Integrative genomics positions MKRN1 as a novel ribonucleoprotein within the embryonic stem cell gene regulatory network

Abstract

In embryonic stem cells (ESCs), gene regulatory networks (GRNs) coordinate gene expression to maintain ESC identity; however, the complete repertoire of factors regulating the ESC state is not fully understood. Our previous temporal microarray analysis of ESC commitment identified the E3 ubiquitin ligase protein Makorin-1 (MKRN1) as a potential novel component of the ESC GRN. Here, using multilayered systems-level analyses, we compiled a MKRN1-centered interactome in undifferentiated ESCs at the proteomic and ribonomic level. Proteomic analyses in undifferentiated ESCs revealed that MKRN1 associates with RNA-binding proteins, and ensuing RIP-chip analysis determined that MKRN1 associates with mRNAs encoding functionally related proteins including proteins that function during cellular stress. Subsequent biological validation identified MKRN1 as a novel stress granule-resident protein, although MKRN1 is not required for stress granule formation, or survival of unstressed ESCs. Thus, our unbiased systems-level analyses support a role for the E3 ligase MKRN1 as a ribonucleoprotein within the ESC GRN.

Keywords: RNA metabolism; embryonic stem cells; makorin‐1; stress granules.

Figure 1. MKRN1 protein expression is correlated with the undifferentiated ESC state

1. qPCR analysis of fold change in Mkrn1, Oct4, Nanog, and Sox2 mRNA expression in R1 ESCs upon differentiation induced by LIF withdrawal (−LIF) or RA treatment (+RA) relative to undifferentiated R1 ESCs collected at 0 h. Data are means ± standard error of the mean (SEM) from three biological replicate experiments (*P < 0.05, **P < 0.01, ***P < 0.0001 vs. +LIF; two‐way ANOVA).

2. Quantitative immunoblot analysis of MKRN1 and OCT4 expression in R1 ESCs cultured for 48 or 72 h in self‐renewal (+LIF) or differentiation conditions (+RA). The OCT4 immunoblot was first probed with anti‐OCT4 antibodies and subsequently probed with anti‐GAPDH antibodies. MKRN1 and OCT4 protein abundance was normalized to GAPDH and reported relative to undifferentiated R1 ESCs collected at 0 h. MKRN1 =  ˜53‐kDa and ˜42‐kDa bands, OCT4 = ˜45‐kDa band, and GAPDH = ˜37‐kDa band. Data are means ± SEM of two biological replicate experiments (*P < 0.05 vs. +LIF; two‐way ANOVA).

3. Co‐immunofluorescent staining of endogenous MKRN1 and OCT4 in R1 ESCs cultured in LIF + serum. MKRN1 is predominantly localized to the cytoplasm. Scale bars: 10 μm.

4. Intracellular flow cytometry analysis for MKRN1 and OCT4 expression from single R1 ESCs cultured in +LIF, −LIF, or +RA for 72 h. Quantile contour plots of OCT4‐ and MKRN1‐positive/negative cells in each culture condition. Mean fluorescent intensity of MKRN1 was quantified from the OCT4⁺ and OCT4⁻ subpopulations in each culture condition. MKRN1 is significantly more abundant in the OCT4⁺ subpopulation than in the OCT4⁻ subpopulation irrespective of culture conditions. Data are means ± SEM from three biological replicates (P = 0.0083, two‐way ANOVA).

Source data are available online for this figure.

Figure EV1. Validation of MKRN1 overexpression and knockdown transgenic ESC clones

1. A, B

   Twenty micrograms of total protein from whole‐cell lysates derived from undifferentiated MKRN1 overexpression (FLAG:MKRN1) (A) and MKRN1 knockdown (shMKRN1) clones (B) and their respective controls (including R1 parental ESCs) was resolved with SDS–PAGE. Blots were probed with commercial antibodies directed against MKRN1 (top) to test the specificity of the antibody, and OCT4 (bottom) to verify that lysates were obtained from undifferentiated ESC populations. The MKRN1 antibody (ab72054) detects two MKRN1‐specific bands at ˜53 and ˜42 kDa, and two non‐specific bands of size 80 and 25 kDa. Both the 53‐kDa and 42‐kDa bands were depleted in stable MKRN1 knockdown ESC lysates, but not the non‐specific 80‐kDa and 25‐kDa bands (B). The 53‐kDa band corresponds to the full‐length endogenous MKRN1 protein and the second ˜42‐kDa band may represent a degraded by‐product of the full‐length 53‐kDa MKRN1, or potentially the putative MKRN1 splice variant, Mkrn1‐004, which encodes a putative protein of 47.3 kDa. The MKRN1 antibody also detects an additional ˜63‐kDa band exclusively in FLAG:MKRN1 ESCs that corresponds to recombinant FLAG:MKRN1 (A). Total MKRN1 and OCT4 protein expression was quantified and reported relative to GAPDH. OCT4 protein abundance was comparable between MKRN1 transgenic and control clones (A, B). Total MKRN1 expression was highest in FLAG:MKRN1 clones B7 and C7 (***P < 0.001), followed by C4 (**P < 0.01) and C2 (*P < 0.05), whereas MKRN1 was depleted to the greatest extent in shMKRN1 clones C4 (**P < 0.01) followed by C12, F5, and C9 (*P < 0.01). Data are means ± SEM from three biological replicate experiments (one‐way ANOVA).

2. C

   Alkaline phosphatase‐positive ESC colonies formed from stable shGFP control and stable MKRN1 knockdown ESC clones cultured in LIF + serum in feeder‐free conditions. Scale bars: 60 μm.

3. D

   R1 ESCs cultured in LIF + serum immunostained with anti‐MKRN1 antibodies and counterstained with Hoechst. Scale bars: 10 μm.

4. E

   FLAG:MKRN1 ESCs cultured for 72 h in self‐renewal (top) or RA‐induced differentiation conditions (bottom) were immunostained with anti‐FLAG antibodies and counterstained with Hoechst. Scale bars: 10 μm.

5. F

   Distribution of MKRN1⁺ and MKRN1⁻ cells in the OCT4⁺ and OCT4⁻ fraction of ESC cultures as determined by high‐content imaging analysis. Association between OCT4 and MKRN1 expression in individual cells was significant in both self‐renewal (72 h +LIF) and differentiation (72 h −LIF) conditions (P < 0.001; chi‐square test).

Figure EV2. Validation of the FLAG:MKRN1 AP‐MS data set

1. A

   Anti‐FLAG immunoblot of representative anti‐FLAG IP immunoprecipitates (lanes 1–4) and total protein lysates (lanes 5–8) derived from MG132‐treated or untreated ESCs expressing recombinant FLAG:MKRN1 protein or an empty FLAG vector. Anti‐FLAG immunoprecipitates purified in this experiment were subjected to LC‐MS/MS analysis.

2. B, C

   Reciprocal anti‐IGF2BP1 (B) and anti‐HuR (C) IPs from undifferentiated ESC lysates verify the presence of MKRN1 in the respective mRNP complexes.

3. D, E

   Steady‐state abundance of MKRN1‐associated proteins, IGF2BP1 and HuR, in MKRN1 knockdown (D) and MKRN1 overexpression (E) ESC populations. Quantified protein abundance is reported relative to GAPDH. Neither IGF2BP1 nor HuR protein abundance was affected by the manipulation of MKRN1 expression. Data are means ± SEM from two biological replicate experiments.

4. F

   Autoradiogram of ³²P‐labeled RNA obtained by CLIP with either anti‐FLAG antibodies or IgG isotype control antibodies from FLAG:MKRN1 or FLAG:Ctrl ESC lysates, as indicated. Prior to lysis, ESCs were either UV cross‐linked, or left uncross‐linked (no X‐link). Data represent findings from two biological replicate experiments.

Figure 2. AP‐MS analysis identifies MKRN1 as a component of mRNPs in ESCs

1. List of the top 20 enriched proteins co‐purified with FLAG:MKRN1 from MG132‐treated and untreated ESC lysates ranked according to mean spectral count values. Data are means ± SEM of three biological replicate experiments. The XY scatter plot (top right insert) indicates a strong positive correlation (r ² = 0.9905) in mean spectral count values for all 48 FLAG:MKRN1‐associated proteins purified from MG132‐treated and untreated lysates.

2. Enrichment map representation of enriched cellular processes within the FLAG:MKRN1 AP‐MS data set (P < 0.001; FDR < 0.1). Nodes and edges are represented as described in Materials and Methods. Proteins within labeled gene sets are listed in Table EV2.

3. Proteins identified with enriched total spectral counts by LC‐MS/MS from RNase A‐treated and untreated FLAG:MKRN1 IPs from two biological replicate experiments. Proteins are ranked along the x‐axis according to whether they are predicted to interact with MKRN1 in a RNA‐independent or RNA‐dependent manner.

4. Predicted RNA‐independent (PABP), RNA‐dependent (HuR), and RNase‐sensitive (IGF2BP1) associations tested by FLAG:MKRN1 co‐IP Western blots. Data are representative of two independent experiments. Note that due to limited amount of IP material, the IGF2BP1 immunoblot was re‐probed with anti‐HuR without stripping the blot. The ˜37‐kDa band corresponding to HuR was visible in FLAG:MKRN1 IPs but not control IPs.

5. Protein–RNA complexes are observed from FLAG:MKRN1 IPs, but not control IPs. Note the radioactive smear from FLAG:MKRN1 IPs treated with a low concentration of RNase (1:100,000) that is resolved to a focused band at the expected size of MKRN1–RNA complexes with moderate RNase A treatment (1:25,000) and strongly reduced with a high dose (1:1,000) of RNase A (n = 2). CLIP data from additional RNase dilutions are provided in Fig EV2F.

6. Anti‐MKRN1 immunoblot (left and center) and corresponding RNA autoradiogram (right) identify MKRN1–RNA complexes uniquely purified from FLAG:MKRN1 IPs. At the optimized RNase concentration of 1:25,000, ³²P‐labeled RNA–protein complexes purified from FLAG:MKRN1 ESC lysates resolved to a mass that corresponded to supershifted MKRN1 proteins that may reflect the RNA‐bound fraction of MKRN1. Data are from CLIP samples obtained from the same experiment and are representative of two biological replicate experiments.

Source data are available online for this figure.

Figure 3. MKRN1 is mobilized to SGs, but is not required for SG formation

1. Recruitment of endogenous MKRN1 in SGs in R1 ESCs stressed for 1 h with 1 mM ${\text{NaAsO}}_2^{-}$ (+NaAsO₂). SG formation was monitored with HuR and TIAR immunostaining. Yellow arrows indicate MKRN1/HuR/TIAR‐positive SGs; white arrows indicate SGs devoid of MKRN1. Enlargements of boxed regions are indicated as 5× zoom.

2. MKRN1 is not absolutely required for SG assembly in undifferentiated ESCs. MKRN1 knockdown and shGFP control ESC clones were stressed as in (A). Yellow arrows indicate TIAR‐positive SGs in OCT4⁺ ESCs; white arrows indicate TIAR‐positive SGs in OCT4⁻ ESCs. Scale bars: 10 μm. Enlargements of boxed regions are indicated as 5× zoom.

Figure EV3. MKRN1 is mobilized to SGs in response to environmental stress

1. Endogenous MKRN1 is targeted to SGs in ESCs following 1‐h treatment with 10 μM thapsigargin. Yellow arrows indicate MKRN1/HuR‐positive SGs. Scale bars: 10 μm.

2. Overexpression of MKRN1 does not spontaneously induce SG assembly in unstressed ESCs and does not impair SG formation in NaAsO₂‐stressed ESCs. Yellow arrows indicate MKRN1/HuR/TIAR‐positive SGs. Scale bars: 10 μm. Enlargements of boxed regions are indicated as 5× zoom.

3. Ectopic FLAG:MKRN1 is localized to SGs upon NaAsO₂‐induced environmental stress. Yellow arrows indicate G3BP‐positive SGs in NaAsO₂‐treated FLAG:MKRN1 ESC populations. Scale bars: 10 μm. Enlargements of boxed regions are indicated as 5× zoom.

Figure 4. Integrative genomic analysis of the MKRN1–mRNA network

1. Histograms comparing the distribution of mRNA abundance for transcripts in the MKRN1–mRNA network with > 20,000 transcripts representing the ESC transcriptome. Further details are provided in Materials and Methods.

2. Venn diagram shows the overlap of transcripts in the MKRN1–mRNA network with the 411 differentially expressed transcripts identified from the transient MKRN1 knockdown microarray data set.

3. Network diagram of the 45 transcripts identified in (B). Node color corresponds to the extent each transcript was differentially expressed in the transient MKRN1 knockdown microarray data set.

4. qPCR data from stable MKRN1 knockdown ESCs validate the microarray data set. Colored bars represent the fold change in abundance of eight FLAG:MKRN1‐associated mRNAs from (C) in the stable shMKRN1 ESC clone C4 relative to a stable shGFP control clone (gray bars). Bar color scheme is the same as (C) and corresponds to the extent the mRNA was differentially expressed in the transient MKRN1 knockdown microarray data set. Data are means ± SEM from three biological replicate experiments (P‐values are indicated, two‐tailed t‐test).

5. RIP‐qPCR experiments were performed in triplicate to corroborate the findings from the RIP‐chip analysis. RIP‐qPCR experiments confirmed that the predicted FLAG:MKRN1 RIP targets (blue bars) were enriched in FLAG:MKRN1 RIPs versus a subset of transcripts not detected as enriched from the RIP‐chip analysis (gray bars) (P < 0.001; Mann Whitney U‐test). Asterisks denote the four of the five predicted FLAG:MKRN1 RIP targets with mean fold enrichment values above the specified threshold (red line) calculated as two standard deviations (SD) from the mean fold enrichment of the negative control group (gray bars). Data are means ± SD from three biological replicate experiments.

Figure EV4. Subcellular fractionation analysis of MKRN1‐associated mRNAs predicted to localize to the ER

1. qPCR analysis verifies that Mkrn1 expression was repressed in shMKRN1‐transfected ESC populations relative to shGFP‐transfected ESC populations prior to performing microarray analysis. Data are means ± SEM from four biological replicate experiments (*P < 0.0001, two‐tailed t‐test).

2. Alkaline phosphatase‐stained, shRNA‐transfected ESC populations following four days of puromycin selection. Only positively transfected cells remained following puromycin selection as indicated by the absence of cells in the mock‐transfected population. Transient knockdown of Mkrn1 did not induce spontaneous differentiation of ESCs cultured in LIF + serum. Scale bars: 100 μm.

3. Immunoblot analysis of the ER‐enriched and the soluble (cytoplasmic) fractions derived from stable MKRN1 knockdown and shGFP control ESC populations. Calnexin, an ER luminal protein marker, was identified exclusively in the ER‐enriched fraction of both shGFP and MKRN1 knockdown lysates irrespective of EDTA treatment, whereas trimethylated H3K27 (a nuclear marker) was not detected in ER‐enriched or soluble fractions. EDTA treatment evoked a marked release of mRNPs from membrane‐bound ribosomes as evident by the decrease of PABP and the RNA helicase, MOV10, in the ER‐enriched fraction. MKRN1 is present in ER‐enriched fractions, yet comparatively it is more abundant in the cytosolic fraction of undifferentiated ESC lysates. Similar to PABP and MOV10, MKRN1 is sensitive to EDTA treatment, suggesting that MKRN1 localization to ER membranes is dependent on its association with mSMERPs. These data confirm that our subcellular fractionation technique accurately partitioned each subcellular compartment and, furthermore, could distinguish between ribosome‐dependent and ribosome‐independent mRNP translocation events.

4. Subcellular ER‐to‐cytosol distribution of seven representative MKRN1 target mSMERPs in shMKRN1 and shGFP ESC lysates. mRNA from the ER‐enriched and soluble fraction was quantified with qPCR. The seven representative MKRN1 target mSMERPs are labeled in black. E‐cadherin, Epcam, and Lamp1 are non‐MKRN1 target mRNAs encoding signal‐peptide‐containing proteins (positive controls; blue labels). Oct4 mRNA is a non‐MKRN1 target mRNA encoding a soluble nuclear protein (negative control; red label). Data are means ± SEM from three biological replicate experiments. Asterisks indicate the transcripts whose association in the ER‐enriched fraction was sensitive to EDTA treatment (two‐way ANOVA).

5. qPCR data comparing the relative distribution of the indicated mRNAs (labels are consistent with D) in MKRN1 knockdown and shGFP control ESC lysates. Data are means of two MKRN1 knockdown clones (C4 and F5) and two shGFP control clones (D5 and D8) in each group ± SEM from three biological replicate experiments. Differences in relative distributions between MKRN1 knockdown and shGFP control clones are not significant.

Figure 5. The MKRN1–mRNA network is enriched for transcripts encoding functionally related proteins

Enrichment map representation of enriched cellular and biological processes for low‐ and high‐abundance mRNAs in the MKRN1–mRNA network (P < 0.005; FDR < 0.1). Nodes and edges are represented as described in Material and Methods. Additional information related to the labeled nodes is provided in Table 2.

Figure 6. ESC populations depleted of MKRN1 exhibit increased PARP cleavage following recovery from oxidative stress

1. A, B

   Control and MKRN1 overexpression (A) or knockdown (B) ESC clones were either untreated (−NaAsO₂) or stressed for 30 min with 1 mM ${\text{NaAsO}}_2^{-}$ and allowed to recover for 4 h (+NaAsO₂) prior to lysis. Total p53 served as an indicator of genotoxic stress. MKRN1, OCT4, cleaved caspase‐3, and cleaved PARP are quantified relative to GAPDH and are presented to the right of the respective immunoblots. Data are means of four independent clones in each group from two biological replicate experiments ± SEM. Statistically significant differences in mean protein abundance between populations are indicated by *P < 0.05, **P < 0.01, or ***P < 0.001 (two‐way ANOVA).

Source data are available online for this figure.

Figure EV5. SGs do not form in response to ADR‐induced genotoxic stress

1. A, B

   Wild‐type R1 ESCs treated for 4 h with 0.5 μM ADR (+ADR) or left untreated (−ADR). Cells were immunostained with either anti‐MKRN1 (A) or anti‐HuR (B) antibodies and counterstained with Hoechst. MKRN1 does not mobilize to stress granules upon ADR‐induced genotoxic stress in ESCs (A), nor does the known SG‐resident protein, HuR (B). Scale bars: 10 μm. Enlargements of boxed regions are indicated as zoom.

Figure 7. MKRN1 overexpression ESC populations exhibit increased expression of early apoptotic markers downstream of ADR‐induced genotoxic stress

1. A, B

   Control and MKRN1 knockdown (A) or overexpression (B) ESC clones were either untreated (−ADR) or stressed with 0.5 μM ADR for 6 h (+ADR) prior to lysis. MKRN1, p53, cleaved caspase‐3, and cleaved PARP are quantified relative to GAPDH and are presented to the right of the respective immunoblots. Data are means of four independent clones in each group from two biological replicate experiments ± SEM. Statistically significant differences in mean protein abundance between populations are indicated by *P < 0.05, **P < 0.01, or ***P < 0.001 (two‐way ANOVA).

Source data are available online for this figure.

Figure 8. A granular apoptotic gene regulatory module embedded in the MKRN1–mRNA network

Seventy‐six apoptosis‐related transcripts identified in the MKRN1–mRNA network are shown as circular nodes. Blue edges denote MKRN1–mRNA associations. Green edges specify occupancy of ESC‐associated transcription factors (blue squares) at the respective gene's promoter based on published data 10, 54. Circular node color indicates whether the transcript was upregulated (red), downregulated (green), or not differentially expressed (yellow) in ADR‐treated R1 ESCs 54.