Argonaute Proteins Affect siRNA Levels and Accumulation of a Novel Extrachromosomal DNA from the Dictyostelium Retrotransposon DIRS-1

• Published in: The Journal of biological chemistry Vol. 289; no. 51; pp. 35124 - 35138
• Main Authors: Boesler, Benjamin, Meier, Doreen, Förstner, Konrad U., Friedrich, Michael, Hammann, Christian, Sharma, Cynthia M., Nellen, Wolfgang
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 19.12.2014; American Society for Biochemistry and Molecular Biology
• Subjects: Argonaute; Argonaute Proteins - genetics; Argonaute Proteins - metabolism; Blotting, Western; Dictyostelium - genetics; Dictyostelium - metabolism; DNA, Protozoan - genetics; DNA, Protozoan - metabolism; DNA, Single-Stranded - genetics; DNA, Single-Stranded - metabolism; Gene Deletion; Gene Expression; Gene Regulation; Gene Silencing; Mutation; Protozoan Proteins - genetics; Protozoan Proteins - metabolism; Retroelements - genetics; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA Interference (RNAi); RNA, Small Interfering - genetics; RNA, Small Interfering - metabolism; Small Interfering RNA (siRNA); Transformation, Genetic; Transposable Element (TE); Transposable Element (TE); Small Interfering RNA (siRNA); RNA Interference (RNAi); Argonaute; Gene Silencing
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.612663

The retrotransposon DIRS-1 is the most abundant retroelement in Dictyostelium discoideum and constitutes the pericentromeric heterochromatin of the six chromosomes in D. discoideum. The vast majority of cellular siRNAs is derived from DIRS-1, suggesting that the element is controlled by RNAi-related mechanisms. We investigated the role of two of the five Argonaute proteins of D. discoideum, AgnA and AgnB, in DIRS-1 silencing. Deletion of agnA resulted in the accumulation of DIRS-1 transcripts, the expression of DIRS-1-encoded proteins, and the loss of most DIRS-1-derived secondary siRNAs. Simultaneously, extrachromosomal single-stranded DIRS-1 DNA accumulated in the cytoplasm of agnA− strains. These DNA molecules appear to be products of reverse transcription and thus could represent intermediate structures before transposition. We further show that transitivity of endogenous siRNAs is impaired in agnA− strains. The deletion of agnB alone had no strong effect on DIRS-1 transposon regulation. However, in agnA−/agnB− double mutant strains strongly reduced accumulation of extrachromosomal DNA compared with the single agnA− strains was observed. Background: Retroelements are frequently under stringent control by RNAi mechanisms. Results: Disruption of the Argonaut AgnA in Dictyostelium leads to loss of retroelement siRNAs, retroelement-encoded proteins, and accumulation of a cytoplasmic cDNA that is abolished with additional deletion of AgnB. Conclusion: Two Argonautes with different functions are involved in retroelement regulation. Significance: AgnA is required to minimize retroelement expression.