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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE104856 | GEO | 2019/01/01

REPOSITORIES: GEO

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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.

2022-12-01 | GSE188445 | GEO

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

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

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

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NORAD-induced phase separation of PUMILIO proteins is required for genome maintenance

Project description:Long noncoding RNAs (lncRNAs) can regulate the activity of interacting RNA binding proteins (RBPs), but how lncRNAs efficiently compete against other transcripts to bind and regulate RBP activity is poorly understood. This problem is exemplified by NORAD, a lncRNA that maintains genomic stability by inhibiting PUMILIO (PUM) proteins. Here we show that NORAD is able to out-compete other PUM-binding transcripts by nucleating the formation of phase-separated PUM condensates, termed NP bodies. Dual mechanisms of PUM recruitment, involving multivalent PUM-NORAD and PUM-PUM interactions, enable NORAD to sequester a super-stoichiometric amount of PUM in these condensates. Accordingly, synthetic RNAs that induce PUM phase separation rescue genome instability in NORAD-deficient cells. These results illuminate mechanisms of RBP regulation by RNA-driven phase separation and uncover an essential role for this process in genome maintenance.

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Project description:Although numerous long noncoding RNAs (lncRNAs) have been identified, our understanding of their roles in mammalian physiology remains limited. Here we investigated the physiologic function of the conserved lncRNA Norad in vivo. Deletion of Norad in mice results in genomic instability and mitochondrial dysfunction, leading to a dramatic multi-system degenerative phenotype resembling premature aging. Loss of tissue homeostasis in Norad-deficient animals is attributable to augmented activity of PUMILIO proteins, which act as post-transcriptional repressors of target mRNAs to which they bind. Norad is the preferred RNA target of PUMILIO2 (PUM2) in mouse tissues and, upon loss of Norad, PUM2 hyperactively represses key genes required for mitosis and mitochondrial function. Accordingly, enforced Pum2 expression fully phenocopies Norad deletion, resulting in rapid-onset aging-associated phenotypes. These findings provide new insights and open new lines of investigation into the roles of noncoding RNAs and RNA binding proteins in normal physiology and aging.

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Project description:Thousands of long noncoding RNA (lncRNA) genes are encoded in the human genome, and hundreds of them are evolutionary conserved, but their functions and modes of action remain largely obscure. Particularly enigmatic lncRNAs are those that are exported to the cytoplasm, including NORAD - an abundant and highly conserved cytoplasmic lncRNA. Most of the sequence of NORAD is comprised of repetitive units that together contain at least 17 functional binding sites for the two Pumilio homologs in mammals. Through binding to PUM1 and PUM2, NORAD modulates the mRNA levels of their targets, which are enriched for genes involved in chromosome segregation during cell division. Our results suggest that some cytoplasmic lncRNAs function by modulating the activities of RNA binding proteins, an activity which positions them at key junctions of cellular signaling pathways.

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Identification of RNAs bound to PUM2 in Norad+/+ and Norad-/- brains [CLIP-seq]

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SAM68 promotes Lung adenocarcinoma tumorigenesis by regulating cancer metabolic conversion through alternative splicing

Project description:The metabolic conversion of oxidative phosphorylation to glycolysis provides tumor cells with energy and biosynthetic substrates, thereby promoting tumorigenesis and malignant progression. However, the mechanisms controlling the tumor metabolic switch is still not entirely clear. Here we demonstrate that SAM68 (gene name: KHDRBS1) as a splicing regulatory factor is frequently overexpressed in Lung adenocarcinoma (LUAD) and negatively correlated with the prognosis of LUAD patients. we find SAM68 promotes LUAD cells tumorigenesis and metastasis both in vitro and in vivo by regulating cancer metabolic switch. SAM68 drives cancer metabolism by mediating alternative splicing of Pyruvate kinase (PKM) pre-mRNAs, finally promoting the formation of PKM2. Mechanically, Sam68 interacted with the splicing repressor hnRNP A1, and depletion of hnRNP A1 or mutations that impair this interaction attenuated the PKM splicing regulation. Together, our work demonstrates key roles of SAM68 in the cancer metabolic conversion by regulating alternative splicing and SAM68 may be a promising therapeutic target for treating LUAD.This project looks into how SAM68 levels affect cancer cell phenotype in vitro

2020-11-04 | GSE149120 | GEO

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 (noncoding RNA activated by DNA damage) is a recently characterized, abundant, and highly conserved lncRNA that is required for proper mitotic divisions in human cells. NORAD acts in the cytoplasm and 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. These interactions provide a paradigm for how repeated elements in a lncRNA facilitate function.

| S-EPMC5828396 | biostudies-literature

NORAD-induced phase separation of PUMILIO proteins is required for genome maintenance

Project description:NORAD-induced phase separation of PUMILIO proteins is required for genome maintenance

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