Mammalian neurogenesis is a key aspect of embryogenesis and persists throughout lifetime. Extensive studies have discovered various regulators of neurogenesis. But little is known about the post-transcriptional regulation that delivers much more rapid and subcellularly localized control of gene expression.
The purpose of this project is to study the post-transcriptional regulation of mouse neurogenesis by two RNA binding proteins, pumilio (PUM) 1 and 2—the two murine members of the evolutionarily conserved PUF protein family. This family is known to mediate post-transcriptional regulation. PUF proteins are required for germline stem cell maintenance in Drosophila and C. elegans and for spermatogenesis in mice, but their function in neurogenesis has not been well established.
To investigate the function of PUM1 and PUM2 in neurogenesis, we generated double conditional knockout mice by crossing Pum1 and Pum2 double loxP mice (with loxP sites flanking certain exons) with a mouse line carrying a Cre-expressing transgene driven by the nestin promoter. PUM1/2 are depleted by the Nes-cre in the entire central nervous system by E15.5. In these mice, we observed severe atrophy of the dentate gyrus (DG) in neonatal and adult brains along with drastically increased apoptosis in neonatal brains. This is very exciting as adult neurogenesis occurs in DG to generate new neurons for the hippocampus--the learning and memory center. Consistent with this phenotype, our behavioral tests revealed that Pum mutants are largely impaired in learning and memory. Using markers for different cell types along neurogenesis we found that removal of PUM1 and PUM2 led to increased EOMES (Tbr2)-positive neuronal progenitors but decreased DCX-positive immature neurons. Moreover, the cultured neural stem cells from the double knockout DG are deficient in forming neurospheres and display defects in proliferation and survival. All these lines of evidence suggest that PUM1 and PUM2 together regulate neurogenesis by preventing immature differentiation and apoptosis, which sustains the homeostasis of newborn neurons.
To identify PUM target mRNAs and downstream pathways, we performed iCLIP (cross-linking immunoprecipitation) using PUM1/2 antibody and neonatal brain lysates. The RNA targets and binding sites are identified; they are involved in pathways that are crucial to neurogenesis such as cell proliferation, differentiation, apoptosis, and cytoskeleton.
Overall the results have shown that PUM1 and PUM2 together play a vital role in regulating mouse neurogenesis at post-transcriptional level by regulating their RNA targets in various pathways.