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Sequence determinants of dsRNA processing by DICER [Massively Parallel Assay]

ABSTRACT: Sequence determinants of dsRNA processing by DICER [Massively Parallel Assay]

PROVIDER: PRJNA836500 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR19143014.fastq.gz	Fastqsanger.gz
	SRR19143015.fastq.gz	Fastqsanger.gz
	SRR19143016.fastq.gz	Fastqsanger.gz
	SRR19143017.fastq.gz	Fastqsanger.gz
	SRR19143018.fastq.gz	Fastqsanger.gz

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Sequence determinants of dsRNA processing by DICER [Massively Parallel Assay]

Project description:RNA silencing relies on specific and efficient processing of dsRNA by DICER which produces microRNAs (miRNAs) and small interfering RNAs (siRNAs). However, our current knowledge of DICER’s specificity is restricted to the secondary structures of its substrates: a dsRNA than 22 bp with a 2-nt 3′ overhang. We recently found evidence pointing to additional sequence-dependent determinant(s) beyond these features. To systematically interrogate the features of precursor miRNAs (pre-miRNAs), we carried out massively parallel assays with over a million pre-miRNA variants. Our analyses revealed a highly conserved cis-acting element, termed the “GYM” motif (paired G, paired pyrimidine, and mismatched C or A) at the cleavage site, which strongly promotes processing at a specific position. We find that the C-terminal double-stranded RNA binding domain (dsRBD) of DICER recognizes the GYM motif. Mutation of the dsRBD alters pre-miRNA cleavage sites and impairs processing in a motif-dependent fashion, which in turn affects the miRNA repertoire in cells. Consistently, integrating the GYM motif into short hairpin RNA (shRNA) or Dicer substrate siRNA (DsiRNA) potentiates RNA interference.

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Project description:Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA silencing mechanism in many eukaryotes. In Drosophila, an RNase III enzyme Dicer-2 (Dcr-2), aided by its cofactor Loquacious-PD (Loqs-PD), plays major role in generating 21 base-pair (bp) siRNA duplexes from long double-stranded RNAs (dsRNAs). The ATP hydrolysis by the helicase domain of Dcr-2 is critical to the successful processing of a long dsRNA into consecutive siRNA duplexes. Here we report the cryo-EM structures of Dcr-2/Loqs-PD in the apo state and in multiple processing states of a 50-bp dsRNA substrate. The structures elucidated interactions between Dcr-2 and Loqs-PD, and significant conformational changes of Dcr-2 during a dsRNA processing cycle. The N-terminal helicase and DUF283 domains undergo conformational change upon initial dsRNA binding, forming an ATP binding pocket and a 5’-phosphate binding pocket. The overall conformation of Dcr-2/Loqs-PD is relatively rigid during translocating along the dsRNA in the presence of ATP, while the interaction between DUF283 and RIIIDb domains prevents non-specific cleavage during translocation by blocking the access of dsRNA to the RNase active center. Additional ATP-dependent conformational changes are required to form an active-dicing state and precisely cleave the dsRNA into 21-bp siRNA duplex confirmed by the structure in the post-dicing state. Collectively, this study revealed the molecular mechanism for the full-cycle of ATP-dependent dsRNA processing by Dcr-2/Loqs-PD.

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