Effects of Dicer and Argonaute down-regulation on mRNA levels in human HEK293 cells.
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.
Schmitter
Daniela
D
Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4002 Basel, Switzerland.
Filkowski
Jody
J
Sewer
Alain
A
Pillai
Ramesh S
RS
Oakeley
Edward J
EJ
Zavolan
Mihaela
M
Svoboda
Petr
P
Filipowicz
Witold
W
D020413:3' Untranslated Regions / Q000737:chemistry; D060565:Argonaute Proteins; D002460:Cell Line; D015536:Down-Regulation; D015852:Eukaryotic Initiation Factor-2; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D010448:Peptide Initiation Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D034622:RNA Interference; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D043244:Ribonuclease III / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*
D020413:3' Untranslated Regions; C466422:AGO2 protein, human; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; D035683:MicroRNAs; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; C487931:mirnlet7 microRNA, human; D043244:Ribonuclease III