Post-transcriptional regulation of mouse neurogenesis by Pumilio proteins

Abstract

Despite extensive studies on mammalian neurogenesis, its post-transcriptional regulation remains under-explored. Here we report that neural-specific inactivation of two murine post-transcriptional regulators, Pumilio 1 (Pum1) and Pum2, severely reduced the number of neural stem cells (NSCs) in the postnatal dentate gyrus (DG), drastically increased perinatal apoptosis, altered DG cell composition, and impaired learning and memory. Consistently, the mutant DG neurospheres generated fewer NSCs with defects in proliferation, survival, and differentiation, supporting a major role of Pum1 and Pum2 in hippocampal neurogenesis and function. Cross-linking immunoprecipitation revealed that Pum1 and Pum2 bind to thousands of mRNAs, with at least 694 common targets in multiple neurogenic pathways. Depleting Pum1 and/or Pum2 did not change the abundance of most target mRNAs but up-regulated their proteins, indicating that Pum1 and Pum2 regulate the translation of their target mRNAs. Moreover, Pum1 and Pum2 display RNA-dependent interaction with fragile X mental retardation protein (FMRP) and bind to one another's mRNA. This indicates that Pum proteins might form collaborative networks with FMRP and possibly other post-transcriptional regulators to regulate neurogenesis.

Keywords: FMRP; Pumilio; hippocampus; mRNA; mouse; neural stem cell; post-transcriptional regulation.

Figure 1.

Pum1 and Pum2 are expressed in mouse brains. (A) Western blot using lysate of whole brains at E13.5 and lysates of microdissected cortices and hippocampi at P1, P7, P15, P30, and 5 mo. (Pum1) 126 kDa; (Pum2) 114 kDa; (Gapdh) 37 kDa. (B,C) Immunofluorescence of PUM1 (B)/PUM2 (C) and markers for NSCs (SOX2), neural progenitor cells (TBR2/EOMES), and neurons (neuronal nuclei [NeuN]). The right four columns are enlarged pictures of the boxed regions in the left column. White arrowheads point to representative cells with Pum1/2 localized in the cytoplasm of the marked cells. (Green) Pum1/Pum2; (red) SOX2/TBR2/NeuN; (blue) DAPI. Bar, 50 µm.

Figure 2.

Nestin-Cre-mediated Pum1;Pum2 double-knockout animals. (A) Schematic diagram for the generation of Nestin-Cre-mediated Pum1;Pum2 double-knockout mice. Exons 8 and 9 of the Pum1 allele and exon 3 of the Pum2 allele are flanked by LoxP sites and depleted in the presence of Cre recombinase, resulting in null alleles. (B) RNA-seq in neonatal brain lysates confirmed the depletion of exons 8 and 9 in the Pum1 transcript in Pum1 global knockout (P1KO) mice and exon 3 in the Pum2 transcript in P2KO mice. (C) Highly efficient depletion of PUM1 and PUM2 protein from the nervous system in Nestin-Cre-mediated conditional knockout mice as shown by Western blot using neonatal brain lysates. (Ctrl) Pum1^+/f;Pum2^+/f; Nestin-Cre⁺; (P1cko) Pum1^f/f;Pum2^+/f;Nestin-Cre⁺; (P2cko) Pum1^+/f;Pum2^f/f;Nestin-Cre⁺; (Ndcko) Pum1^f/f;Pum2^f/f;Nestin-Cre⁺. (D) Quantification of the Western blot in C. The intensity of the Pum1 and Pum2 bands was normalized to the intensity of the respective Gapdh bands. For comparison, each data set was normalized to the control. n = 3.

Figure 3.

Depletion of Pum1 and Pum2 resulted in neurogenesis deficiency. (A) Representative images of P60 control and Ndcko brain coronal sections. The horizontal check mark-shaped dense granule cell zones defined by red dotted lines are the DG. (Left to right) Anterior to posterior. (O) Statum oriens; (P) pyrimidal cell layer; (R) stratum radiatum; (LM) lacunosum molecular; (blue) DAPI. Bar, 100 µm. (B,C) Quantification of the absolute DG volume (B) and the DG volume normalized to the corresponding brain weight (C) in control and Ndcko mice at different time points. (**) P-value < 0.01. n = 4–6. (D) Quantification of the whole stratum layer and the pyramidal cell layer area of the CA1 (cornu ammonis region 1) region normalized to the corresponding brain weight in control and Ndcko mice at P1 and P60 as representative time points. n = 4–6. (E) Illustration of cell lineage during neurogenesis: NSCs give rise to NPCs, which eventually differentiate into neurons. (F,H) Representative TBR2 (F) and Doublecortin (DCX) (H) staining on P30 control and Ndcko brains. The white dotted line defines the DG. (Green) TBR2; (red) DCX; (blue) DAPI. Bar, 100 µm. (G,I) Quantification of TBR2-positive and DCX-positive cells normalized to the DG area at different time points. For each animal, four to six serial sections of the brain were scored and averaged. (*) P-value < 0.05; (**) P-value < 0.01. n = 4–6. (J,K) Increased apoptosis in neonatal Ndcko brains compared with control in the cortex and hippocampus. (Green) Activated Caspase3; (blue) DAPI. Bar, 100 µm. (**) P-value < 0.01. n = 5.

Figure 4.

Ndcko NSPCs are deficient in self-renewal and differentiation. (A) Experimental procedure of neurosphere assays. (B) Representative images and quantification of primary and secondary neurospheres from control and Ndcko NSPCs. Bar, 0.1 mm. (**) P-value < 0.01. n = 5. (C,D) Growth curve (C) and cell viability (D) of the cultured NSPCs based on the total cell numbers (C) and Trypan blue staining-negative cell numbers divided by total cell numbers (D) prior to seeding the cells at P0 (harvest day), P1 (passage 1), P2, and P3. (*) P-value < 0.05. n = 5. (E) Apoptosis in control and Ndcko neurospheres by TUNEL staining. (Green) TUNEL; (blue) DAPI. Bar, 100 µm. (**) P-value < 0.01. n = 3. (F) Proliferation in control and Ndcko neurospheres by 24 h of BrdU labeling. (Red) BrdU; (blue) DAPI. Bar, 100 µm. (*) P-value < 0.05. n = 3. (G) Differentiation of control and Ndcko NSPCs into neurons and astrocytes. (Green) β-tubulin III (β-TubIII); (red) GFAP; (blue) DAPI. Bar, 100 µm. (*) P-value < 0.05. n = 3.

Figure 5.

Ndcko mice have impaired learning and memory. (A) Schematic diagram of the Lashley maze. (B) Days of learning for young (4- to 6-mo-old) and aged (12- to 14-mo-old) cohorts of control and Ndcko animals. None of the aged Ndcko mice learned the maze by the end of the 15-d test. (C,D) The learning index (correct entries/total entries) on the first four test days for young (C) and aged (D) control and Ndcko animals. Dotted lines represent the linear regression of each data set. (E) The slopes of the learning index linear regression from C and D. (**) P-value < 0.01. n = 6 for young groups; n = 9 for aged groups.

Figure 6.

iCLIP-seq (iCLIP combined with high-throughput sequencing) identification of Pum1 and Pum2 targets. (A) Reproducibility among biological triplicates for Pum1 and Pum2 iCLIP, indicated by significant overlapping of the binding site-associated genes. (Rep) Biological repeat (the numbers of genes are indicated). (B) iCLIP peaks on the Git1 and Rps9 transcript in Pum1 iCLIP and Pum2 iCLIP. Git1 is a target for Pum1 and Pum2, and Rps9 is a nontarget. (C) Pum1 and Pum2 RIP-qPCR validation of the target Git1 and nontarget Rps9. (D) Distribution of the Pum1-binding sites (left) and Pum2-binding sites (right) in different genomic sections. (E) MEME results for de novo discovery of the binding motif for Pum1 (top) and Pum2 (bottom). (F) Comparison between Pum1 and Pum2 target genes. Pum1 has 1874 target genes, and Pum2 has 875 target genes, 694 of which are common targets. (G,H) Biological processes identified by gene ontology analysis of Pum1 (G) and Pum2 (H) target genes. (I) Reproducibility among biological repeats for RNA-seq, indicated by the coefficient of variation. (Four repeats for wild type, P1KO, and P2KO and three repeats for Ndcko). (J) RNA-seq heat map showing the expression of the 694 iCLIP-identified Pum1 and Pum2 common target transcripts in wild type, Ndcko, P2KO, and P1KO. (K) RT-qPCR analysis of iCLIP-identified Pum1 and Pum2 common targets in wild type, Ndcko, P2KO, and P1KO. (Red) Targets; (blue) nontargets. Data were normalized to Ubqln1. n = 3. (L) Western blot analysis of iCLIP-identified Pum1 and Pum2 common targets revealed regulation of these targets at the protein level. (Red) Targets; (blue) nontargets; (black) loading control. (M) Quantification of the Western blot in L. Gapdh bands or prestained gels (Supplemental Fig. S6G) were used for normalization. For each protein, the results were normalized to wild type. (*) P-value < 0.05; (**) P-value < 0.01. n = 9.

Figure 7.

Pum proteins interact with Fmrp in an RNA-dependent manner. (A) Venn diagram of Pum1 iCLIP-identified, Pum2 iCLIP-identified, and Fmrp HITS-CLIP-identified (Darnell et al. 2011) target genes. (B) Fraction of a wild-type neonatal mouse brain into cytoplasmic, nucleoplasmic, and chromatin compartments. The proteins used as markers are color-coded with the corresponding compartments. (C) Polysome fractionation of wild-type neonatal mouse brains. Every third fraction was used for RNA and protein analysis. (D–F) Immunoprecipitation of Pum1 (D), Pum2 (E), and Fmrp (F). (FT) Flow-through. (G,H) Pum1 (G) and Pum2 (H) RIP in Fmr1 heterozygous (Het) and homozygous knockout (KO) neonatal brains. RT-qPCR was used to analyze the immunoprecipitation/input enrichment of the Pum1, Pum2, and Fmrp common targets (red); Pum-unique target (yellow); Fmrp-unique target (green); and nontarget (blue) in the knockout compared with heterozygous. Two biological repeats, each with two technical repeats. (I) Fmrp RIP in wild-type, P1KO, P2KO, and Ndcko neonatal brains. RT-qPCR was used to analyze the immunoprecipitation/input enrichment of the Pum1, Pum2, and Fmrp common targets (red); Fmrp-unique targets (green); and nontargetd (blue) in the mutants compared with wild type. Three biological repeats, each with two technical repeats.