Unknown,Transcriptomics,Genomics,Proteomics

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Ptbp2 represses adult-specific splicing to regulate the generation of neuronal precursors in the embryonic brain [HITS-CLIP]

ABSTRACT: Two polypyrimidine tract RNA-binding proteins (PTBs), one near-ubiquitously expressed (Ptbp1) and another highly tissue-restricted (Ptbp2), regulate RNA in interrelated but incompletely understood ways. Ptbp1, a splicing regulator, is replaced in the brain and differentiated neuronal cell lines by Ptbp2. To define the roles of Ptbp2 in the nervous system, we generated two independent Ptbp2-null strains, unexpectedly revealing that Ptbp2 is expressed in neuronal progenitors and is essential for postnatal survival. A HITS-CLIP (high-throughput sequencing crosslinking immunoprecipitation)-generated map of reproducible Ptbp2–RNA interactions in the developing mouse neocortex, combined with results from splicing-sensitive microarrays, demonstrated that the major action of Ptbp2 is to inhibit adult-specific alternative exons by binding pyrimidine-rich sequences upstream of and/or within them. These regulated exons are present in mRNAs encoding proteins associated with control of cell fate, proliferation, and the actin cytoskeleton, suggesting a role for Ptbp2 in neurogenesis. Indeed, neuronal progenitors in the Ptbp2-null brain exhibited an aberrant polarity and were associated with regions of premature neurogenesis and reduced progenitor pools. Thus, Ptbp2 inhibition of a discrete set of adult neuronal exons underlies early brain development prior to neuronal differentiation and is essential for postnatal survival. Eight Ptbp2 HITS-CLIP libraries generated from mouse embryonic brain (four libraries from each of two biologic replicates).

ORGANISM(S): Mus musculus

SUBMITTER: Donny Licatalosi

PROVIDER: E-GEOD-47564 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Ptbp2 represses adult-specific splicing to regulate the generation of neuronal precursors in the embryonic brain.

Licatalosi Donny D DD Yano Masato M Fak John J JJ Mele Aldo A Grabinski Sarah E SE Zhang Chaolin C Darnell Robert B RB

Genes & development 20120701 14

Two polypyrimidine tract RNA-binding proteins (PTBs), one near-ubiquitously expressed (Ptbp1) and another highly tissue-restricted (Ptbp2), regulate RNA in interrelated but incompletely understood ways. Ptbp1, a splicing regulator, is replaced in the brain and differentiated neuronal cell lines by Ptbp2. To define the roles of Ptbp2 in the nervous system, we generated two independent Ptbp2-null strains, unexpectedly revealing that Ptbp2 is expressed in neuronal progenitors and is essential for p ...[more]

PMID: 22802532

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Project description:The splicing regulator PTBP2 controls a program of embryonic splicing required for neuronal maturation. The splicing regulatory proteins PTBP1 and PTBP2 show distinct temporal expression profiles in the developing brain. Neuronal progenitor cells predominantly express PTBP1, whereas developing neurons express high levels of PTBP2, which are subsequently reduced late in neuronal maturation. We show here that PTBP2 and the program of splicing it controls are essential to proper neuronal maturation and survival. To investigate its in vivo function, we generated conditional PTBP2 null alleles in mice. Loss of PTBP2 in neuronal progenitor cells leads to neonatal death without gross defects in brain architecture. Mice with specific depletion of PTBP2 in the cortex and forebrain are viable. However over the first three postnatal weeks, when the normal cortex expands and develops mature circuits, the PTBP2 null cortices degenerate. We find that PTBP2-/- neurons cultured from embryonic brain show the same initial viability as wild type cells with proper early marker expression and neurite outgrowth. Strikingly, between 10 and 20 days in culture PTBP2 null neurons undergo a catastrophic failure to mature and die. To assess the target transcripts leading to these phenotypes, we examined the genomewide splicing changes in the PTBP2 null brains. This identified a large number of mis-regulated exons that share a temporal pattern of regulation; in the absence of PTBP2 many isoforms normally found in adults are precociously expressed in the developing brain. Transcripts following this pattern encode essential neuronal proteins affecting neurite growth, pre- and post-synaptic assembly, and synaptic transmission. Our results define a new genetic regulatory program essential for neuronal survival and maturation, where PTBP2 acts to temporarily repress expression of protein isoforms until the final maturation of the neuron. Mice carrying a conditional floxed PTBP2 allele of PTBP2 were crossed to mice carrying Cre recombinase driven by the nestin promoter. The resulting knockout mutant mouse brains were analyzed for changes in gene expression and alternative splicing. Knockout mice were compared to wildtype littermates. Whole mouse brain polyA plus RNA was isolated from three Nestin-cre knockout embryos at embryonic day 18 and compared to three wildtype littermates. RNA was converted to cDNA and used to probe Affymetrix MJAY splicing sensitive microarrays and analysed by Omniviewer to identify changes in splicing.

Project description:The splicing regulator PTBP2 controls a program of embryonic splicing required for neuronal maturation. The splicing regulatory proteins PTBP1 and PTBP2 show distinct temporal expression profiles in the developing brain. Neuronal progenitor cells predominantly express PTBP1, whereas developing neurons express high levels of PTBP2, which are subsequently reduced late in neuronal maturation. We show here that PTBP2 and the program of splicing it controls are essential to proper neuronal maturation and survival. To investigate its in vivo function, we generated conditional PTBP2 null alleles in mice. Loss of PTBP2 in neuronal progenitor cells leads to neonatal death without gross defects in brain architecture. Mice with specific depletion of PTBP2 in the cortex and forebrain are viable. However over the first three postnatal weeks, when the normal cortex expands and develops mature circuits, the PTBP2 null cortices degenerate. We find that PTBP2-/- neurons cultured from embryonic brain show the same initial viability as wild type cells with proper early marker expression and neurite outgrowth. Strikingly, between 10 and 20 days in culture PTBP2 null neurons undergo a catastrophic failure to mature and die. To assess the target transcripts leading to these phenotypes, we examined the genomewide splicing changes in the PTBP2 null brains. This identified a large number of mis-regulated exons that share a temporal pattern of regulation; in the absence of PTBP2 many isoforms normally found in adults are precociously expressed in the developing brain. Transcripts following this pattern encode essential neuronal proteins affecting neurite growth, pre- and post-synaptic assembly, and synaptic transmission. Our results define a new genetic regulatory program essential for neuronal survival and maturation, where PTBP2 acts to temporarily repress expression of protein isoforms until the final maturation of the neuron. Mice carrying a conditional "floxed" PTBP2 allele of PTBP2 were crossed to mice carrying Cre recombinase driven by either the nestin or Emx1 promoters. The resulting knockout mutant mouse brains were analyzed for changes in gene expression and alternative splicing. Knockout mice were compared to wildtype littermates. Whole mouse brain polyA plus RNA was isolated from two Nestin-cre knockout embryos at embryonic day 18 and compared to two wildtype littermates. Total polyA plus RNA was isolated from the cortices of an Emx-cre knockout and its wildtype littermate at postnatal day 1. This RNA was converted to standard Illumina paired end libraries using the Truseq kit. The Emx sample and control libraries were strand specific through elimination of the second strand of cDNA using the USER enzyme. Libaries were sequenced on an Illumina HiSeq using the standard paired end protocol. Data was analyzed using the Cufflinks pipeline. Splicing analysis was carried out using the SpliceTrap program.

Dataset Information

Ptbp2 represses adult-specific splicing to regulate the generation of neuronal precursors in the embryonic brain [HITS-CLIP]

Publications

Ptbp2 represses adult-specific splicing to regulate the generation of neuronal precursors in the embryonic brain.

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