Developmentally synchronized expression of two Bombyx mori Piwi subfamily genes, SIWI and BmAGO3 in germ-line cells

Kawaoka, Shinpei; Minami, Kosuke; Katsuma, Susumu; Mita, Kazuei; Shimada, Toru

doi:10.1016/j.bbrc.2008.01.013

Cited by 61 publications

(75 citation statements)

References 23 publications

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“…Reverse transcription for mRNAs and the following quantitative PCR analyses were performed essentially as described previously (Kawaoka et al 2008 piRNA library construction and bioinformatic analyses piRNA libraries were constructed as described previously (Kawaoka et al 2009). To analyze the change of piRNA abundance by 17-AAG, we used the number of reads matching 5S ribosomal RNA fragments for normalization because they were relatively unsusceptible to 17-AAG treatment; miRNAs were unsuitable for normalization purpose, since Hsp90 plays a critical role in their biogenesis (Iwasaki et al 2010;Johnston et al 2010).…”

Section: Western Blottingmentioning

confidence: 99%

Hsp90 facilitates accurate loading of precursor piRNAs into PIWI proteins

Izumi

Kawaoka

Yasuhara

et al. 2013

RNA

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PIWI-interacting RNAs (piRNAs) defend the genome against transposon activity in animal gonads. The Hsp90 chaperone machinery has been implicated in the piRNA pathway, but its exact role remains obscure. Here, we examined the effect of 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), an Hsp90-specific inhibitor, on the piRNA pathway. In the silkworm ovary-derived BmN4 cells, 17-AAG treatment reduced the level of piRNAs and PIWI proteins. In vitro, the 5 ′ -nucleotide preference upon precursor piRNA loading was compromised by 17-AAG, whereas 3 ′ -end trimming and 2 ′ -O-methylation were unaffected. Our data highlight a role of Hsp90 in accurate loading of precursor piRNAs into PIWI proteins.

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Section: Western Blottingmentioning

confidence: 99%

Hsp90 facilitates accurate loading of precursor piRNAs into PIWI proteins

Izumi

Kawaoka

Yasuhara

et al. 2013

RNA

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“…The piRNA biogenesis initiates with fragmentation of putative long, single-stranded piRNA precursors. Resulting RNA fragments, perhaps longer than mature piRNAs, are incorporated into a subset of PIWI proteins, Siwi in silkworm (Kawaoka et al 2008a), with a specific nucleotide preference for uracil (1U) at the 59 end of associated RNAs (Brennecke et al 2007;Gunawardane et al 2007;Girard and Hannon 2008;Klattenhoff and Theurkauf 2008;Ghildiyal and Zamore 2009;Kawaoka et al 2009Kawaoka et al , 2011a. 39 ends of PIWI-associated RNAs are further trimmed by a 39 to 59 exonuclease named Trimmer (Kawaoka et al 2011a) to the mature length, followed by 29-O-methylation catalyzed by Hen1 (Horwich et al 2007;Houwing et al 2007; Kirino and Mourelatos 2007;Ohara et al 2007;Saito et al 2007;Kawaoka et al 2011a).…”

Section: Introductionmentioning

confidence: 99%

“…PIWI and primary piRNA complexes then cleave their complementary targets across from positions 10 and 11 from the guide piRNAs (Brennecke et al 2007;Gunawardane et al 2007). 39 fragments of cleavage products are then incorporated into another subset of PIWI proteins, BmAgo3 in silkworm (Kawaoka et al 2008a), which do not show a firstnucleotide bias, and again processed into mature secondary piRNAs with adenine at the position 10 (10A), overlapping with primary 1U piRNAs precisely by 10 nt (Brennecke et al 2007;Gunawardane et al 2007;Girard and Hannon 2008;Klattenhoff and Theurkauf 2008;Ghildiyal and Zamore 2009;Kawaoka et al 2009Kawaoka et al , 2011a. These secondary 10A piRNAs can in turn generate secondary 1U piRNAs-in theory indistinguishable from primary 1U piRNAs-by cleaving their complementary targets.…”

Section: Introductionmentioning

confidence: 99%

A role for transcription from a piRNA cluster in de novo piRNA production

Kawaoka¹,

Mitsutake²,

Kiuchi³

et al. 2011

RNA

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PIWI-interacting RNAs (piRNAs) are at the heart of the nucleic acid-based adaptive immune system against transposons in animal gonads. To date, how the piRNA pathway senses an element as a substrate and how de novo piRNA production is initiated remain elusive. Here, by utilizing a GFP transgene, we screened and obtained clonal silkworm BmN4 cell lines producing massively amplified GFP-derived piRNAs capable of silencing GFP in trans. In multiple independent cell lines where GFP expression was silenced by the piRNA pathway, we detected a common transcript from an endogenous piRNA cluster, in which a part of the cluster is uniquely fused with an antisense GFP sequence. Bioinformatic analyses suggest that the fusion transcript is a source of GFP primary piRNAs. Our data implicate a role for transcription from a piRNA cluster in initiating de novo piRNA production against a new insertion.

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“…4). We have previously found a rapid decrease of PIWI mRNAs at 20 hpf (Kawaoka et al 2008b), whereas protein expressions did not alter until 96 hpf during embryogensis. Collectively, we suggest that PIWI/piRNA complexes are highly stable during embryogenesis.…”

Section: Discussionmentioning

confidence: 78%

“…We revealed by Western blotting that Siwi and BmAgo3, two silkworm PIWI proteins (Kawaoka et al 2008b) were detected before zygotic activation (Fig. 1B).…”

Section: Maternal Deposition Of Piwi/pirna Complexes In Silkworm Embryosmentioning

confidence: 95%

Zygotic amplification of secondary piRNAs during silkworm embryogenesis

Kawaoka¹,

Kadota²,

Suzuki³

et al. 2011

RNA

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PIWI-interacting RNAs (piRNAs) are 23-30-nucleotide-long small RNAs that act as sequence-specific silencers of transposable elements in animal gonads. In flies, genetics and deep sequencing data have led to a hypothesis for piRNA biogenesis called the ping-pong cycle, where antisense primary piRNAs initiate an amplification loop to generate sense secondary piRNAs. However, to date, the process of the ping-pong cycle has never been monitored at work. Here, by large-scale profiling of piRNAs from silkworm ovary and embryos of different developmental stages, we demonstrate that maternally inherited antisense-biased piRNAs trigger acute amplification of secondary sense piRNA production in zygotes, at a time coinciding with zygotic transcription of sense transposon mRNAs. These results provide on-site evidence for the ping-pong cycle.

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Developmentally synchronized expression of two Bombyx mori Piwi subfamily genes, SIWI and BmAGO3 in germ-line cells

Cited by 61 publications

References 23 publications

Hsp90 facilitates accurate loading of precursor piRNAs into PIWI proteins

Hsp90 facilitates accurate loading of precursor piRNAs into PIWI proteins

A role for transcription from a piRNA cluster in de novo piRNA production

Zygotic amplification of secondary piRNAs during silkworm embryogenesis

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