ABSTRACT
Given their central role in translation, splicing, localization and stability of transcripts, RNA binding proteins (RBPs) are key regulators of several cellular processes. While experimental efforts have been put to study how RBPs bind to transcripts, very little is known about the RNA contributions to the interaction. Here, we review the most common RNA-centric methods to reveal interactions with RBPs: both in vitro (SELEX, SEQR, RNA-compete and RBNS) and in silico (MEME, SeAMotE, GLAM2, iDeep, MEMERIS, RNA context, RCK, RNApromo and GraphProt). We emphasize the main advantages and disadvantages of each technique and highlight the key physico-chemical features contributing to the identification of RNA motifs involved in RBP recognition. We discuss extrinsic determinants influencing protein-RNA binding, such as post-transcriptional and post-translational modifications as well as expression and location of transcripts.
Subject(s)
Nucleic Acid Conformation , RNA-Binding Proteins/isolation & purification , RNA/isolation & purification , Binding Sites/genetics , Computer Simulation , Nucleotide Motifs/genetics , Protein Binding/genetics , RNA/genetics , RNA-Binding Proteins/geneticsABSTRACT
How pluripotent stem cells differentiate into the main germ layers is a key question of developmental biology. Here, we show that the chromatin-related factor Whsc1 (also known as Nsd2 and MMSET) has a dual role in pluripotency exit and germ layer specification of embryonic stem cells. On induction of differentiation, a proportion of Whsc1-depleted embryonic stem cells remain entrapped in a pluripotent state and fail to form mesendoderm, although they are still capable of generating neuroectoderm. These functions of Whsc1 are independent of its methyltransferase activity. Whsc1 binds to enhancers of the mesendodermal regulators Gata4, T (Brachyury), Gata6 and Foxa2, together with Brd4, and activates the expression of these genes. Depleting each of these regulators also delays pluripotency exit, suggesting that they mediate the effects observed with Whsc1. Our data indicate that Whsc1 links silencing of the pluripotency regulatory network with activation of mesendoderm lineages.
Subject(s)
Cell Differentiation/physiology , Endoderm/cytology , Histone-Lysine N-Methyltransferase/metabolism , Pluripotent Stem Cells/cytology , Animals , Cell Differentiation/genetics , Cell Lineage , Embryonic Stem Cells/cytology , Germ Layers/cytology , Mice , Neural Plate/cytology , Nuclear Proteins/metabolism , Transcription Factors/metabolismABSTRACT
The hierarchical relationships between stem cells and progenitors that guide mammary gland morphogenesis are still poorly defined. While multipotent basal stem cells have been found within the myoepithelial compartment, the in vivo lineage potential of luminal progenitors is unclear. Here we used the expression of the Notch1 receptor, previously implicated in mammary gland development and tumorigenesis, to elucidate the hierarchical organization of mammary stem/progenitor cells by lineage tracing. We found that Notch1 expression identifies multipotent stem cells in the embryonic mammary bud, which progressively restrict their lineage potential during mammary ductal morphogenesis to exclusively generate an ERαneg luminal lineage postnatally. Importantly, our results show that Notch1-labelled cells represent the alveolar progenitors that expand during pregnancy and survive multiple successive involutions. This study reveals that postnatal luminal epithelial cells derive from distinct self-sustained lineages that may represent the cells of origin of different breast cancer subtypes.
