Trim71 cooperates with microRNAs to repress Cdkn1a expression and promote embryonic stem cell proliferation

Abstract

Pluripotent embryonic stem cells have a shortened cell cycle that enables their rapid proliferation. The embryonic stem cell-specific miR-290 and miR-302 microRNA families promote proliferation whereas let-7 microRNAs inhibit self-renewal, and promote cell differentiation. Lin28 suppresses let-7 expression in embryonic stem cells. Here to gain further insight into mechanisms controlling embryonic stem cell self-renewal, we explore the molecular and cellular role of the let-7 target Trim71 (mLin41). We show that Trim71 associates with Argonaute2 and microRNAs, and represses expression of Cdkn1a, a cyclin-dependent kinase inhibitor that negatively regulates the G1-S transition. We identify protein domains required for Trim71 association with Argonaute2, localization to P-bodies, and for repression of reporter messenger RNAs. Trim71 knockdown prolongs the G1 phase of the cell cycle and slows embryonic stem cell proliferation, a phenotype that was rescued by depletion of Cdkn1a. Thus, we demonstrate that Trim71 is a factor that facilitates the G1-S transition to promote rapid embryonic stem cell self-renewal.

Figure 1. Trim71 is highly expressed in undifferentiated cells and is a let-7 target

(a)quantitative real time PCR (qRT-PCR) of Trim71 mRNA levels (normalized to β-Actin levels). (b) Western blot of Trim71 protein (arrowhead) using a rabbit polyclonal antibody. Flag-Trim71 expressed in HEK293 cells serves as a positive control. β-Tubulin was used as a loading control. (c) qRT-PCR for Oct-4 and Trim71 (normalized to β-Actin) and TaqMan qRT-PCR for mature let-7g and miR-295 (normalized to snoRNA142) during ESC differentiation to embryoid bodies (EB). RNA was prepared from V6.5 ESCs on day 0 (undifferentiated) and on day 5, 8, 10, and 12 of differentiation. (d) Whole cell extracts from J1 ESCs transfected with control, Lin28 or Trim71 siRNA were examined by Western blot 60 hours post-transfection. (e) TaqMan PCR of mature let-7 levels from samples in d. (f) Samples from J1 ESCs transfected with Lin28 siRNA, anti-let-7a, or both were examined by TaqMan PCR for mature let-7a (top) and by Western blot (bottom). (g) Luciferase reporter assays performed on HeLa cells using a firefly luciferase that contains two let-7 binding sites from the mouse Trim71 3′ UTR. All Signals were normalized to Renilla luciferase levels. Error bars represent mean +/− S.D. with n=3. The p value is from a two-tailed t test.

Figure 2. Trim71 interacts with Argonaute2

(a) Cell extracts were prepared from KH2 ESCs expressing either Flag-Lin28 or Flag-Trim71. Flag-affinity purifications were washed with buffer containing the indicated KCl concentration. Where indicated, RNase A was added to the lysates for IP. The input and affinity eluates were analyzed by Western blot. (b) Analysis of miRNA and Argonaute protein in RNase-treated extracts. TaqMan qRT-PCR for the indicated miRNA (Top), Error bars represent mean +/− S.D. with n=3, and Western blot for Ago2 (bottom). (c) Flag-affinity purifications on whole cell extracts from HEK293 (left) and P19 (right) that were transfected to express Flag-Trim71 or the indicated control proteins. Western blots performed using α-Flag and α-Ago2 antibodies. (d) Flag IPs performed as in (a) (100 mM KCl washes). The eluates were analyzed by Western blot. (e,f) Endogenous co- immunoprecipitation from J1 whole cell extracts performed with the indicated antibodies. The immunoprecipitated proteins are indicated by arrowheads.

Figure 3. Trim71 ribonucleoprotein complex contains miRNAs

(a) UV crosslinking IP (CLIP) on Dox-inducible Flag-Trim71 ESCs. RNA analysis for the indicated miRNAs or control RNA performed using TaqMan q.RT-PCR and the pull down efficiency each RNA is represented as % of Input. Error bars represent mean +/− S.D. with n=3. (b) Slicer assays performed on Flag-affinity eluates (100 mM KCl washes) to monitor Ago2-mediated RNA cleavage activity. Left: Western blot of the Flag-Lin28 and Flag-Trim71 IP eluates used in slicer assays. Right: reaction products resolved by 15% denaturing PAGE. Arrowhead indicates the specific 5′ cleavage product of the target RNA. (c) Flag-IP Flag-Trim71 expressing ESCs transfected with wild type or PAZ10 Myc-Ago2 construct. Affinity eluates were probed with α-Flag and α-Myc antibodies.

Figure 4. Trim71 domains required for interaction with Ago2

Top: schematic representation of Trim71 deletion constructs. Bottom: The constructs were assessed for their ability to co-precipitate endogenous Ago2 in HeLa cells. Flag-IPs were performed in the presence or absence of RNase A.

Figure 5. Trim71 domains required for P-body localization

(a) KH2 ESCs transfected with a DCP1a::GFP construct are fixed and probed with α-Trim71 antibody. Nuclei are stained with DAPI. (b) HeLa cells are co-transfected with DCP1a::GFP and mCherry::Trim71 fusions. (c) Individual mCherry::Trim71 constructs were co-transfected with DCP1a::GFP into HeLa cells and visualized by fluorescence microscopy. For each panel the scale bar is 5 μm.

Figure 6. Trim71 cooperates with miR-302 to inhibit Cdkn1a expression

(a) Trim71 depletion leads to upregulation of Cdkn1a. The same cell extracts from Fig. 1(d) were blotted with α-Cdkn1a antibody. (b) TaqMan PCR for mature miR-295 and miR-302a levels (normalized to snoRNA142). Error bars represent mean +/− S.D. with n=3. (c) J1 ESCs transfected with Trim71 siRNA or control siRNA, together with anti-miR-302a or control inhibitor, as indicated. 60 hours post-transfection total RNA was analyzed by qRT-PCR for Cdkn1a mRNA levels (normalized to β-Actin). Error bars represent mean +/− S.D. with n=3. (d) Reporter assays were performed using a firefly luciferase that contains a miR-302 binding site from the mouse Cdkn1a 3′ UTR. A control pre-miRNA or pre-miR-302a was transfected into HeLa cells that do not express ESC-specific miRNAs. Signals were normalized to Renilla luciferase levels. Normalized signals for the pre-miR control were set to 100. (e) Reporter assays were performed 60 hours post-transfection using the same luciferase constructs as in (d) as well as with a mutant reporter (mut) in which nucleotides corresponding to the miR-302 seed sequence were mutated. Normalized signals for the mutant construct were set to 100 in each condition. Error bars represent mean +/− S.D. with n=3. The p values are from two-tailed t test.

Figure 7. Posttranscriptional gene repression by Trim71

(a) Tethering assays. Wild type and DGCR8 KO ESCs were transfected with a Renilla luciferase, a firefly luciferase with or without MS2 binding sites in the 3′ UTR, and a plasmid expressing either MS2 protein or MS2-fusion, as indicated. Firefly/Renilla ratios were calculated. Relative luciferase activity represents the ratio of the reporter containing MS2 binding sites to the reporter lacking a site. Normalized signals for the MS2 control were set to 1 (b) Tethering assays in wild type V6.5 ESCs were performed as in (a) except Rck, TNRC6B, or Trim71 not fused to MS2 (and hence not tethered to the firefly luciferase mRNA 3′ UTR). (c) Tethering assays in Hela cells performed as in (a) with the indicated Trim71 deletion MS2 fusion proteins. Normalized signals for the MS2 control were set to 1. Error bars represent mean +/− S.D. with n=3 in. The p values are from two-tailed t test result. (d) Schematic representation of functional domains of Trim71.

Figure 8. Trim71 facilitates the cell cycle G1–S transition to promote rapid ESC proliferation

(a–d) ESCs were transfected with the indicated expression vectors or siRNAs Trim71 (siRNA#2). Equal numbers of cells were plated and cells counted daily starting from 48 hours posttransfection (except for c which is starting from 24 hours). Error bars represent mean +/− S.D. of biological triplicates. The p values are from two-tailed t test result. (e) Cell cycle profile analysis of DGCR8 knockout ESCs, V6.5 ESCs (WT), and V6.5 transfected with siRNA targeting Trim71, Cdkn1a, or both. For each phase of the cell cycle the data is presented as the percent change in populations compared to untransfected V6.5 ESCs. (f) Model of a Trim71 pathway in ESCs that leads to unrestricted cell cycle G1–S transition. Dashed lines indicate steps that are blocked in ESCs.