An alpha motif at Tas3 C terminus mediates RITS cis spreading and promotes heterochromatic gene silencing.
RNA interference (RNAi) plays a pivotal role in the formation of heterochromatin at the fission yeast centromeres. The RNA-induced transcriptional silencing (RITS) complex, composed of heterochromatic small interfering RNAs (siRNAs), the siRNA-binding protein Ago1, the chromodomain protein Chp1, and the Ago1/Chp1-interacting protein Tas3, provides a physical tether between the RNAi and heterochromatin assembly pathways. Here, we report the structural and functional characterization of a C-terminal Tas3 alpha-helical motif (TAM), which self-associates into a helical polymer and is required for cis spreading of RITS in centromeric DNA regions. Site-directed mutations of key residues within the hydrophobic monomer-monomer interface disrupt Tas3-TAM polymeric self-association in vitro and result in loss of gene silencing, spreading of RITS, and a dramatic reduction in centromeric siRNAs in vivo. These results demonstrate that, in addition to the chromodomain of Chp1 and siRNA-loaded Ago1, Tas3 self-association is required for RITS spreading and efficient heterochromatic gene silencing at centromeric repeat regions.
Li
Haitao
H
Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
Motamedi
Mohammad R
MR
Yip
Calvin K
CK
Wang
Zhanxin
Z
Walz
Thomas
T
Patel
Dinshaw J
DJ
Moazed
Danesh
D
D020816:Amino Acid Motifs; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D002503:Centromere / Q000235:genetics / Q000378:metabolism; D042002:Chromatin Assembly and Disassembly; D047369:Chromatin Immunoprecipitation; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D020868:Gene Silencing / Q000502:physiology*; D006570:Heterochromatin / Q000235:genetics*; D006657:Histones / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D008745:Methylation; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000378:metabolism* / Q000502:physiology; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D002352:Carrier Proteins; D006570:Heterochromatin; D006657:Histones; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; D029702:Schizosaccharomyces pombe Proteins; C499720:Tas3 protein, S pombe; D008239:Lysine