The Bombyx ovary-derived cell line endogenously expresses PIWI/PIWI-interacting RNA complexes

Abstract

Genetic studies and large-scale sequencing experiments have revealed that the PIWI subfamily proteins and PIWI-interacting RNAs (piRNAs) play an important role in germ line development and transposon control. Biochemical studies in vitro have greatly contributed to the understanding of small interfering RNA (siRNA) and microRNA (miRNA) pathways. However, in vitro analyses of the piRNA pathway have been thus far quite challenging, because their expression is largely restricted to the germ line. Here we report that Bombyx mori ovary-derived cultured cell line, BmN4, endogenously expresses two PIWI subfamily proteins, silkworm Piwi (Siwi) and Ago3 (BmAgo3), and piRNAs associated with them. Siwi-bound piRNAs have a strong bias for uridine at their 59 end and BmAgo3-bound piRNAs are enriched for adenine at position 10. In addition, Siwi preferentially binds antisense piRNAs, whereas BmAgo3 binds sense piRNAs. Moreover, we identified many pairs in which Siwi-bound antisense and BmAgo3-bound sense piRNAs are overlapped by precisely 10 nt at their 59 ends. These signatures are known to be important for secondary piRNA biogenesis in other organisms. Taken together, BmN4 is a unique cell line in which both primary and secondary steps of piRNA biogenesis pathways are active. This cell line would provide useful tools for analysis of piRNA biogenesis and function.

The MID-PIWI module of Piwi proteins specifies nucleotide- and strand-biases of piRNAs

“…Another complex aspect within piRNA populations is the preferential incorporation of piRNAs with specific transposon strand-orientations into distinct Piwi proteins. This is exemplified by Bombyx Siwi and Ago3 expressed in BmN4 cells (Kawaoka et al 2009). Siwi incorporates primary piRNAs with a 1U-bias (∼80%) that are predominantly antisense in orientation to the transposon consensus sequences.…”

“…Finally, given a chance to bind RNAs containing any of the 4 nt at the 5 ′ end, some Piwi proteins prefer to enrich only those carrying a 5 ′ U. This specificity is evident in the case of the two Piwi proteins expressed in the Bombyx mori (silkworm) ovary-derived BmN4 cells that participate in secondary processing via the Ping-pong cycle (Kawaoka et al 2009). Within BmN4 cells, Siwi binds primary piRNAs with a 1U-bias, while Ago3 has secondary piRNAs lacking such a preference.…”

Abundant primary piRNAs, endo-siRNAs, and microRNAs in a Drosophila ovary cell line

“…While this paper was in review, Kawaoka et al (2009) described an ovary-derived cell line from Bombyx mori with endogenous expression of piRNAs and Piwi-class proteins.…”

Sex hormone-dependent tRNA halves enhance cell proliferation in breast and prostate cancers

“…In the course of investigating PIWI-interacting RNAs (piRNAs), a germ-line-specific class of small regulatory RNAs (18), we serendipitously detected the expression of tRNA halves in BmN4 cells. BmN4 cells are Bombyx mori ovary-derived cultured germ cells that endogenously express piRNAs and their bound PIWI proteins; therefore, they present a unique model system for piRNA research (19,20). When we examined the expression of piRNA-a, an abundant piRNA expressed in BmN4 cells (20) (DNA Databank of Japan no.…”