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Homo sapiens

ABSTRACT: Gene expression profiles in NORAD knockout and PUMILIO overexpressing cells

PROVIDER: PRJNA304717 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR2969250_1.fastq.gz	Fastqsanger.gz
	SRR2969250_2.fastq.gz	Fastqsanger.gz
	SRR2969260_1.fastq.gz	Fastqsanger.gz
	SRR2969260_2.fastq.gz	Fastqsanger.gz
	SRR2969251_1.fastq.gz	Fastqsanger.gz

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Gene expression profiles in NORAD knockout and PUMILIO overexpressing cells

Project description:Purpose: PUMILIO proteins are known to repress target genes by specifically binding to PUMILIO response elements (PREs) in target mRNAs. NORAD is a noncoding RNA that negatively regulates PUMILIO activity. The goal of this study was to determine the gene expression changes that result from knockout of NORAD or overexpression of PUMILIO and to test whether NORAD knockout causes PUMILIO hyperactivity. Methods: RNA-seq libraries were prepared using the TruSeq Stranded Total RNA with Ribo-Zero Human/Mouse/Rat Sample Preparation kit (Illumina) and sequenced using the 100 bp paired-end protocol on an Illumina HiSeq 2000. For comparing NORAD+/+ and NORAD-/- HCT116 cells, 3 biological replicates per genotype were sequenced. For PUM overexpression experiments, 3 replicates of GFP-expressing HCT116 cells (negative control) and 2 independent PUM1- or PUM2-overexpressing clones (2 replicates each) were sequenced. Results: Gene expression profiles show that PUMILIO target genes are downregulated in both NORAD knockout cells and PUMILIO overexpressing cells. Conclusions: These data indicate that NORAD sequesters PUMILIO, preventing excessive repression of PUMILIO target genes that are important for maintaining genomic stability.

2015-12-03 | GSE75440 | GEO

Identification of RNAs bound to endogenous PUM2 in HCT116 cells

Project description:Purpose: PUMILIO proteins are known to repress target genes by binding to PUMILIO response elements (PREs) in target mRNAs. The goal of this study was to demonstrate binding of endogenous PUM2 to the noncoding RNA NORAD and to identify PUM2 target genes in NORAD wild-type and knockout HCT116 cells. Methods: PAR-CLIP was performed with endogenous PUM2 in HCT116 cells and isogenic NORAD knockout cells. Results: Endogenous PUM2 binds to NORAD in HCT116 cells. In addition, PUM2 target genes were identified in HCT116 cells. Conclusions: PUM2 binds to NORAD through multiple PREs on NORAD. Compared to all other PUM2 target genes in HCT116, NORAD is the preferred binding partner of endogenous PUM2.

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SAM68 is required for regulation of Pumilio by the NORAD long noncoding RNA

Project description:The number of known long noncoding RNA (lncRNA) functions is rapidly growing, but how those functions are encoded in their sequence and structure remains poorly understood. NORAD is a recently characterized, abundant, and highly conserved cytoplasmic lncRNA that is required for proper mitotic divisions in human cells. NORAD antagonizes repressors from the Pumilio family that bind at least 17 sites spread through 12 repetitive units in NORAD sequence. Here we study conserved sequences in NORAD repeats, identify additional interacting partners, and characterize the interaction between NORAD and the RNA binding protein SAM68 (KHDRBS1), which is required for NORAD function in antagonizing Pumilio. The interactions between NORAD, Pumilio and SAM68 provide a paradigm for how specific repeated and structured elements with a lncRNA can facilitate its function.

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Structural features within the NORAD long noncoding RNA underlie efficient repression of Pumilio activity

Project description:It is increasingly appreciated that long non-coding RNAs (lncRNAs) carry out important functions in mammalian cells, but how these are encoded in their sequences, and manifested in their structures remains largely unknown. Some lncRNAs bind to and modulate the availability of RNA binding proteins, but the structural principles that underlie this mode of regulation are underexplored. Here, we focused on the NORAD lncRNA, which binds Pumilio proteins and modulates their ability to repress hundreds of mRNA targets. We probed the RNA structure and long-range RNA-RNA interactions formed by NORAD inside cells, under different stressful conditions. We discovered that NORAD structure is highly modular, and consists of well-defined domains that contribute independently to NORAD function. The structure adopted by NORAD spatially clusters the Pumilio binding sites along NORAD in a manner that contributes to de-repression of Pumilio target proteins. Following arsenite stress, the majority of NORAD structure undergoes relaxation and forms inter-molecular interactions with RNAs that are targeted to stress granules. NORAD sequence thus dictates elaborated structural domain organisation that facilitates its function on multiple levels, and which helps explain the extensive evolutionary sequence conservation of NORAD regions that are not predicted to directly bind Pumilio proteins.

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