Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Miguel Brown
PROVIDER: E-GEOD-44616 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Human LIN28A and B are RNA-binding proteins (RBPs) conserved in animals with important roles during development and stem cell reprogramming. We used Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in HEK293 cells and identified a largely overlapping set of ~3,000 mRNAs at ~9,500 sites located in the 3M-bM-^@M-^YUTR and CDS. In vitro and in vivo, LIN28 preferentially bound single-stranded RNA containing a uridine-rich element and one or more flanking guanosines, and appeared to be able to disrupt base-pairing to access these elements when embedded in predicted secondary structure. In HEK293 cells, LIN28 protein binding mildly stabilized target mRNAs and increased protein abundance. The top targets were its own mRNAs and those of other RBPs and cell-cycle regulators. Alteration of LIN28 protein levels also negatively regulated the abundance of some, but not all let-7 miRNA family members, indicating sequence-specific binding of let-7 precursors to LIN28 proteins and competition with cytoplasmic miRNA biogenesis factors. To assess whether miRNAs are regulated by LIN28B we analyzed the miRNA levels of LIN28B overexpressing and LIN28B-depleted cells using small RNA cDNA library sequencing. The RBP LIN28B was depleted by siRNAs and the expression levels was compared to mock-transfected HEK293 cells.
2013-02-26 | E-GEOD-44568 | biostudies-arrayexpress
Project description:Human LIN28A and B are RNA-binding proteins (RBPs) conserved in animals with important roles during development and stem cell reprogramming. We used Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in HEK293 cells and identified a largely overlapping set of ~3,000 mRNAs at ~9,500 sites located in the 3M-bM-^@M-^YUTR and CDS. In vitro and in vivo, LIN28 preferentially bound single-stranded RNA containing a uridine-rich element and one or more flanking guanosines, and appeared to be able to disrupt base-pairing to access these elements when embedded in predicted secondary structure. In HEK293 cells, LIN28 protein binding mildly stabilized target mRNAs and increased protein abundance. The top targets were its own mRNAs and those of other RBPs and cell-cycle regulators. Alteration of LIN28 protein levels also negatively regulated the abundance of some, but not all let-7 miRNA family members, indicating sequence-specific binding of let-7 precursors to LIN28 proteins and competition with cytoplasmic miRNA biogenesis factors. To assess whether the transcripts identified by PAR-CLIP are regulated by LIN28B we analyzed the mRNA levels of LIN28B overexpressing and LIN28B-depleted cells using microarrays. Transcripts crosslinked to LIN28B were slightly downregulated upon LIN28B knockdown compared to LIN28B overexpression indicating that LIN28B stabilizes transcripts. The RBP LIN28B was depleted by siRNAs and the expression levels was compared to mock-transfected HEK293 cells The RBP LIN28B was depleted by siRNAs and the expression levels was compared to mock-transfected HEK293 cells
2013-02-26 | E-GEOD-44613 | biostudies-arrayexpress
Project description:Human LIN28A and B are RNA-binding proteins (RBPs) conserved in animals with important roles during development and stem cell reprogramming. We used Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in HEK293 cells and identified a largely overlapping set of ~3,000 mRNAs at ~9,500 sites located in the 3M-bM-^@M-^YUTR and CDS. In vitro and in vivo, LIN28 preferentially bound single-stranded RNA containing a uridine-rich element and one or more flanking guanosines, and appeared to be able to disrupt base-pairing to access these elements when embedded in predicted secondary structure. In HEK293 cells, LIN28 protein binding mildly stabilized target mRNAs and increased protein abundance. The top targets were its own mRNAs and those of other RBPs and cell-cycle regulators. Alteration of LIN28 protein levels also negatively regulated the abundance of some, but not all let-7 miRNA family members, indicating sequence-specific binding of let-7 precursors to LIN28 proteins and competition with cytoplasmic miRNA biogenesis factors. LIN28 protein PAR-CLIP
2013-02-26 | E-GEOD-44615 | biostudies-arrayexpress
Project description:We applied 4-thiouridine to cultured cells expressing the FLAG/HA-tagged RNA-binding proteins (RBPs) followed by UV 365 nm irradiation. The crosslinked RNA-protein complexes were isolated by immunoprecipitation, and the covalently bound RNA was partially digested with RNase T1 and radiolabeled. The radiolabeled RNPs were subsequenctly separated by SDS-PAGE, the crosslinked RNA segments recovered and converted into a cDNA library and sequenced. RBPs were UV-crosslinked to their RNA targets containing 4-thiouridine. The RNA segments were recovered after immunoprecipitation and seqeunced by Solexa.
2010-05-21 | E-GEOD-21918 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE21574: Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP: QKI data GSE21575: Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP: IGF2BP data GSE21577: Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP: miRNA inhibition data GSE21918: Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP: sequencing data Refer to individual Series
2010-04-29 | E-GEOD-21578 | biostudies-arrayexpress
Project description:RNA-binding proteins coordinate the fates of multiple RNAs, but the principles underlying these global interactions remain poorly understood. We elucidated regulatory mechanisms of the RNA-binding protein HuR, by integrating data from diverse high-throughput targeting technologies, specifically PAR-CLIP, RIP-chip, and whole-transcript expression profiling. The number of binding sites per transcript, degree of HuR-association, and degree of HuR-dependent RNA stabilization were positively correlated. Pre-mRNA and mature mRNA containing both intronic and 3' UTR binding sites were more highly stabilized than transcripts with only 3' UTR or only intronic binding sites, suggesting that HuR couples pre-mRNA processing with mature mRNA stability. We also observed HuR-dependent splicing changes and substantial binding of HuR in poly-pyrimidine tracts of pre-mRNAs. Comparison of the spatial patterns surrounding HuR and miRNA binding sites provided functional evidence for HuR-dependent antagonism of proximal miRNA-mediated repression. We conclude that HuR coordinates gene expression outcomes at multiple interconnected steps of RNA processing. HuR (ELAVL1) PAR-CLIP
2011-08-19 | E-GEOD-29779 | biostudies-arrayexpress
Project description:To test the influence of IGF2BPs on the stability of their interacting mRNAs, as reported previously for some targets (Yisraeli, 2005), we simultaneously depleted all three IGF2BP family members using siRNAs and compared the cellular RNA from knockdown and mock-transfected cells on microarrays. The levels of transcripts identified by PAR-CLIP decreased in IGF2BP-depleted cells, indicating that IGF2BP proteins stabilize their target mRNAs. Moreover, transcripts that yielded clusters with the highest T to C mutation frequency were most destabilized (Figure 4G of PMID 20371350), indicating that the ranking criterion that we derived based on the analysis of PUM2 and QKI data generalizes to other RNA-binding proteins (RBPs). The RBPs IGF2BP1-3 were depleted by siRNAs and the expression level was compared to mock-transfected HEK 293 cells.
2010-05-27 | E-GEOD-21575 | biostudies-arrayexpress
Project description:To assess whether the transcripts identified by PAR-CLIP are regulated by the RNA-binding protein (RBP) Quaking (QKI), we analyzed the mRNA levels of mock-transfected and QKI-specific siRNA-transfected cells with microarrays. Transcripts crosslinked to QKI were significantly upregulated upon siRNA transfection, indicating that QKI negatively regulates bound mRNAs (Figure 3H of PMID 20371350), consistent with previous reports of QKI being a repressor. The RBP QKI was depleted by siRNAs and the expression level was compared to mock-transfected HEK 293 cells.
2010-05-27 | E-GEOD-21574 | biostudies-arrayexpress
Project description:To obtain evidence that Argonaute (AGO) crosslink-centered regions (CCRs) indeed contain functional miRNA-binding sites, we blocked 25 of the most abundant miRNAs in HEK 293 cells (Figure 5C of PMID 20371350) by transfection of a cocktail of 2'-O-methyl-modified antisense oligoribonucleotides and monitored the changes in mRNA stability by microarrays (Figure 7A of PMID 20371350). Consistent with previous studies of individual miRNAs (Grimson et al., 2007), the magnitude of the destabilization effects of transcripts containing at least one CCR depended on the length of the seed-complementary region and dropped from 9-mer to 8-mer to 7-mer to 6-mer matches (Figure 7B of PMID 20371350). We did not find evidence for significant destabilization of transcripts that only contained imperfectly paired seed regions. The top 25 expressed miRNAs expressed in HEK 293 cells were inhibited by transfection of a cocktail of 25 antisense 2'-O-Me oligoribonucleotides and the gene expression was compared to mock-transfected cells.
2010-05-27 | E-GEOD-21577 | biostudies-arrayexpress
Project description:Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation and translation. We developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing. Application to a human embryonic kidney cell line identified close to 800 proteins. Close to one third of these proteins, were neither previously annotated nor could be functionally predicted to bind RNA. Protein occupancy profiling provides a transcriptome-wide catalog of potential cis-regulatory regions on mammalian mRNAs and showed that large stretches in 3' UTRs can be contacted by the mRNA-bound proteome, with numerous putative binding sites in regions harboring disease-associated nucleotide polymorphisms. Our observations indicate the presence of a large number of unexpected mRNA-binders with novel molecular functions participating in combinatorial post-transcriptional gene-expression networks. PARCLIP was performed as in Hafner et al. 2010 Cell 141, 129M-bM-^@M-^S141, with HEK293 cell lines stably expressing HIS/FLAG/HA-tagged ALKBH5, C17orf85, C22orf28, CAPRIN1, and ZC3H7B. We used 4-thiouridine (4SU) to enhance the crosslink and generated two PAR-CLIP cDNA libraries per protein.
2012-06-01 | E-GEOD-38201 | biostudies-arrayexpress