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

Abstract

SUMMARY 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-level IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3′UTR-enriched targets. RNA Bind-N-Seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes, including a reduction in cell adhesion and an increase in cell death. For cell adhesion, in hPSCs we find IMP1 maintains levels of integrin mRNA, specifically regulating RNA stability of ITGB5. Additionally, we show IMP1 can be linked to hPSC survival via direct target BCL2. Thus, transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles.

IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts

“…We observed enrichment of BCL11A transcripts after RNA immunoprecipitation with IGF2BP1 antibody, but the broad list of potential binding partners identified by RIP-Seq reported here and elsewhere (21) suggest that better methods are needed to predict which mRNA species are specifically and functionally affected by IGF2BP1 binding. IGF2BP1 binding to BCL11A mRNA was reported previously to motifs located in both coding and noncoding regions (21).…”

“…Initially, to identify mRNAs that potentially interact with IGF2BP1, IGF2BP1 immunoprecipitation was performed followed by RNASeq analysis of coprecipitating RNA. As predicted by the prevalence of the IGF2BP1-binding motif as well as earlier studies of mRNA binding in nonerythroid cells (21)(22)(23), a broad list of candidate genes was identified (Dataset S1). Due to the large number of potential binding partners in erythroid cells, additional studies were performed upon both mRNA and protein expression levels of IGF2BP1-binding partners.…”

“…IGF2BP3 overexpression demonstrated weaker HbF effects, which will require further study, and could be a reflection of its lower expression levels. Alternatively, the HbF effects may have a structural basis because the two proteins have only a 73% similarity (13) and have been reported to exhibit distinct binding preferences (21). Increased gamma-globin mRNA and suppressed adult deltaand beta-globin genes were identified in the adult cells, which are a pattern of changes predicted for a reversion to a more fetal-like phenotype.…”

“…Prior studies also validated developmental silencing by LIN28B and activation of let-7 in erythroid cells (10,11). IGF2BPs function primarily in the binding and posttranscriptional regulation of multiple RNA targets (12,21,(34)(35)(36), as a consequence having multiple biological functions, including pluripotency of stem cells and regulation of glucose metabolism (37,38). Because let-7 miRNAs are downstream targets of LIN28B, and the IGF2BPs mRNAs are targets of let-7, a developmental regulatory pathway for fetal hemoglobin expression during ontogeny emerges.…”

Ancient antagonism between CELF and RBFOX families tunes mRNA splicing outcomes

“…Finally, we utilized publicly available eCLIP peaks for RBFOX2 from a variety of cell types including HEK293, human embryonic stem cells (H1ES), HepG2, and K562 cells (Conway et al 2016;Sundararaman et al 2016;Van Nostrand et al 2016) to see if there was evidence of in vivo binding of RBFOX2 in regions proximal to CELF2-regulated cassette exons. In line with the sequence enrichment data, we find enrichment of RBFOX2 eCLIP binding sites around CELF2-repressed exons, compared to unresponsive exons, in all four cell types examined (from ∼14% to 25%) ( Fig.…”