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Drosophila melanogaster retrotransposon and inverted repeat-derived endogenous siRNAs are differentially processed in distinct cellular locations

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Since the discovery of endogenous small interfering (esiRNAs) in Drosophila, very little progress in understanding their biogenesis and molecular mechanisms of action has been made. Here we provide evidence that components of two major RNA processing pathways, 3’ end processing and esiRNA biogenesis, interact in Drosophila somatic cells, a connection not previously reported. Importantly, we also show that esiRNAs processed from retroTns have different physical characteristics than those generated from hairpins. Double-stranded retroTn RNAs are retained in the nucleus while Esi1/2 hps are exported to the cytoplasm. Together these data support a novel model in which retroTns and hps, both double stranded RNAs cleaved by Dcr2, are differentially processed in Drosophila somatic cells. This is the first evidence that precursor secondary structures potentially contribute to Dcr2 activity in vivo.

mRNA 3’ end processing performed by the CCC is co-transcriptional and therefore occurs in the nucleus [

47

,

48

]. The RNA pol II CTD phosphatase Ssu72 interacts with the N-terminal region of Symplekin to direct processing of mRNAs with a 3’ poly(A) tail [

49

] and with the stem loop binding protein for replication dependent histone mRNAs (Dan Michalski, data not shown). Here, we show that this N-terminal region of Symplekin can also interact with esiRNA processing factor Dcr2 (Fig. 

1

) in the nuclear compartment (Fig. 

3

), although, Dcr2 is not required for proper mRNA 3’ end formation (Fig. 

2

). The Symplekin C-terminal region binds CPSF73 and CPSF100 to form the CCC [

20

], therefore leaving the N-terminal region free to bridge the CCC and other cellular factors. While previous work shows that regulation of Tns by piRNAs in the

Drosophila

germline is a nuclear process [

50

54

] and researchers have documented a nuclear pool of Dcr2 that associates with heat shock loci and transcription machinery in

Drosophila

[

23

], potential nuclear functions of Dcr2 in

Drosophila

somatic cells have not been extensively investigated [

6

]. Our data support a model in which the N-terminal region of Symplekin mediates Dcr2-CCC complex formation, but only when the CCC is not actively engaged in co-transcriptional mRNA 3’ end processing (Fig. 

8

).

Fig. 8

EsiRNAs are differnentially processed in D. melanogaster cells. Data support a model in which double stranded retroTn transcripts are retained and processed to esiRNAs in the nucleus while RNAs containing inverted repeats are exported and processed in the cytoplasm. Dcr2 interacts with the N-terminal 271 amino acids of Symplekin in the nucleus, but not in the cytoplasm

To understand the functional implications of CCC-Dcr2 interactions, esiRNA and precursor levels were measured in Symplekin and CPSF73 RNAi-depleted samples. Globally, we observe increased levels of Tn-derived esiRNAs and decreased hp-derived esiRNAs in CCC factor knockdowns (Fig. 4). Examination of specific retroTns and Esi1/2 precursors reveals that changes in esiRNA levels correlate to shifts in precursor abundance in Symplekin and CPSF73 knockdowns (Fig. 4). As we hypothesize that dsRNA retroTn precursor levels are determined AS transcript abundance [17], more AS transcript would lead to an increase in Dcr2 substrates (and more retroTn-derived esiRNAs), while decreased AS transcript would result in less Dcr2 substrate. Esi1/2 precursors consist of only one S mRNA. Therefore, hp Dcr2 substrate concentration is determined by only CG44774 and CR18854 levels. Once again, the observed lower Esi1/2 esiRNA levels in CCC factor depleted cells correlate with decreased CG44774 and CR18854 transcripts in these samples (Fig. 4). These data support a model in which esiRNA levels are partially influenced by Dcr2 substrate concentration. Substrate levels are affected by CCC factor RNAi-depletion indicating that Symplekin and CPSF73 indirectly determine esiRNA abundance. Because Symplekin and CPSF73 RNAi-depleted samples follow the same trends, we concluded that the CCC is involved in this process.

While hp and retroTn esiRNA levels correlate to S and AS transcript abundance, the number of esiRNAs is always less than the Dcr2 substrate concentration in Symplekin and CPSF73 knockdowns indicating that additional mechanisms must be modulating esiRNA levels in these samples. Although, Dcr2 cleavage site selectivity is unaffected in CCC factor RNAi-depleted samples (Additional file 7), Dcr2 activity could be altered by interaction with the CCC. An additional hypothesis for the observed molecular phenotypes is inefficient nuclear export of retroTn and hp RNAs in Symplekin and CPSF73 RNAi-depleted samples. CCC component knockdowns cause global mRNA 3’ end processing defects (Fig. 2) and how this misprocessing affects cellular localization of retroTn dsRNAs is unknown. However, previous work shows that less polyadenylated RNAs are not effectively exported from the nucleus [55]. Additionally, 3’ end misprocessing of RNAs generated from the Esi2 locus (Fig. 5) might lead to changes in secondary structure that unpredictably affect nuclear export. Inefficient nuclear export of hp RNAs with modified 3’ ends might not change total precursor levels, but could result in less Esi2-derived esiRNAs since cytoplasmic hp precursor levels would be reduced. When Symplekin and CPSF73 are RNAi-depleted, both hp and retroTn dsRNAs are enriched in the nucleus (Fig. 7) supporting the hypothesis that non-polyadenylated RNAs are retained in the nucleus. Taken together, these data support a model in which the CCC indirectly affects the abundance of retroTn- and hp-derived esiRNAs by modulating cellular localization and concentration of Dcr2 substrates (Fig. 8).

Bioinformatic analyses of retroTn- and hp-derived esiRNAs reveals physical distinctions between these groups (Fig. 6). Additionally, retroTn precursors and their corrosponding esiRNAs are highly enriched in the nucleus while hp dsRNAs and their corresponding esiRNAs are cytoplasmic similar to mRNAs (Fig. 7). We hypothesize that these observed disparities are directly related to distinct differences in secondary structures (Fig. 5) and compartmentalization of esiRNA biogenesis factors required to process each structure. dsRNAs derived from S and AS transcription of retroTns [17] generally result in fully complementary, blunt-ended dsRNAs as many AS retroTn transcripts are poorly polyadenylated [17]. The secondary structures of hps containing multiple inverted repeats are likely variable and complex with frayed ends (Fig. 5). Previous in vitro assays suggest that Dcr2 alone can bind and processively cleave blunt dsRNAs. However, Dcr2 requires a co-factor, Loqs-PD, to process dsRNAs with frayed termini presumably because Loqs-PD allows Dcr2 to bind a substrate internally [56]; Loqs-PD is cytoplasmic in Drosophila culture cells [57]. Taken together, these data suggest a model in which nuclear retained blunt-ended, fully complementary retroTn precursors can be processed in the nucleus by Dcr2 alone while more complicated hp precursors requiring Loqs-PD are cleaved in the cytoplasm by Dcr2 (Fig. 8). This model is supported by our observations that esiRNAs map the entire length of retroTns (Fig. 3d). Additionally, previous work shows that R2D2 and Dcr2 aggregate in cytoplasmic D2 bodies together with hps [27].

This model predicts that depletion of Loqs-PD would only affect cleavage of hps precursors and the levels of hp-derived esiRNAs, but not esiRNAs generated from retroTns. Zhou et al. previously reported that depletion of Loqs isoforms reduced the number of esiRNAs derived from both hps and Tns [58]; however, close examination of the data reveal that retroTn-mapping esiRNAs were unaffected by Loqs knockdown. The most notably affected Tn, Proto-P, is not regulated by the esiRNA pathway [59].

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