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Structural analysis of Stc1 provides insights into the coupling of RNAi and chromatin modification.

ABSTRACT: Noncoding RNAs can modulate gene expression by directing modifications to histones that alter chromatin structure. In fission yeast, siRNAs produced via the RNAi pathway direct modifications associated with heterochromatin formation. siRNAs associate with the RNAi effector protein Argonaute 1 (Ago1), targeting the Ago1-containing RNA-induced transcriptional silencing (RITS) complex to homologous nascent transcripts. This promotes recruitment of the Clr4 complex (CLRC), which mediates methylation of histone H3 on lysine 9 (H3K9me) in cognate chromatin. A key question is how the RNAi and chromatin modification machineries are connected. Stc1 is a small protein recently shown to associate with both Ago1 and CLRC and to play a pivotal role in mediating the RNAi-dependent recruitment of CLRC to chromatin. To understand its mode of action, we have performed a detailed structural and functional analysis of the Stc1 protein. Our analyses reveal that the conserved N-terminal region of Stc1 represents an unusual tandem zinc finger domain, with similarities to common LIM domains but distinguished by a lack of preferred relative orientation of the two zinc fingers. We demonstrate that this tandem zinc finger domain is involved in binding Ago1, whereas the nonconserved C-terminal region mediates association with CLRC. These findings elucidate the molecular basis for the coupling of RNAi to chromatin modification in fission yeast.

SUBMITTER: He C

PROVIDER: S-EPMC3666757 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Structural analysis of Stc1 provides insights into the coupling of RNAi and chromatin modification.

He Chao C Pillai Sreerekha S SS Taglini Francesca F Li Fudong F Ruan Ke K Zhang Jiahai J Wu Jihui J Shi Yunyu Y Bayne Elizabeth H EH

Proceedings of the National Academy of Sciences of the United States of America 20130423 21

Noncoding RNAs can modulate gene expression by directing modifications to histones that alter chromatin structure. In fission yeast, siRNAs produced via the RNAi pathway direct modifications associated with heterochromatin formation. siRNAs associate with the RNAi effector protein Argonaute 1 (Ago1), targeting the Ago1-containing RNA-induced transcriptional silencing (RITS) complex to homologous nascent transcripts. This promotes recruitment of the Clr4 complex (CLRC), which mediates methylation ...[more]

PMID: 23613586

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Project description:Aggregation of neuronal protein ?-synuclein leads to the formation of amyloid fibrils, which are associated with the development of Parkinson's disease. The mechanism of ?-synuclein pathology is not fully understood and is a subject of active research in the field. To tackle this problem, the fusions of fluorescent proteins to ?-synuclein C-terminus are often used in cellular and animal studies. The effects induced by such ?-synuclein sequence extension on ?-synuclein aggregation propensity are, however, not systematically examined despite the evidence that the negatively charged C-terminus plays a critical role in the regulation of ?-synuclein aggregation. In this work, we investigated how the charge and length variations of the C-terminus affect the aggregation propensity of ?-synuclein. To address these questions, we prepared mutants of ?-synuclein carrying additional moieties of different charge and length at the protein C-terminus. We determined the rates of two different aggregation stages (primary nucleation and elongation) based on a thioflavin T kinetic assay. We observed that all mutants bearing neutrally charged moieties of different length fibrilized slower than wild-type ?-synuclein. The primary nucleation and elongation rates strongly decreased with increase of the C-terminal extension length. Meanwhile, charge variation of the C-terminus significantly changed the rate of ?-synuclein nucleation, but did not markedly affect the rate of fibril elongation. Our data demonstrate that both the charge and length of the C-terminus play an important role at the stage of initial fibril formation, but the stage of fibril elongation is affected mainly by the length of C-terminal extension. In addition, our results suggest that there are at least two steps of incorporation of ?-synuclein monomers into the amyloid fibril: namely, the initial monomer binding to the fibril end (charge-dependent, relatively fast), and the subsequent conformational change of the protein (charge-independent, relatively slow, and thus the rate-limiting step).

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Structural analysis of Stc1 provides insights into the coupling of RNAi and chromatin modification.

Publications

Structural analysis of Stc1 provides insights into the coupling of RNAi and chromatin modification.

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