MINDY1 Is a Downstream Target of the Polyamines and Promotes Embryonic Stem Cell Self-Renewal.

Embryonic stem cells have the ability to self-renew or differentiate and these processes are under tight control. We previously reported that the polyamine regulator AMD1 is critical for embryonic stem cell self-renewal. The polyamines putrescine, spermidine, and spermine are essential organic cations that play a role in a wide array of cellular processes. Here, we explore the essential role of the polyamines in the promotion of self-renewal and identify a new stem cell regulator that acts downstream of the polyamines: MINDY1. MINDY1 protein levels are high in embryonic stem cells (ESCs) and are dependent on high polyamine levels. Overexpression of MINDY1 can promote ESC self-renewal in the absence of the usually essential cytokine Leukemia Inhibitory Factor (LIF). MINDY1 protein is prenylated and this modification is required for its ability to promote self-renewal. We go on to show that Mindy1 RNA is targeted for repression by mir-710 during Neural Precursor cell differentiation. Taken together, these data demonstrate that high polyamine levels are required for ESC self-renewal and that they function, in part, through promotion of high MINDY1 levels. Stem Cells 2018;36:1170-1178.

Cytoplasmic long noncoding RNAs are frequently bound to and degraded at ribosomes in human cells.

Recent footprinting studies have made the surprising observation that long noncoding RNAs (lncRNAs) physically interact with ribosomes. However, these findings remain controversial, and the overall proportion of cytoplasmic lncRNAs involved is unknown. Here we make a global, absolute estimate of the cytoplasmic and ribosome-associated population of stringently filtered lncRNAs in a human cell line using polysome profiling coupled to spike-in normalized microarray analysis. Fifty-four percent of expressed lncRNAs are detected in the cytoplasm. The majority of these (70%) have >50% of their cytoplasmic copies associated with polysomal fractions. These interactions are lost upon disruption of ribosomes by puromycin. Polysomal lncRNAs are distinguished by a number of 5' mRNA-like features, including capping and 5'UTR length. On the other hand, nonpolysomal "free cytoplasmic" lncRNAs have more conserved promoters and a wider range of expression across cell types. Exons of polysomal lncRNAs are depleted of endogenous retroviral insertions, suggesting a role for repetitive elements in lncRNA localization. Finally, we show that blocking of ribosomal elongation results in stabilization of many associated lncRNAs. Together these findings suggest that the ribosome is the default destination for the majority of cytoplasmic long noncoding RNAs and may play a role in their degradation.

p27 is regulated independently of Skp2 in the absence of Cdk2.

Cyclin-dependent kinase 2 (Cdk2) is dispensable for mitotic cell cycle progression and Cdk2 knockout mice are viable due to the compensatory functions of other Cdks. In order to assess the role of Cdk2 under limiting conditions, we used Skp2 knockout mice that exhibit increased levels of Cdk inhibitor, p27(Kip1), which is able to inhibit Cdk2 and Cdk1. Knockdown of Cdk2 abrogated proliferation of Skp2(-/-) mouse embryonic fibroblasts, encouraging us to generate Cdk2(-/-)Skp2(-/-) double knockout mice. Cdk2(-/-)Skp2(-/-) double knockout mice are viable and display similar phenotypes as Cdk2(-/-) and Skp2(-/-) mice. Unexpectedly, fibroblasts generated from Cdk2(-/-)Skp2(-/-) double knockout mice proliferated at normal rates. The increased stability of p27 observed in Skp2(-/-) MEFs was not observed in Cdk2(-/-)Skp2(-/-) double knockout fibroblasts indicating that in the absence of Cdk2, p27 is regulated by Skp2-independent mechanisms. Ablation of other ubiquitin ligases for p27 such as KPC1, DDB1, and Pirh2 did not restore stability of p27 in Cdk2(-/-)Skp2(-/-) MEFs. Our findings point towards novel and alternate pathways for p27 regulation.

The Polyamine Regulator AMD1 Upregulates Spermine Levels to Drive Epidermal Differentiation.

Maintaining tissue homeostasis depends on a balance between cell proliferation, differentiation, and apoptosis. Within the epidermis, the levels of the polyamines putrescine, spermidine, and spermine are altered in many different skin conditions, yet their role in epidermal tissue homeostasis is poorly understood. We identify the polyamine regulator, Adenosylmethionine decarboxylase 1 (AMD1), as a crucial regulator of keratinocyte (KC) differentiation. AMD1 protein is upregulated on differentiation and is highly expressed in the suprabasal layers of the human epidermis. During KC differentiation, elevated AMD1 promotes decreased putrescine and increased spermine levels. Knockdown or inhibition of AMD1 results in reduced spermine levels and inhibition of KC differentiation. Supplementing AMD1-knockdown KCs with exogenous spermidine or spermine rescued aberrant differentiation. We show that the polyamine shift is critical for the regulation of key transcription factors and signaling proteins that drive KC differentiation, including KLF4 and ZNF750. These findings show that human KCs use controlled changes in polyamine levels to modulate gene expression to drive cellular behavior changes. Modulation of polyamine levels during epidermal differentiation could impact skin barrier formation or can be used in the treatment of hyperproliferative skin disorders.

Psychometric properties of the NEPSY-II affect recognition subtest in a preschool sample: a Rasch modeling approach.

OBJECTIVE: Understanding a child's ability to decode emotion expressions is important to allow early interventions for potential difficulties in social and emotional functioning. This study applied the Rasch model to investigate the psychometric properties of the NEPSY-II Affect Recognition subtest, a U.S. normed measure for 3-16 year olds which assesses the ability to recognize facial expressions of emotion. METHOD: Data were collected from 1222 children attending preschools in Singapore. We first performed the Rasch analysis with the raw item data, and examined the technical qualities and difficulty pattern of the studied items. We subsequently investigated the relation of the estimated affect recognition ability from the Rasch analysis to a teacher-reported measure of a child's behaviors, emotions, and relationships. Potential gender differences were also examined. RESULTS: The Rasch model fits our data well. Also, the NEPSY-II Affect Recognition subtest was found to have reasonable technical qualities, expected item difficulty pattern, and desired association with the external measure of children's behaviors, emotions, and relationships for both boys and girls. CONCLUSIONS: Overall, findings from this study suggest that the NEPSY-II Affect Recognition subtest is a promising measure of young children's affect recognition ability. Suggestions for future test improvement and research were discussed.

Polyamine Regulator AMD1 Promotes Cell Migration in Epidermal Wound Healing.

Wound healing is a dynamic process involving gene-expression changes that drive re-epithelialization. Here, we describe an essential role for polyamine regulator AMD1 in driving cell migration at the wound edge. The polyamines, putrescine, spermidine, and spermine are small cationic molecules that play essential roles in many cellular processes. We demonstrate that AMD1 is rapidly upregulated following wounding in human skin biopsies. Knockdown of AMD1 with small hairpin RNAs causes a delay in cell migration that is rescued by the addition of spermine. We further show that spermine can promote cell migration in keratinocytes and in human ex vivo wounds, where it significantly increases epithelial tongue migration. Knockdown of AMD1 prevents the upregulation of urokinase-type plasminogen activator/urokinase-type plasminogen activator receptor on wounding and results in a failure in actin cytoskeletal reorganization at the wound edge. We demonstrate that keratinocytes respond to wounding by modulating polyamine regulator AMD1 in order to regulate downstream gene expression and promote cell migration. This article highlights a previously unreported role for the regulation of polyamine levels and ratios in cellular behavior and fate.

Analysis of mRNA Translation Rate in Mouse Embryonic Stem Cells.

Regulation of gene expression is essential to enable embryonic stem cells (ESCs) to either self-renew or to differentiate. Translational regulation of mRNA plays a major role in regulating gene expression and has been shown to be important for ESC differentiation. Sucrose gradients can be used to separate mRNAs based on the number of associated ribosomes and this can be used as a readout of the rate of translation. Following centrifugation through a sucrose gradient, mRNAs can be recovered, purified, and analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) to determine their ribosomal load in different cell states. Here, we describe how to differentiate mouse ESCs to Neural Precursor Cells (NPCs) and analyze the rate of translation of individual mRNAs by qRT-PCR following polysome fractionation.