Genome-wide analysis of mRNAs bound to the histone stem-loop binding protein

The replication-dependent histone mRNAs are cell-cycle-regulated and expressed only during S phase. In contrast to all other eukaryotic mRNAs, the histone mRNAs end in a highly conserved 16-nucleotide stem–loop rather than a poly(A) tail. The stem–loop is necessary and sufficient for the post-transcriptional regulation of histone mRNA during the cell cycle. The histone mRNA 3′ stem–loop is bound by the stem–loop binding protein (SLBP) that is involved in pre-mRNA processing, translation, and stability of histone mRNA. Immunoprecipitation (IP) of RNA-binding proteins (RBPs) followed by microarray analysis has been used to identify the targets of RNA-binding proteins. This method is sometimes referred to as RIP-Chip (RNA IP followed by microarray analysis). Here we introduce a variation on the RIP-Chip method that uses a recombinant RBP to identify mRNA targets in a pool of total RNA; we call this method recombinant, or rRIP-Chip. Using this method, we show that recombinant SLBP binds exclusively to all five classes of histone mRNA. We also analyze the messages bound to the endogenous SLBP on polyribosomes by immunoprecipitation. We use two different microarray platforms to identify enriched mRNAs. Both platforms demonstrate remarkable specificity and consistency of results. Our data suggest that the replication-dependent histone mRNAs are likely to be the sole target of SLBP

INCREASING FOOD ACCESSIBILITY AND MENTAL HEALTH RESOURCES TO PROMOTE ADOLESCENT WELL-BEING IN A RURAL COMMUNITY

The Neighborhood and Built Environment is one of five Social Determinants of Health domains and is defined as where one lives, works, plays, and learns — all factors that affect one’s health. Cleveland County, North Carolina has a high percentage of school-age children who live in poverty, and face greater adversities including financial hardships, food insecurity, and adverse childhood experiences. The objective of this proposal is to increase the proportion of schools with policies and practices that promote health and safety. To address these issues, the nutrition-focused recommendation is to increase access to healthy foods by providing free meals to all participating County School students by expanding the Community Eligibility Provision of the National School Lunch Program. The policy-focused recommendation is to implement the “Require All Schools to Staff Mental Health Counselors” policy, encouraging a sustainable mental health support system in schools.Master of Public Healt

INCREASING FOOD ACCESSIBILITY AND MENTAL HEALTH RESOURCES TO PROMOTE ADOLESCENT WELL-BEING IN A RURAL COMMUNITY

The Neighborhood and Built Environment is one of five Social Determinants of Health domains and is defined as where one lives, works, plays, and learns — all factors that affect one’s health. Cleveland County, North Carolina has a high percentage of school-age children who live in poverty, and face greater adversities including financial hardships, food insecurity, and adverse childhood experiences. The objective of this proposal is to increase the proportion of schools with policies and practices that promote health and safety. To address these issues, the nutrition-focused recommendation is to increase access to healthy foods by providing free meals to all participating County School students by expanding the Community Eligibility Provision of the National School Lunch Program. The policy-focused recommendation is to implement the “Require All Schools to Staff Mental Health Counselors” policy, encouraging a sustainable mental health support system in schools.Master of Public Healt

INCREASING FOOD ACCESSIBILITY AND MENTAL HEALTH RESOURCES TO PROMOTE ADOLESCENT WELL-BEING IN A RURAL COMMUNITY

The Neighborhood and Built Environment is one of five Social Determinants of Health domains and is defined as where one lives, works, plays, and learns — all factors that affect one’s health. Cleveland County, North Carolina has a high percentage of school-age children who live in poverty, and face greater adversities including financial hardships, food insecurity, and adverse childhood experiences. The objective of this proposal is to increase the proportion of schools with policies and practices that promote health and safety. To address these issues, the nutrition-focused recommendation is to increase access to healthy foods by providing free meals to all participating County School students by expanding the Community Eligibility Provision of the National School Lunch Program. The policy-focused recommendation is to implement the “Require All Schools to Staff Mental Health Counselors” policy, encouraging a sustainable mental health support system in schools.Master of Public Healt

INCREASING FOOD ACCESSIBILITY AND MENTAL HEALTH RESOURCES TO PROMOTE ADOLESCENT WELL-BEING IN A RURAL COMMUNITY

The Neighborhood and Built Environment is one of five Social Determinants of Health domains and is defined as where one lives, works, plays, and learns - all factors that affect one’s health. Cleveland County, North Carolina has a high percentage of school-age children who live in poverty, and face greater adversities including financial hardships, food insecurity, and adverse childhood experiences. The objective of this proposal is to increase the proportion of schools with policies and practices that promote health and safety. To address these issues, the nutrition-focused recommendation is to increase access to healthy foods by providing free meals to all participating County School students by expanding the Community Eligibility Provision of the National School Lunch Program. The policy-focused recommendation is to implement the “Require All Schools to Staff Mental Health Counselors” policy, encouraging a sustainable mental health support system in schools.Master of Public Healt

PHD2/3-dependent hydroxylation tunes cardiac response to β-adrenergic stress via phospholamban

Ischemic heart disease is the leading cause of heart failure. Both clinical trials and experimental animal studies demonstrate that chronic hypoxia can induce contractile dysfunction even before substantial ventricular damage, implicating a direct role of oxygen in the regulation of cardiac contractile function. Prolyl hydroxylase domain (PHD) proteins are well recognized as oxygen sensors and mediate a wide variety of cellular events by hydroxylating a growing list of protein substrates. Both PHD2 and PHD3 are highly expressed in the heart, yet their functional roles in modulating contractile function remain incompletely understood. Here, we report that combined deletion of Phd2 and Phd3 dramatically decreased expression of phospholamban (PLN), resulted in sustained activation of calcium/calmodulin-activated kinase II (CaMKII), and sensitized mice to chronic β-adrenergic stress–induced myocardial injury. We have provided evidence that thyroid hormone receptor-α (TR-α), a transcriptional regulator of PLN, interacts with PHD2 and PHD3 and is hydroxylated at 2 proline residues. Inhibition of PHDs increased the interaction between TR-α and nuclear receptor corepressor 2 (NCOR2) and suppressed Pln transcription. Together, these observations provide mechanistic insight into how oxygen directly modulates cardiac contractility and suggest that cardiac function could be modulated therapeutically by tuning PHD enzymatic activity

Genome-wide analysis of mRNAs bound to the histone stem–loop binding protein

The replication-dependent histone mRNAs are cell-cycle-regulated and expressed only during S phase. In contrast to all other eukaryotic mRNAs, the histone mRNAs end in a highly conserved 16-nucleotide stem–loop rather than a poly(A) tail. The stem–loop is necessary and sufficient for the post-transcriptional regulation of histone mRNA during the cell cycle. The histone mRNA 3′ stem–loop is bound by the stem–loop binding protein (SLBP) that is involved in pre-mRNA processing, translation, and stability of histone mRNA. Immunoprecipitation (IP) of RNA-binding proteins (RBPs) followed by microarray analysis has been used to identify the targets of RNA-binding proteins. This method is sometimes referred to as RIP-Chip (RNA IP followed by microarray analysis). Here we introduce a variation on the RIP-Chip method that uses a recombinant RBP to identify mRNA targets in a pool of total RNA; we call this method recombinant, or rRIP-Chip. Using this method, we show that recombinant SLBP binds exclusively to all five classes of histone mRNA. We also analyze the messages bound to the endogenous SLBP on polyribosomes by immunoprecipitation. We use two different microarray platforms to identify enriched mRNAs. Both platforms demonstrate remarkable specificity and consistency of results. Our data suggest that the replication-dependent histone mRNAs are likely to be the sole target of SLBP

PHD2/3-dependent hydroxylation tunes cardiac response to β-adrenergic stress via phospholamban

Ischemic heart disease is the leading cause of heart failure. Both clinical trials and experimental animal studies demonstrate that chronic hypoxia can induce contractile dysfunction even before substantial ventricular damage, implicating a direct role of oxygen in the regulation of cardiac contractile function. Prolyl hydroxylase domain (PHD) proteins are well recognized as oxygen sensors and mediate a wide variety of cellular events by hydroxylating a growing list of protein substrates. Both PHD2 and PHD3 are highly expressed in the heart, yet their functional roles in modulating contractile function remain incompletely understood. Here, we report that combined deletion of Phd2 and Phd3 dramatically decreased expression of phospholamban (PLN), resulted in sustained activation of calcium/calmodulin-activated kinase II (CaMKII), and sensitized mice to chronic β-adrenergic stress–induced myocardial injury. We have provided evidence that thyroid hormone receptor-α (TR-α), a transcriptional regulator of PLN, interacts with PHD2 and PHD3 and is hydroxylated at 2 proline residues. Inhibition of PHDs increased the interaction between TR-α and nuclear receptor corepressor 2 (NCOR2) and suppressed Pln transcription. Together, these observations provide mechanistic insight into how oxygen directly modulates cardiac contractility and suggest that cardiac function could be modulated therapeutically by tuning PHD enzymatic activity