A Slicer-Mediated Mechanism for Repeat-Associated siRNA 5' End Formation in Drosophila

Abstract

In Drosophila, repeat-associated small interfering RNAs (rasiRNAs) are produced in the germ line by a Dicer-independent pathway and function through the PIWI subfamily of Argonautes to ensure silencing of retrotransposons. We sequenced small RNAs associated with the PIWI subfamily member AGO3. Although other members of PIWI, Aubergine (Aub) and Piwi, associated with rasiRNAs derived mainly from the antisense strand of retrotransposons, AGO3-associated rasiRNAs arose mainly from the sense strand. Aub- and Piwi-associated rasiRNAs showed a strong preference for uracil at their 5' ends, and AGO3-associated rasiRNAs showed a strong preference for adenine at nucleotide 10. Comparisons between AGO3- and Aub-associated rasiRNAs revealed pairs of rasiRNAs showing complementarities in their first 10 nucleotides. Aub and AGO3 exhibited Slicer activity in vitro. These data support a model in which formation of a 5' terminus within rasiRNA precursors is guided by rasiRNAs originating from transcripts of the other strand in concert with the Slicer activity of PIWI.

From guide to target: molecular insights into eukaryotic RNA-interference machinery

“…As a slicer, Piwi can cleave targets [107,108]. Subsequently, the RNA products of this reaction may be loaded into the complementary Piwi-clade protein Ago3 for ping-pong amplification [107] (Box 1).…”

“…As a slicer, Piwi can cleave targets [107,108]. Subsequently, the RNA products of this reaction may be loaded into the complementary Piwi-clade protein Ago3 for ping-pong amplification [107] (Box 1). Piwi also appears to initiate transcriptional gene silencing (TGS) by shuttling to the nucleus, recognizing transposon transcripts, and recruiting DNA [109,110] and/or chromatin modifiers, [111,112] which repress transcription of transposon loci (Box 2).…”

“…In the secondary piRNA pathway, transcripts arising from bidirectional transcription are exported from the nucleus with the aid of factors such as UAP56 (blue) and directed to the primary piRNA processing machinery. An important adaptation is the utilization of a signal amplification loop, the ping-pong cycle [41,107], in which pairs of Piwi proteins work in concert to adaptively boost the number of loaded piRNAs. In insects, primary piRNAs are loaded into either Piwi or Aubergine (Aub).…”

Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems

“…The Piwi subfamily is involved in germline-specific events such as stem cell maintenance and meiosis, and is responsible for maintaining incomplete differentiation in stem cells and stable rates of cell division in germ line cells. Piwi proteins are well-conserved, and are present in all studied animals [60].…”

“…PiRNAs are the small RNA guides of Piwi proteins, and do not have any distinct characteristics beyond a uridine at their 5 end and a high likelihood of an adenosine as the tenth nucleotide [60]. While other small RNAs like miRNAs and siRNAs are created from double-stranded hairpin RNA or other dsRNAs after cleavage by the Dicer enzyme, it is believed that piRNA is produced from long polycistronic RNA transcripts via an unknown Dicer-independent mechanism.…”