAi constructs (Fig 3B). In addition, the combined knock-down of Imp and Lin28 results in dramatically shorter-lived flies, with median lifespan of 8 to 10 days at 29°C (Fig 3C). Of note, the combined knock-down does not affect the sensitivity to DSS treatment (S2A Fig), suggesting that ISC proliferation is required for long-term maintenance but dispensable for stress response. These data suggest that Imp and Lin28 cooperate to maintain intestinal homeostasis. Thus, we next tested whether they may act redundantly in ISCs. To this end, we performed genetic rescue experiments using MARCM clones (S2B Fig). As expected, Imp expression can significantly rescue the proliferation of Imp7 homozygous clones and Lin28 expression restores Lin28Δ1 clone growth. However, when Lin28 was expressed in Imp null mutant clones or, conversely, when Imp was expressed in Lin28 null clones, only minimal rescue was observed (Fig 3B), suggesting that Imp and Lin28 cannot optimally compensate for the absence of the other factor. Of note, we observed that over-expression of Imp using the esgGFPts driver leads to a detectable increase in Lin28 in intestinal progenitors and, conversely, that over-expressing Lin28 results in increased Imp protein expression (S3 Fig). This suggests that these two RBPs regulate each other directly or indirectly. Together, these observations lead us to conclude that Imp and Lin28 are not redundant, but rather cooperate to control ISC proliferation and maintenance.

Imp regulates InR expression in ISCs In hub cells, Imp and Lin28 both bind to the upd mRNA and regulate its stability [22]. In ISCs, Lin28 controls cell proliferation, at least in part, by regulating the InR messenger [24]. We then hypothesized that, similarly, Imp regulates InR and that the defects in ISC proliferation observed in Imp loss-of-functions are caused by reduced insulin/IGF-1 signaling. To first test this model, we asked whether increased of InR expression in Imp mutant clones could rescue the strong proliferation defect that we observed (Fig 2A). Using the MARCM technique, we drove the expression of the wild-type InR protein (UAS-InR) or an activated form of the receptor (UAS- InRact) in Imp7 homozygous mutant clones (Fig 4A). We found that expression of either of these InR transgenes is sufficient to fully rescue ISC proliferation, as shown by the significant clone growth observed 7 and 14 days after induction. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Imp regulates InR mRNA expression in the intestinal epithelium. (A) Confocal images of MARCM clones illustrating the growth of Imp7 homozygous mutant clones expressing wild-type and constitutive active forms of the insulin receptor (InR). GFP expression labels ISC clones, Sox21a stains for the progenitors and DNA is stained with Hoechst. The number of cells per clones in the different genetic backgrounds, 7 and 14 days after heat shock induction (AHS), are shown, demonstrating that activating InR signaling is sufficient to restore ISC proliferation in Imp mutants. Student’s t-test **** p-value = <0.0001 Scale bar: 10μm. (B) Relative expression of the InR mRNA, normalized to escargot, in the 2-day old gut from control animals and when Imp and/or Lin28 are expressed in esg-positive cells. Imp+Lin28 co-expression is sufficient to drive the expression of InR, suggesting that both proteins regulate InR synergistically. Student’s t-test **** p-value = <0.0001. (C) qPCR quantification of the InR mRNA, normalized to rp49, in 7-day old gut RNA extracts shows reduced InR transcript levels in Imp heterozygotes and Lin28 homozygous mutants. Student’s t-test *** p-value = 0.0005, ** p = 0.0036. (D) Representative confocal image showing the decreased levels of InR protein detected by immunostaining (green) in intestinal progenitors of Imp and Lin28 mutants, compared to wild-type animals. Dl-positive ISCs are indicated by arrowheads. DNA is stained with Hoechst. (E) Imp and Lin28 bind to InR transcripts in adult guts. 7-day old guts were subjected to RNA-immunoprecipitation using either GFP-Imp or Lin28-venus, followed by qPCR. Significant levels of InR mRNA, normalized to input materials, are pulled down using both proteins, compared to controls. Student’s t-test **** p-value = <0.0001. https://doi.org/10.1371/journal.pgen.1010385.g004 Previously it was shown that Lin28 can physically interact with the 3’UTR of the InR mRNA to regulate its stability [24]. Thus, we reasoned that, like Lin28, Imp may also affect the steady state level of the InR transcript. We performed qRT-PCR in the same conditions where we observed a synergistic effect of Imp and Lin28 co-expression on ISC proliferation (Fig 3A). No significant change in InR expression is observed when Imp or Lin28 are over-expressed individually using the esgGFPts driver. However, a two-day combined induction of the UAS-Imp+UAS-Lin28 transgenes, which does not cause a significant change in the number of esg+ cells (Fig 3A), results in a marked increase in the level of InR mRNA in total intestinal RNA extracts (Fig 4B). This strongly suggests that the levels of InR mRNA are synergistically induced by Imp and Lin28 in ISCs and EBs. Using immunostaining, we confirmed the ability of Imp or Lin28 over-expression to individually cause the accumulation of significantly higher InR protein levels in esg-positive cells, 7 days after transgene induction (S4A and S4B Fig). Conversely, we found that, like Lin28 null animals, Imp7 heterozygotes show reduced levels of InR transcripts in the intestine compared to wild-type controls (Fig 4C) and lower InR protein expression in Dl-positive cells (Fig 4D). Finally, we tested the binding of Imp to the InR mRNA in the adult gut. We performed RNA-Immunoprecipitation followed by qRT-PCR in intestinal extracts from animals expressing either the GFP-Imp or Lin28-Venus protein fusions under their respective endogenous promoters. We found a significant enrichment of InR transcripts in the anti-GFP (GFP-Imp extracts), or anti-Venus (Lin28-Venus extracts) immuno-precipitates compared to control non-specific antibodies (Fig 4E), strongly suggesting that Imp and Lin28 can both bind the InR mRNA in intestinal progenitors. Altogether, these data strongly support the notion that Imp binds to the InR mRNA to promote its stability and allow ISC proliferation.

Lin28 and Imp can directly interact We found that Imp and Lin28 are both expressed in intestinal progenitors, are essential for ISC proliferation and regulate InR mRNA levels. This raised the possibility that the Imp and Lin28 proteins directly interact to control expression of their targets, including InR. Interestingly, previous studies reported the direct interaction between IMP3 and LIN28B in hematopoietic stem and progenitor cells (HSPCs), and IMP1 and LIN28A in neuronal progenitors [13, 14]. To demonstrate the ability of Drosophila Imp and Lin28 proteins to interact, we used several distinct experimental models. Firstly, we carried out a yeast two hybrid assay and found that Lin28 and Imp protein fusions can form a stable complex in yeast. Co-expression of Imp-TAD+Lin28-DBD or Imp-DBD+Lin28-TAD (TAD: Transcriptional Activation Domain of the transcription factor GAL4; DBD: LexA DNA Binding Domain) is sufficient to promote the expression of a LexAop-driven beta-Galactosidase reporter in S. cerevisiae cells (Fig 5A). Next, we performed co-immunoprecipitation in cultured Drosophila S2 cells. We found that, when Flag-tagged Imp and HA-tagged Lin28 proteins are co-expressed, both proteins can be co-precipitated using either an anti-Flag antibody or an anti-HA antibody (S5 Fig). Importantly, when the protein extract is pre-treated with RNAse, the co-precipitation is not affected suggesting the Imp-Lin28 protein interaction is RNA-independent in fly cells (Fig 5B). Finally, we confirmed the ability of Imp and Lin28 proteins to interact by GFP fragment complementation assay in HEK293T cells. Imp and Lin28 fusion proteins with the C-terminal or N-terminal domains of GFP were transfected in cells. Only when the combinations GFP-ImpNter+Lin28-GFPCter or GFP-ImpCter+Lin28-GFPNter are co-expressed, can significant GFP fluorescence signals be detected, indicating the formation of a stable interaction between the two GFP domains mediated by Imp and Lin28 (Fig 5C). Using these three distinct assays, including in yeast and mammalian cells, we demonstrate that Drosophila Imp and Lin28 can interact, and that this interaction is unlikely to be mediated by common mRNA targets, but rather is a direct protein-protein interaction. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Drosophila Imp and Lin28 physically interact. (A) Imp and Lin28 can form a stable complex in yeast cells, as shown by 2-hybrid assay. Quantification of the β-galactosidase activity when Imp and Lin28 protein fusion with the Gal4 Transcriptional Activation Domain (TAD) or the LexA DNA Binding Domain (DBD). Significant reporter activity is detected only when the Imp-TAD+Lin28-DBD or Imp-DBD+Lin28-TAD protein combinations are expressed. Student’s t-test **** p-value = <0.0001. (B) Co-immunoprecipitation of HA-tagged Lin28 and FLAG-tagged Imp proteins expressed in cultured Drosophila S2 cells. Anti-Flag precipitation pulls-down both protein fusions. RNase treatment of the lysate does not affect the stability of the complex. Total RNAs stained with ethidium bromide demonstrate the efficacy of the RNase treatment. (C) Fluorescence images of GFP complementation assay in HEK293T cells show that only when the appropriate combinations of Lin28 and Imp protein fusions (GFP-ImpNter+Lin28-GFPCter or GFP-ImpCter+Lin28-GFPNter) result in significant GFP fluorescence. Quantification of the integrated GFP intensity in different combinations of Imp and Lin28 proteins is shown over hundreds of cells per condition. RFP expression serve as a transfection control and for normalization. Scale bar: 10μm. https://doi.org/10.1371/journal.pgen.1010385.g005

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