Mammalian Pum1 and Pum2 Control Body Size via Translational Regulation of the Cell Cycle Inhibitor Cdkn1b

Abstract

Body and organ size regulation in mammals involves multiple signaling pathways and remains largely enigmatic. Here, we report that Pum1 and Pum2, which encode highly conserved PUF RNA-binding proteins, regulate mouse body and organ size by post-transcriptional repression of the cell cycle inhibitor Cdkn1b. Binding of PUM1 or PUM2 to Pumilio binding elements (PBEs) in the 3' UTR of Cdkn1b inhibits translation, promoting G1-S transition and cell proliferation. Mice with null mutations in Pum1 and Pum2 exhibit gene dosage-dependent reductions in body and organ size, and deficiency for Cdkn1b partially rescues postnatal growth defects in Pum1^-/- mice. We propose that coordinated tissue-specific expression of Pum1 and Pum2, which involves auto-regulatory and reciprocal post-transcriptional repression, contributes to the precise regulation of body and organ size. Hence PUM-mediated post-transcriptional control of cell cycle regulators represents an additional layer of control in the genetic regulation of organ and body size.

Keywords: Cdkn1b; G1S; PUF; PUM; body size; cell cycle; growth regulator; organ size; post-transcriptional regulation; translational control.

Figure 1.. Pum1^−/− Mice Exhibit Prenatal and Postnatal Growth Reduction

(A and C) Representative images of (A) female and (C) male Pum1^−/−, Pum1^+/−, and wild-type mice. (B and D) Postnatal body weight curves (weeks 2–96) of (B) female (17 Pum1^−/−, 27 Pum1^+/−, and 20 Pum1^+/+) and (D) male mice (11 Pum1^−/−, 23 Pum1^+/−, and 11 Pum1^+/+). (E and F) Body length of 2-week-old (E) females (9 Pum1^−/−, 24 Pum1^+/−, and 13 Pum1^+/+) and (F) males (16 Pum1^−/−, 24 Pum1^+/−, and 9 Pum1^+/+). (G and H) Representative images of Pum1^+/+, Pum1^+/−, and Pum1^−/− mice on postnatal day 1 (G) and embryonic day 14.5 (H). (I and J) Growth curve of neonatal (I) female (6 Pum1^−/−, 13 Pum1^+/−, and 8 Pum1^+/+) and (J) male mice (5 Pum1^−/−, 12 Pum1^+/−, and 11 Pum1^+/+). (K) Body weight of fetuses at E13.5, E14.5, and E16.5 for Pum1^−/− (7), Pum1^+/− (18), and Pum1^+/+ (10) mice. Data are presented as mean ± SD. Significant p values are indicated by asterisks and pound signs. Significant differences between Pum1^+/− or Pum1^−/− and wild-type (Pum1^+/+) are marked by asterisks, and significant differences between Pum1^−/− and Pum1^+/− by pound signs (***p < 0.001 and ###p < 0.001, **p < 0.01 and ##p < 0.01, and *p < 0.05).

Figure 2.. Global Organ Size and Cell Number Reduction in Pum1^−/−-Mutant Mice

(A) Organ weights were measured in Pum1^+/+ (n = 7) and Pum1^−/− (n = 12) males at 3 weeks of age. (B) Representative images of organs from 3-week-old mice. (C) Organ weights of adult Pum1^+/+ (n = 5) and Pum1^−/− (n = 5) males. (D) Representative images of adult organs. (E–H) Analysis of cell size distribution in testis (E) and bone marrow (G) by flow cytometry. Three distinct cell populations were gated in a FSC-A by FL2-A dot plot and sorted in a FSC-A histogram. FSC, forward scatter; G1, gate 1; G2, gate 2; G3, gate 3. Cell size distribution analysis using median fluorescence values (FSC-A) of testicular (F) and bone marrow (H) cells for adult Pum1^+/+ (n = 3) and Pum1^−/− (n = 3) males. (I) Total cell count of testis and thymus in Pum1^+/+ (n = 5) and Pum1^−/− (n = 6) males at 3 weeks of age. (J) Total cell count of testis and bone marrow in adult Pum1^+/+ (n = 5) and Pum1^−/− (n = 5) males. Br, brain; He, heart; Ki, kidney; Li, liver; Lu, lung; Sp, spleen; Te, testis; Th, thymus; Ti, thigh. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 3.. Decreased Cell Proliferation in Pum1-Depleted Cells and Mutant Animals

(A) Proliferation of NIH 3T3 cells following lentiviral transduction with a small hairpin RNA against Pum1 (sh-Pum1KD) or control (sh-Con). Cell proliferation rates were determined by CCK8 assay. (B) Pum1 knockdown (sh-Pum1KD) and control (sh-Con) NIH 3T3 cells were incubated with EdU to quantify DNA synthesis. (C) Growth curves of Pum1^+/+ (n = 3) and Pum1^−/− (n = 3) mouse embryonic fibroblasts (MEFs) from passage 2 to 6 (P2–P6). (D and E) Immunostaining for BrdU (D) and statistical analysis (E) of BrdU-positive (BrdU⁺) cells in the testis of 3-week-old Pum1^+/+ (n = 5) and Pum1^−/− (n = 5) mice. (F and G) Immunostaining for phospho-Histone 3 (P-H3) (F) and statistical analysis of P-H3-positive (P-H3+) cells (G) in Pum1^+/+ (n = 4) and Pum1^−/− (n = 5) testes at 3 weeks of age. P-H3⁺ cells are presented as number per ten tubules. Scale bar, 50 μm. (H) Annexin V-FITC/propidium iodide (PI) co-staining for apoptotic cells in Pum1 knockdown (sh-Pum1KD) and control (sh-Con) NIH 3T3 cells. (I and J) Apoptotic cells in passage 3 Pum1^+/+ (n = 3) and Pum1^−/− (n = 3) MEF by annexin V-FITC/PI co-staining in bar graph (I) and two-dimensional dot blot (J) from flow cytometry analysis. (K and L) Typical images of TUNEL staining (K) and quantification of apoptotic cells (L) in testes from 3-week-old Pum1^+/+ (n = 5) and Pum1^−/− (n = 5) mice. TUNEL-positive cells are presented as number per ten tubules. Scale bar, 50 μm. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 (t test).

Figure 4.. Pum1-Depleted Cells and Mutant Tissues Exhibited Cell Cycle Defects and Increased Expression of Cdkn1b

(A) Cell cycle analysis of control (sh-Con) and Pum1-knockdown (sh-Pum1KD) NIH 3T3 cells by FACS. (B) Cell cycle analysis of MEF cells from E13.5 Pum1^+/+ (n = 3) and Pum1^−/− (n = 3) fetuses. (C) Cell cycle progression analysis of three pairs of wild-type and mutant MEFs cells with EdU pulse labeling at different time points after resumption of cell cycle from G0 phase. (D) Western blot analysis of cell cycle and apoptosis regulators in Pum1^+/+ and Pum1^−/− MEFs. (E) Western blot analysis of G1-S transition regulators (CDK1, CDK2, Cyc E2, and CDKN1B) in the adult testis of Pum1^+/+ and Pum1^−/− mice. (F) Immunostaining for PUM1 and CDKN1B in tissue sections from the testis of 3-week-old Pum1^+/+ and Pum1^−/− mice. Scale bar, 50 μm. (G) Western blot analysis of PUM1 and CDKN1B in Pum1 knockdown (sh-Pum1KD) and control (sh-Con) NIH 3T3 cells. Extracts from Pum1^−/− and Pum1^+/+ were loaded for comparison. (H) Western blot analysis of PUM1 and CDKN1B in NIH 3T3 cells overexpressing wild-type mouse Pum1 and mutant Pum1. (I) Cdkn1b expression in Pum1^−/− relative to Pum1^+/+ tissues by densitometric analysis of western signal using ImageJ software (NIH). All tissues were performed from at least two individual samples and are reported as mean ± SD. The mean intensity value of Pum1^+/+ mice was set at 100%. (J) Western blot analysis of PUM1 and CDKN1B protein levels of different tissues from Pum1^+/+ and Pum1^−/− mice at 3 weeks of age. For each tissue, left lane is from wild-type and right lane is from knockout tissue. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5.. Pum1 Binds the 3′ UTR of Cdkn1b mRNA to Repress Its Translation

(A) PUM1 RIP (RNA immunoprecipitation) from NIH 3T3 cell lysate (left). qRT-PCR demonstrates significantly increased levels of Cdkn1b mRNA in Pum1 IP of NIH 3T3 cells in comparison with IgG precipitates. (B) Enrichment of Cdkn1b mRNAs in PUM1 RIP of testis lysates from 3-week-old mice. Input refers to total protein or total tissue RNA, and Pum1 and IgG refer to protein extract or RNA present in the anti-Pum1 and IgG immune complex. (C) Diagram of mouse Cdkn1b-3′ UTR constructs (shown as boxes) containing two PBEs (filled boxes). Numbers correspond to positions of PBEs in the mouse Cdkn1b 3′ UTR. The mutated nucleotides are highlighted in lowercase in red. EMSA using different concentration of PUM1 HD domain with wild-type or mutant PBE1 (171 bp) and PBE2 (156 bp), respectively, showed direct binding of PUM1 to wild-type PBE. (D) Bar graph results from dual-luciferase assay on NIH 3T3 cells expressing the reporter constructs containing either wild-type or mutated Cdkn1b 3′ UTR, PBE1, or PBE2. The cells were also co-transfected with mouse Pum1 vector (Pum1) or the same vector without Pum1 (empty). Cdkn1b 3′ UTR used for each assay contained both wild-type PBE1 and PBE2 or mutated PBE1 and PBE2 or one mutated and one wild-type PBE, as indicated under the graph. (E and F) Cdkn1b translation was reduced in absence of PUM1. (E) Polysome profiles from fractionation experiments of NIH 3T3 cells lysate of Pum1 knockdown (sh-PUM1KD) and control (sh-Con). Peak position of free RNP (1), 40S (2), 60S (3), and 80S ribosome (4) and polysomes (5–9) are indicated. Wild-type (black line) and Pum1^−/−-mutant (red line) profiles are overlaid. The values were normalized against β-actin. The experiments were performed in triplicate. (F) Cdkn1b mRNA distribution among fractions were determined by qRT-PCR using beta-actin as internal control. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 (t test).

Figure 6.. Cdkn1b Mutation Partially Rescued the Smaller Body Weight Phenotype of Pum1^−/−-Mutant Mice

(A) Western blot analysis of PUM1 and CDKN1B protein expression of brain tissues from adult single- or double-mutant mice validated the loss of function nature of all three mutants. (B and C) Body weight of female (B) and male (C) mice was measured from postnatal week 3 to week 12. The body weights of double-knockout females were significantly higher than those of Pum1 single-knockout mice but smaller than wild-type. Data are presented as mean ± SD (N = 5–19 mice/genotype). (D and E) Representative images of age-matched wild-type, Pum1^−/−, Cdkn1b^−/−, and Pum1^−/−;Cdkn1b^−/− female (D) and male (E) mice at the age of 2–3 months. (F) Testis weight (total weight of both testes) was compared among adult male wild-type, Pum1^−/−, Cdkn1b^−/−, and Pum1^−/−;Cdkn1b^−/− mice. Double-mutant testes were again significantly bigger than those of Pum1 mutants but smaller than those of wild-type, indicating a partial rescue in organ weight. N = 7–11 mice/genotype. *p < 0.05, **p < 0.01, and ***p < 0.001 (t test).

Figure 7.. Pum1 and Pum2 Double Mutants Exhibited Discrete Body Weight Reduction, and Reciprocal and Auto-Regulation of PUM Gene Expression May Be Responsible for Such a Precise Control of Body Weight

(A–C) Body weight was measured at the age of 3 weeks for female (A) and male (B) mice of various combinations of Pum1 and Pum2 single or double mutations. A picture of one representative litter showing a remarkable discrete effect of loss of Pum genes at one gene copy interval is shown (C). (D) Western blot analyses of key cell cycle regulators in 3-week-old testis of Pum1^−/− and Pum2^−/−. Left-pointing arrow represents chimeric PUM2-βgeo fusion protein in Pum2^−/− mice. (E and F) Both Pum2 (E) and Pum1 (F) mRNAs were enriched in PUM1 immunoprecipitates. Total cellular RNA (input) and RNA present in the immune complex (anti-Pum1 and anti-IgG, respectively) were used. (G) Western blot of testis PUM2 RNA immunoprecipitation indicated that PUM2 proteins could be pulled down at high efficiency. (H) Pum1, Pum2, and Cdkn1b mRNAs were significantly enriched in the PUM2 immunoprecipitates. (I and J) The relative Pum2 mRNA levels in total RNA (T) and polysome fractions (number) were measured using qRT-PCR on fractionation extracts from adult testis (I) and NIH 3T3 (J). The values are normalized to β-actin. The experiments were performed in triplicate; 1 represents free RNP, and 5–9 represent polysome fractions. (K) Proposed model of PUM expression feedback loop. Expression of Pum1 and Pum2 is co-expressed in most tissues and precisely regulated by auto and reciprocal translation repression via binding of PBEs on their 3′ UTR.