Transcriptomics,Genomics

Dataset Information

28

Identification of cis- and trans-acting factors involved in the localization of MALAT-1 to nuclear speckles


ABSTRACT: MALAT-1 is a long mammalian non-coding transcript of approximately 8500 nucleotides. It is localized to nuclear speckles despite its mRNA-like characteristics, which usually result in the export of transcripts to the cytoplasm. In the present study, we report the identification of several factors that influence the nuclear speckle localization of MALAT-1, and we provide evidence that MALAT-1 is involved in the regulation of gene expression. Heterokaryon assays revealed that MALAT-1 does not shuttle between the nucleus and cytoplasm, but is stably retained within the nucleus. Analysis of MALAT-1 fragments showed that MALAT-1 contains two distinct nuclear speckle-directing elements (between nucleotides 1961 to 3040 and 6008 to 7011 of the MALAT-1 sequence). The knockdown of the nuclear speckle proteins, RNPS1, SRm160 or IBP160, resulted in the diffusion of MALAT-1 to the nucleoplasm. In addition, we have demonstrated that depletion of MALAT-1 represses the expression of several genes. This repression of gene expression also occurs in response to the delocalization of MALAT-1 from the nuclear speckles. These results suggest that RNPS1, SRm160 and IBP160 contribute to the localization of MALAT-1 to nuclear speckles where it is involved in regulating gene expression Overall design: We used DNA microarray analysis to analyze differentially expressed (up- and down-regulated) genes in cells depleted of MALAT-1 by RNAi. Microarray analysis using the Agilent Whole Human Genome 4 x 44K oligo microarray in two independent MALAT-1 knockdown cell lines transfected with different siRNAs. Total RNA (800 ng) was labeled with either Cy3 or Cy5 dye using an Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent). Fluorescently labeled cRNAs, derived from total RNA isolated from control cells, and from MALAT-1 knockdown cells, were hybridized to the same microarray slide carrying 60-mer probes from the whole human genome 4 x 44K oligo microarray kit (Agilent Technologies, Palo Alto, CA, USA). A flip labeling (dye-swap or reverse labeling with Cy3 and Cy5 dyes) procedure was followed to nullify the dye bias associated with unequal incorporation of the two Cy dyes into cDNA.

INSTRUMENT(S): Agilent-014850 Whole Human Genome Microarray 4x44K G4112F (Feature Number version)

SUBMITTER: RANDEEP RAKWAL  

PROVIDER: GSE20506 | GEO | 2010-06-02

SECONDARY ACCESSION(S): PRJNA125093

REPOSITORIES: GEO

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Publications

MALAT-1 enhances cell motility of lung adenocarcinoma cells by influencing the expression of motility-related genes.

Tano Keiko K   Mizuno Rie R   Okada Tomoko T   Rakwal Randeep R   Shibato Junko J   Masuo Yoshinori Y   Ijiri Kenichi K   Akimitsu Nobuyoshi N  

FEBS letters 20101013 22


MALAT-1, a long non-coding RNA, is associated with metastasis, but its role in the metastatic process remains unknown. Here, we show that short-interfering RNA-mediated MALAT-1 silencing impaired in vitro cell motility of lung cancer cells and influenced the expression of numerous genes. In these genes, knockdown of any one of CTHRC1, CCT4, HMMR, or ROD1 clearly inhibited cell migration. In MALAT-1 knockdown cells, pre-mRNA levels were decreased in some but not all genes. Thus, our findings sugg  ...[more]

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