Project description:Tight control of β cell mRNA translation plays a central role in regulating glucose homoeostasis and β cell health. RNA binding proteins (RBPs) impact translational dynamics and function in networks to achieve their regulatory outcomes, yet an understanding of the RBPs and nature of their interplay in directing β cell translation remain limited. We recently established that the RBP PCBP2 is a key post-transcriptional regulator of β cell function. Here, we investigate the relationship of PCBP2 and its sister-isoform PCBP1 in shaping β cell homeostasis and translation. Pcbp co-deletion in primary β cells gave rise to a severe diabetic phenotype due to compromised β cell viability. Single-cell RNA sequencing of Pcbp co-deficient β cells revealed downregulation of a network of core translation initiation factors and ribosomal mRNAs. Motif enrichment analysis, mRNA-protein interaction, and mRNA stability studies identified that the PCBPs co-impact these mRNAs in part through binding and stabilization. Accordingly, protein translational monitoring demonstrated a requirement for the PCBPs in sustaining global mRNA translation in β cells. Together, these findings demonstrate a requirement for the PCBPs in sustaining the global rates of mRNA translation critical for β cell control of glucose homeostasis.
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
Project description:To explore potential functional implications of isoform usage shifts, we examined the effect of PCBP1/PCBP2 expression in human AC16 cardiac cells. PCBP1/PCBP2 are KH-domain-containing RNA-binding proteins (RBP) that have been implicated in transcriptional, post-transcriptional and translational regulations. How PCBP1 and PCBP2 are functionally differentiated remains a subject of interest and few reports have outlined their function in cardiac cells. The two paralogs share close homology (~85% identical sequences) and partially overlapping RNA binding targets, suggesting they may form mutually regulatory relationships. Human and mouse PCBP1 sequences share 100.0% identity, whereas human and mouse PCBP2 are identical except in 5 positions; therefore we expressed the human/mouse PCBP1 and human PCBP2 coding sequences in human AC16 cardiac cells. Immunofluorescence imaging confirms broad cytonuclear localization of both paralogs under overexpression. The cells were then subjected to RNA sequencing to determine the effects of PCBP1/2 on gene expression.
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.
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.
Project description:Iron is important for normal cellular function and posttranscriptional mechanisms regulate the cellular response to iron deficiency. In this study we explore how RNA binding proteins, PCBP1 and PCBP2, exhibit iron-sensitive RNA regulation.
Project description:Iron is important for normal cellular function and posttranscriptional mechanisms regulate the cellular response to iron deficiency. In this study we explore how RNA binding proteins, PCBP1 and PCBP2, exhibit iron-sensitive RNA regulation.
Project description:Iron is important for normal cellular function and posttranscriptional mechanisms regulate the cellular response to iron deficiency. In this study we explore how RNA binding proteins, PCBP1 and PCBP2, exhibit iron-sensitive RNA regulation.
Project description:Iron is important for normal cellular function and posttranscriptional mechanisms regulate the cellular response to iron deficiency. In this study we explore how RNA binding proteins, PCBP1 and PCBP2, exhibit iron-sensitive RNA regulation.