Unknown,Transcriptomics,Genomics,Proteomics

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SiRNA of human HeLa cells reveals Staufen1 regulates a variety of mammalian transcripts

ABSTRACT: It is currently unknown how extensively the double-stranded RNA binding protein Staufen (Stau)1 is utilized by mammalian cells to regulate gene expression. To date, Stau1 binding to the 3â untranslated region (3âUTR) of ARF1 mRNA has been shown to target ARF1 mRNA for Stau1-mediated mRNA decay (SMD). ARF1 SMD depends on translation and recruitment of the nonsense-mediated mRNA decay factor Upf1 to the ARF1 3âUTR by Stau1. Here, we use microarray analyses to examine changes in the abundance of cellular mRNAs that occur when Stau1 is depleted. Results indicate that 1.1% and 1.0% of the 11,569 HeLa-cell transcripts that were analyzed are, respectively, upregulated and downregulated at least two-fold in three independently performed experiments. Additionally, we localize the Stau1 binding site to the 3âUTR of four mRNAs that we define as natural SMD targets. Together, these and substantiating results suggest that Stau1 influences the expression of a wide variety of physiologic transcripts and metabolic pathways. Experiment Overall Design: We report the results of three independently performed microarray analyses that examined changes in the abundance of transcripts from HeLa-cell genes upon Stau1 depletion. HeLa cells were transiently transfected with either a nonspecific Control small interfering (si)RNA or Stau1 siRNA. Stau1 siRNA reduced the level of cellular Stau1 to as little as 4% of normal, where normal is defined as the level in the presence of Control siRNA (data not shown). RNA from three independently performed transfections was separately hybridized to microarrays.

ORGANISM(S): Homo sapiens

SUBMITTER: Luc DesGroseillers

PROVIDER: E-GEOD-6679 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Staufen1 regulates diverse classes of mammalian transcripts.

Kim Yoon Ki YK Furic Luc L Parisien Marc M Major François F DesGroseillers Luc L Maquat Lynne E LE

The EMBO journal 20070517 11

It is currently unknown how extensively the double-stranded RNA-binding protein Staufen (Stau)1 is utilized by mammalian cells to regulate gene expression. To date, Stau1 binding to the 3'-untranslated region (3'-UTR) of ADP ribosylation factor (ARF)1 mRNA has been shown to target ARF1 mRNA for Stau1-mediated mRNA decay (SMD). ARF1 SMD depends on translation and recruitment of the nonsense-mediated mRNA decay factor Upf1 to the ARF1 3'-UTR by Stau1. Here, we demonstrate that Stau1 binds to a com ...[more]

PMID: 17510634

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Project description:Several of the thousands of human long non-coding RNAs (lncRNAs) have been functionally characterized; however, potential roles for lncRNAs in somatic tissue differentiation remain poorly understood. Here we show that a 3.7kb lncRNA, terminal differentiation-induced ncRNA (TINCR), controls human epidermal differentiation by a post-transcriptional mechanism. TINCR is required for high mRNA abundance of key differentiation genes, many of which are mutated in human skin diseases, including FLG, LOR, ALOXE3, ALOX12B, ABCA12, CASP14 and ELOVL3. TINCR-deficient epidermis lacked terminal differentiation ultrastructure, including keratohyalin granules and intact lamellar bodies. Genome-scale RNA interactome analysis revealed that TINCR interacts with a suite of differentiation mRNAs. TINCR-mRNA interaction occurs through a 25 nucleotide “TINCR box” motif which is strongly enriched in interacting mRNAs \and required for TINCR binding. A high-throughput screen to analyze TINCR binding capacity to ~9,400 human recombinant proteins revealed direct binding of TINCR RNA to the Staufen1 (STAU1) protein. STAU1-deficient tissue recapitulated the impaired differentiation seen with TINCR depletion. Loss of UPF1 and UPF2, both of which are required for STAU1-mediated RNA decay (SMD), however, lacked differentiation impacts. Instead, the TINCR/STAU1 complex seems to mediate stabilization of differentiation mRNAs, such as KRT80. These data identify TINCR as a key lncRNA required for somatic tissue differentiation, which occurs through inducible lncRNA binding to differentiation mRNAs to ensure their expression. Gene expression analysis: To establish a gene expression signature for regenerated human epidermis depleted of TINCR, as well as STAU1, total RNA was isolated in biologic duplicate from organotypic cultures of cells with a non-targeting siRNA, or an siRNA to the gene of interest. This RNA was processed and then hybridized to Affymetrix HG-U133 2.0 Plus arrays.

Dataset Information

SiRNA of human HeLa cells reveals Staufen1 regulates a variety of mammalian transcripts

Publications

Staufen1 regulates diverse classes of mammalian transcripts.

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