Enhanced CLIP Uncovers IMP Protein-RNA Targets in Human Pluripotent Stem Cells Important for Cell Adhesion and Survival.

Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using enhanced UV crosslinking and immunoprecipitation (eCLIP), we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region and binding site levels, IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3' UTR-enriched targets.

LIN28 binds messenger RNAs at GGAGA motifs and regulates splicing factor abundance.

LIN28 is a conserved RNA-binding protein implicated in pluripotency, reprogramming, and oncogenesis. It was previously shown to act primarily by blocking let-7 microRNA (miRNA) biogenesis, but here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Through crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28, we have defined discrete LIN28-binding sites in a quarter of human transcripts.

Somatic coding mutations in human induced pluripotent stem cells.

Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods, it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein-coding point mutations per exome).

Genome-wide approaches in the study of microRNA biology.

MicroRNAs (miRNAs), a class of ∼21-23 nucleotide long non-coding RNAs (ncRNAs), have critical roles in diverse biological processes that encompass development, proliferation, apoptosis, stress response, and fat metabolism. miRNAs recognize their target mRNA transcripts by partial sequence complementarity and collectively have been estimated to regulate the majority of human genes. Consequently, misregulation of miRNAs or disruption of their target sites in genes has been implicated in a variety of human diseases ranging from cancer metastasis to neurological disorders.

Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans.

MicroRNAs (miRNAs) regulate gene expression by guiding Argonaute proteins to specific target mRNA sequences. Identification of bona fide miRNA target sites in animals is challenging because of uncertainties regarding the base-pairing requirements between miRNA and target as well as the location of functional binding sites within mRNAs. Here we present the results of a comprehensive strategy aimed at isolating endogenous mRNA target sequences bound by the Argonaute protein ALG-1 in C.