Abstract
BackgroundRNA silencing is a common term for pathways utilizing small RNAs as sequence-specific guides to repress gene expression. Components of the RNA silencing machinery are involved in different aspects of chromatin function in numerous organisms. However, association of RNA silencing with chromatin in mammalian cells remains unclear.Methodology/Principal FindingsImmunostaining of mitotic chromosomes with antibodies visualizing either endogenous or ectopically expressed Dicer in mammalian cells revealed association of the protein with ribosomal DNA (rDNA) repeats. Chromatin immunoprecipitations and bisulfite sequencing experiments indicated that Dicer is associated with transcribed regions of both active and silenced genes in rDNA arrays of interphase chromosomes. Metabolic labeling of the mouse embryonic stem (ES) cells lacking Dicer did not reveal apparent defect in rRNA biogenesis though pre-rRNA synthesis in these cells was decreased, likely as a consequence of their slower growth caused by the loss of miRNAs. We analyzed in detail chromatin structure of rDNA but did not find any epigenetic changes at rDNA loci in Dicer−/− ES cells. Instead, we found that rDNA methylation is rather low in primary tissues, contrasting with rDNA methylation patterns in transformed cell lines.Conclusion/SignificanceWe found that Dicer, a key component of RNA silencing pathways, can be detected in association with rDNA chromatin in mammalian cells. The role of this particular localization of Dicer is not readily apparent since the enzyme is associated with rDNA genes regardless of their transcriptional activity. However, localization of Dicer to the transcribed region suggests that transcription may contribute to the Dicer deposition at rDNA chromatin. We hypothesize that Dicer functions in maintaining integrity of rDNA arrays.
Highlights
RNA interference (RNAi) and microRNA pathways represent RNA silencing mechanisms utilizing short RNA molecules, produced by RNAse III family enzyme Dicer, to guide sequence-specific silencing of gene expression
To verify that Dicer is localized at ribosomal DNA (rDNA) loci, we co-stained mitotic chromosomes with antibodies against UBF, an RNA polymerase I factor involved in ribosomal RNA (rRNA) transcription [16]
We have found that mammalian Dicer is physically associated with rDNA chromosomal in mammalian cells
Summary
RNA interference (RNAi) and microRNA (miRNA) pathways represent RNA silencing mechanisms utilizing short RNA molecules, produced by RNAse III family enzyme Dicer, to guide sequence-specific silencing of gene expression. Components of RNA silencing were implicated in transcriptional silencing and heterochromatin in animals but, unlike in plants and S. pombe, the mechanistic aspects of the connection remain unclear [3]. Mammalian Dicer was implicated in the formation of centromeric heterochromatin [7,8], and in regulation of intergenic transcription at the human and chicken b-globin locus [9,10]. RNA silencing is a common term for pathways utilizing small RNAs as sequence-specific guides to repress gene expression. Association of RNA silencing with chromatin in mammalian cells remains unclear
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