Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Chromatin immunoprecipitation profiling of human HEK293 cells transiantly transfected with plasmids coding for Staufen2-HA isoforms

ABSTRACT: In human cells, Staufen2 is a double-stranded RNA-binding protein involved in several cellular functions. Although 51% identical to Staufen1, these proteins are nevertheless found in different RNA particles. In addition, differential splicing events generate Staufen2 isoforms that only differ at their N-terminal extremities. We used a genome wide approach to identify and compare the mRNA targets of mammalian Staufen2 isoforms. The mRNA content of Staufen mRNPs was identified by probing DNA microarrays with probes derived from mRNAs isolated from immunopurified Staufen2-containing complexes following transfection of HEK293T cells with Stau2-HA (59kDa) or Stau2-HA (62kDa) expressors. Our results indicate that 11% of the cellular RNAs expressed in HEK293T cells are found in Stau2-containing mRNPs. There is a predominance of mRNAs involved in cell metabolism, transport, transcription, regulation of cell processes and catalytic activity. Experiment Overall Design: HEK293 cells were transiantly transfected with plasmids coding for Staufen2-HA isoforms. Cell extracts were immunoprecipitated using anti-HA antibodies and co-immunoprecipitated mRNAs were purified and used to hybridize microarrays. As control, cell extract from mock transfected cells were used.

ORGANISM(S): Homo sapiens

SUBMITTER: Luc DesGroseillers

PROVIDER: E-GEOD-8437 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

A genome-wide approach identifies distinct but overlapping subsets of cellular mRNAs associated with Staufen1- and Staufen2-containing ribonucleoprotein complexes.

Furic Luc L Maher-Laporte Marjolaine M DesGroseillers Luc L

RNA (New York, N.Y.) 20071219 2

Messenger RNAs are associated with multiple RNA-binding proteins to form ribonucleoprotein (mRNP) complexes. These proteins are important regulators of the fate of their target mRNAs. In human cells, Staufen1 and Staufen2 proteins, coded by two different genes, are double-stranded RNA-binding proteins involved in several cellular functions including mRNA localization, translation, and decay. Although 51% identical, these proteins are nevertheless found in different RNA particles. In addition, di ...[more]

PMID: 18094122

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Project description:CarD is an essential mycobacterial protein that we had previously shown to bind the RNA polymerase (RNAP) and affect the transcriptional profile of M. smegmatis and Mycobacterium tuberculosis. For this reason, we suspected that CarD was directly regulating transcriptional complexes but we did not know at what stage of CarD was functioning and at which genes CarD interacted with the RNAP. To determine in which stage of the transcription cycle (initiation, elongation, or termination) CarD acts, we used Chromatin Immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD throughout the M. smegmatis chromosome. Specific antibodies targeting core RNAPb, RNAPσ, or a hemagglutinin (HA) epitope fused to CarD (CarD-HA) were used to co-immunoprecipitate associated DNA. CarD-HA was immunoprecipitated from the M. smegmatis Mc2155 ΔcarD attB::tetcarD-HA strain and unfused HA was immunoprecipitated from the Mc2155 attB::pmsg431 strain with monoclonal antibodies specific for HA (Sigma). RNAP β and σ were immunoprecipitated from M. smegmatis ΔcarD attB::tetcarD-HA with monoclonal antibodies specific for these subunits (Neoclone, Madison, WI; 8RB13 for β, 2G10 for σ). Co-precipitated DNA was sequenced using a SOLiD sequencer (Life Technologies), which provided sufficient reads for 100-fold coverage of the genome. The number of sequence reads per base pair was normalized to the total number of reads and expressed as a log2 value. The reads per base pair from the HA-alone sample served as the background and was subtracted from the other datasets. We found that CarD was never present on the genome in the absence of RNAP. However, whereas RNAP core enzyme was found throughout transcribed regions of the genome, CarD was primarily associated with promoter regions and highly correlated with RNAPσ. The colocalization of σA and CarD led us to propose that in vivo, CarD associates with RNAP initiation complexes at most promoters and is therefore a global regulator of transcription initiation. The genome sequences associated with M. smegmatis CarD, RNAPb, and RNAPs were determined by ChIP-seq analysis. Samples were done in duplicate, except for RNAPs. And sequencing was performed using a SOLiD sequencer (Life Technologies).

Dataset Information

Chromatin immunoprecipitation profiling of human HEK293 cells transiantly transfected with plasmids coding for Staufen2-HA isoforms

Publications

A genome-wide approach identifies distinct but overlapping subsets of cellular mRNAs associated with Staufen1- and Staufen2-containing ribonucleoprotein complexes.

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