Coupling of double-stranded RNA synthesis and siRNA generation in fission yeast RNAi.
The fission yeast centromeric repeats are transcribed and ultimately processed into small interfering RNAs (siRNAs) that are required for heterochromatin formation. siRNA generation requires dsRNA synthesis by the RNA-directed RNA polymerase complex (RDRC) and processing by the Dicer ribonuclease. Here we show that Dcr1, the fission yeast Dicer, is physically associated with RDRC. Dcr1 generates siRNAs in an ATP-dependent manner that requires its conserved N-terminal helicase domain. Furthermore, C-terminal truncations of Dcr1 that abolish its interaction with RDRC, but can generate siRNA in vitro, abolish siRNA generation and heterochromatic gene silencing in vivo. Finally, reconstitution experiments show that the association of Dcr1 with RDRC strongly stimulates the dsRNA synthesis activity of RDRC. Our results suggest that heterochromatic dsRNA synthesis and siRNA generation are physically coupled processes. This coupling has implications for cis-restriction of siRNA-mediated heterochromatin assembly and for mechanisms that give rise to siRNA strand polarity.
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Serafin U
SU
Colmenares
Shane M
SM
Buker
Marc
M
Buhler
Mensur
M
Dlakić
Danesh
D
Moazed
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002503:Centromere / Q000378:metabolism; D042002:Chromatin Assembly and Disassembly / Q000502:physiology*; D020090:Chromosome Segregation / Q000235:genetics / Q000502:physiology; D006570:Heterochromatin / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D034622:RNA Interference / Q000502:physiology*; D012330:RNA, Double-Stranded / Q000502:physiology*; D034741:RNA, Small Interfering / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D006570:Heterochromatin; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D029702:Schizosaccharomyces pombe Proteins; D000255:Adenosine Triphosphate; D012324:RNA-Dependent RNA Polymerase; D000251:Adenosine Triphosphatases; C416926:Rdp1 protein, S pombe