Next Generation Sequencing to determine the roles of RNA-binding proteins PCBP1 and PCBP2 on murine erythropoiesis
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA737990 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14819277_1.fastq.gz | Fastqsanger.gz | |||
| SRR14819277_2.fastq.gz | Fastqsanger.gz | |||
| SRR14819278_1.fastq.gz | Fastqsanger.gz | |||
| SRR14819278_2.fastq.gz | Fastqsanger.gz | |||
| SRR14819279_1.fastq.gz | Fastqsanger.gz |
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Project description:E14.5 fetal liver cells from CD-1 mice were enriched for hematopoietic progenitors. The purified hematopoietic progenitor cells were infected immediately with retroviruses encoding shRNAs that target Pcbp1, Pcbp2, as well as two sets of control shRNAs; Luciferase and scrambled sequence. The transduced cells were grown for 48 hours. These cells were collected (‘Day 0’ cells), or washed and resuspended in differentiation medium and incubated for an additional 2 days (‘Day 2’ cells). 1ug of total RNA isolated from ‘Day 0’ and ‘Day 2’ cells from each Pcbp1 or Pcbp2 depleted sample was used for cDNA library construction after polyA selection. A total of 9 samples were generated from each of the two time points: 3 controls; Pcbp1 knockdowns with three distinctly targeting shRNAs (sh1,3,4); Pcbp2 knockdown with three distinctly targeting shRNAs (sh1,3,4). Sequencing was carried out using a 150nt paired-end sequencing protocol.
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Project description:RNA-binding proteins participate in a complex array of post-transcriptional controls essential to cell-type specification and somatic development. Despite their detailed biochemical characterizations, the degree to which each RNA-binding protein impacts on mammalian embryonic development remains incompletely defined and the level of functional redundancy among subsets of these proteins remains open to question. The poly-(C) binding proteins, Pcbp's (aCPs, hnRNPEs), are encoded by a highly conserved and broadly expressed gene family. The two major Pcbp isoforms, Pcbp2 and Pcbp1, are robustly expressed in a wide range of tissues and exert both nuclear and cytoplasmic controls over gene expression. Here we report that Pcbp1-null embryos are rendered nonviable in the peri-implantation stage. In contrast, Pcbp2-null embryos survive until mid-gestation at which time they undergo a loss in viability associated with cardiovascular and hematopoietic abnormalities. Adult mice heterozygous for either Pcbp1 or Pcbp2 null alleles display a mild and non-disruptive growth defect. These data reveal that Pcbp1 and Pcbp2 are individually essential for mouse embryonic development and post-natal growth, reveal a non-redundant in vivo role for Pcpb2 in hematopoiesis, and provide direct evidence that Pcbp1, a retrotransposed derivative of Pcpb2, has evolved essential function(s) in the mammalian genome. mRNA-seq on fetal liver tissue from 12.5 days post coitum. 4 replicates of WT and 3 replicates of PCBP2 Knockout
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Project description:RNA-binding proteins participate in a complex array of post-transcriptional controls essential to cell-type specification and somatic development. Despite their detailed biochemical characterizations, the degree to which each RNA-binding protein impacts on mammalian embryonic development remains incompletely defined and the level of functional redundancy among subsets of these proteins remains open to question. The poly-(C) binding proteins, Pcbp’s (aCPs, hnRNPEs), are encoded by a highly conserved and broadly expressed gene family. The two major Pcbp isoforms, Pcbp2 and Pcbp1, are robustly expressed in a wide range of tissues and exert both nuclear and cytoplasmic controls over gene expression. Here we report that Pcbp1-null embryos are rendered nonviable in the peri-implantation stage. In contrast, Pcbp2-null embryos survive until mid-gestation at which time they undergo a loss in viability associated with cardiovascular and hematopoietic abnormalities. Adult mice heterozygous for either Pcbp1 or Pcbp2 null alleles display a mild and non-disruptive growth defect. These data reveal that Pcbp1 and Pcbp2 are individually essential for mouse embryonic development and post-natal growth, reveal a non-redundant in vivo role for Pcpb2 in hematopoiesis, and provide direct evidence that Pcbp1, a retrotransposed derivative of Pcpb2, has evolved essential function(s) in the mammalian genome.
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Project description:We previously demonstrated that the two paralogous RNA-binding proteins PCBP1 and PCBP2 are individually essential for mouse development: Pcbp1-null embryos are peri-implantation lethal, while Pcbp2-null embryos lose viability at midgestation. Midgestation Pcbp2-/- embryos revealed a complex phenotype that included loss of certain hematopoietic determinants. Whether PCBP2 directly contributes to erythropoietic differentiation and whether PCBP1 has a role in this process remained undetermined. Here, we selectively inactivated the genes encoding these two RNA-binding proteins during differentiation of the erythroid lineage in the developing mouse embryo. Individual inactivation of either locus failed to impact viability or blood formation. However, combined inactivation of the two loci resulted in midgestational repression of erythroid/hematopoietic gene expression, loss of blood formation, and fetal demise. Orthogonal ex vivo analyses of primary erythroid progenitors selectively depleted of these two RNA-binding proteins revealed that they mediate a combination of overlapping and isoform-specific impacts on hematopoietic lineage transcriptome, impacting both mRNA representation and exon splicing. These data lead us to conclude that PCBP1 and PCBP2 mediate functions critical to differentiation of the erythroid lineage.
| S-EPMC8384066 | biostudies-literature