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PMID: 16971455 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Effects of Dicer and Argonaute down-regulation on mRNA levels in human HEK293 cells.

Nucleic acids research ·Vol. 34 ·No. 17 ·2006-00-00 ·Pages 4801-15

Schmitter D, Filkowski J, Sewer A, Pillai RS, Oakeley EJ, Zavolan M, Svoboda P, Filipowicz W

Abstract

RNA interference and the microRNA (miRNA) pathway can induce sequence-specific mRNA degradation and/or translational repression. The human genome encodes hundreds of miRNAs that can post-transcriptionally repress thousands of genes. Using reporter constructs, we observed that degradation of mRNAs bearing sites imperfectly complementary to the endogenous let-7 miRNA is considerably stronger in human HEK293 than HeLa cells. The degradation did not result from the Ago2-mediated endonucleolytic cleavage but it was Dicer- and Ago2-dependent. We used this feature of HEK293 to address the size of a pool of transcripts regulated by RNA silencing in a single cell type. We generated HEK293 cell lines depleted of Dicer or individual Ago proteins. The cell lines were used for microarray analyses to obtain a comprehensive picture of RNA silencing. The 3'-untranslated region sequences of a few hundred transcripts that were commonly up-regulated upon Ago2 and Dicer knock-downs showed a significant enrichment of putative miRNA-binding sites. The up-regulation upon Ago2 and Dicer knock-downs was moderate and we found no evidence, at the mRNA level, for activation of silenced genes. Taken together, our data suggest that, independent of the effect on translation, miRNAs affect levels of a few hundred mRNAs in HEK293 cells.

MeSH Terms
3' Untranslated Regions/chemistry Argonaute Proteins Cell Line Down-Regulation Eukaryotic Initiation Factor-2 Genes, Reporter HeLa Cells Humans MicroRNAs/metabolism Oligonucleotide Array Sequence Analysis Peptide Initiation Factors/antagonists & inhibitors,genetics,physiology RNA Interference RNA, Messenger/chemistry,metabolism Ribonuclease III/antagonists & inhibitors,genetics,physiology
Chemicals
3' Untranslated Regions AGO2 protein, human Argonaute Proteins Eukaryotic Initiation Factor-2 MicroRNAs Peptide Initiation Factors RNA, Messenger mirnlet7 microRNA, human Ribonuclease III
Authors & Affiliations
8 authors, click to expand affiliations / ORCID
Schmitter Daniela
Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4002 Basel, Switzerland.
Filkowski Jody
Sewer Alain
Pillai Ramesh S
Oakeley Edward J
Zavolan Mihaela
Svoboda Petr
Filipowicz Witold
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
1362-4962
DOI
10.1093/nar/gkl646
Published
2006-00-00
Epub
2006-00-13
Pages
4801-15
Language
English
Region
England
NLM ID
0411011
PMCID
PMC1635286
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/gkl646 PMC Full Text View journal details
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