Drosophila exoribonuclease nibbler is a tumor suppressor, acts within the RNAi machinery and is not enriched in the nuage during early oogenesis
© The Author(s) 2017
Received: 26 July 2017
Accepted: 11 September 2017
Published: 29 September 2017
micro RNAs (miRNAs) are important regulators of many biological pathways. A plethora of steps are required to form, from a precursor, the mature miRNA that eventually acts on its target RNA to repress its expression or to inhibit translation. Recently, Drosophila nibbler (nbr) has been shown to be an important player in the maturation process of miRNA and piRNA. Nbr is an exoribonuclease which helps to shape the 3′ end of miRNAs by trimming the 3′ overhang to a final length.
In contrast to previous reports on the localization of Nbr, we report that 1) Nbr is expressed only during a short time of oogenesis and appears ubiquitously localized within oocytes, and that 2) Nbr was is not enriched in the nuage where it was shown to be involved in piwi-mediated mechanisms. To date, there is little information available on the function of nbr for cellular and developmental processes. Due to the fact that nbr mutants are viable with minor deleterious effects, we used the GAL4/UAS over-expression system to define novel functions of nbr. We disclose hitherto unknown functions of nbr 1) as a tumor suppressor and 2) as a suppressor of RNAi. Finally, we confirm that nbr is a suppressor of transposon activity.
Our data suggest that nbr exerts much more widespread functions than previously reported from trimming 3′ ends of miRNAs only.
In eukaryotes, three main RNAi pathways have received considerable attention in the past: microRNAs (miRNAs), small interfering (siRNAs) and Piwi-interacting RNAs (piRNAs) . All three pathways reveal difference in their biogenesis, type of Argonaute family proteins, mode of target regulation and substrates . The RNAi machinery and mechanisms associated with it are evolutionarily conserved in most eukaryotic organisms, including insects .
During the last decade, microRNAs (miRNAs) were found to be important regulators of development, pathology and physiology of plant, as well as humans (reviewed by [22, 48]. Despite their small size of about 22 nucleotide (nt), these RNA molecules exert complex functions by binding preferentially to the 3′ untranslated region (UTR) of target RNAs to block their function. miRNAs are initially synthesized by RNA polymerase II to yield a precursor miRNA of about 70 nt length which are 5′ capped and which are also 3′ polyadenylated which subsequently folds into a structure with a partially-paired stem, a single-stranded loop and a 2 nucleotide 3′ overhang. These 3 features are characteristic for the primary miRNA.
As a next step, these primary miRNA are exported from the nucleus to the cytoplasm. There, Dicer, a RNase III processes the pre-miRNA to a 22 nt mature miRNA/miRNA* duplex [20, 27]. Subsequently, miRNA duplexes are assembled in a complex with Argonaute (Ago) to form the precursor RNA-induced silencing complex (RISC) . This complex formation appears to be uncoupled from the synthesis of the miRNA. It is also within the RISC complex where one of the strands is chosen as the active silencing complex. Finally, the active miRNA within RISC binds preferentially to the 3′ UTR of their target mRNAs which leads either to repression of the transcription or by inhibiting its translation .
When measuring the length of the end of the 3′ overhangs, it was noted that there was an unusually high heterogeneity within the per-miRNA molecules which was ascribed to a sloppy mode of action of RNase III. This necessitated to postulate the presence of yet another enzyme that would account for the precise outcome of the 2 nt overhang. In 2011, two groups presented Nibbler (Nbr) protein in Drosophila as a candidate enzyme belonging to the class of exoribonucleases which likely represented the missing link [23, 32]. in vitro assays showed that Nbr trims many miRNAs to a 22 nt product, when bound to Ago , exemplified on a preferred target, miR-34 [23, 32] which itself exists in several isoforms. In absence of nbr, all smaller isoforms of miR-34 are lost , indicating that Nbr has a specific function on trimming miR-34, but it would not exclude that nbr would have a broader set of targets. Interestingly, Nbr was predicted to contain no RNA-binding activity, therefore, it was suggested that Ago would exert this job, and only the binding of Nbr to Ago in an complex would allow to act on miRNAs. nbr flies were first reported to be semi-lethal and sterile [23, 32]. Later nbr flies were found to be viable, but showing accelerated age-related effects [15, 24, 49].
Recently, research on the Piwi protein, a protein functionally and structurally close to Ago, and the associated piwi pathway furthered our understanding on the mechanisms of the biogenesis of small interference RNAs . The piwi pathway and the associated piRNAs have mainly been studied in Drosophila. piRNAs are 23–29-nt small RNAs expressed predominantly in the oocyte [6, 34]. Concomitantly, piRNAs were discovered as master regulators to repress transposable elements (TEs) in Drosophila as well as in mice, rats, nematodes, and zebra fish [4, 19, 21, 31, 37, 39, 47]. It appears that there are thousands of distinct piRNA sequences present in the genomes of Drosophila . To date, no structural or sequence similarity between the sequences of different piRNAs was found, except for a stronger bias for uracil in the first nucleotide . In Drosophila, piRNAs recognize their targets, which predominantly are mRNAs of TEs through perfect or nearly perfect antisense matching. Hence, interfering with the piwi system may change the activity of transposon which may have deleterious effects on the organisms. piRNAs undergo several steps of maturation including formation of the primary piRNAs which are loaded onto Piwi . As a further maturation process, the “Ping-Pong” cycle, reported to occur in the nuage of Drosophila germ cells , amplifies secondary piRNAs and thereby silences targets [12, 43]. Due to the amplification of the piRNAs, it is thought that the process consumes transcripts of TEs, thereby leading to a silencing of TEs. Conversely, interfering with the Ping-Pong cycle has likely the opposite effect, i. e. TE transcripts are present at unusually high levels. This in turn increases the probability of TEs to insert into developmentally-important genes or tumor-suppressor genes which may have deleterious effects such as generating cancer in tissues which otherwise would not happen if the regulation of TE activity was in balance.
Given the importance of nbr, little information is available as to the overall function of nbr for development or cellular mechanisms in a broader context. We therefore thought to shed some light onto possible mechanisms. Instead of using classical mutants where information on the function is very limited, we used inducible nbr RNAi and dominant-negative versions of Nbr, and employed the GAL4/UAS over-expression system . Using these approaches, we disclose hitherto novel functions of nbr in (i) regulating TE activity, and in (ii) suppressing tumors. Moreover, we show that Nrb is expressed very early during oogenesis and that nbr is involved in regulating small interfering RNA (siRNA) activity. Taken together, our data suggests that nbr reveals a broader involvement in regulative cellular processes than just trimming specific miRNAs.
All transgenic stocks were obtained using conventional transformation techniques  and were maintained as balanced stocks. MS1096-GAL4, apterous-GAL4, tubulin-GAL4, paired-GAL4, en-GAL4 and UAS-nbr were obtained from the Bloomington stock center. UAS-Dg i flies were obtained from Martina Schneider .
Total RNA was isolated from ovaries or adult males and females using TRIzol (Invitrogen) and treated with DNase I (Ambion) to remove DNA. First strand cDNA was prepared using the SuperScript II reverse transcriptase kit (Invitrogen) according to the manufacturer’s instructions with 500 ng total RNA and 50 ng random hexamer primers in a 10 μl reaction.
The primers for amplification of copia were: (5′-ATTCAACCTACAAAAATAACG-3′) and (5′-ATTACGTTTAGCCTTGT-3′), producing a product of 438 bp. The primers for amplification of the control, the ribosomal protein-encoding gene rp 49 were: (5′-GACCATCCGCCCAGCATACAGGC-3′) and (5′-GAGAACGCAGGCGACCGTTGG-3′) producing a product of 388 bp. In order to compensate for the distinct abundance of transcripts, primers for copia were used at 200 nM and for rp49 at 40 nM.
In situ hybridization
Riboprobes were generated using a DIG-labeling kit (Roche). Two templates were amplified from cDNA and cloned into pBS (KS). The 5′ template including the signal peptide sequence of nbr was amplified with primers (5′-ATGGTACCTCGCAATGAGTGATTTAC-3′) and (5′-TATGGATCCTGCAGTTGGTTCTCTAGT-3′) generating a 467 base pair probe. The 3′ template from the cytoplasmic part of the gene was amplified with the primers, (5′-ACAAGTCGTCGTACAAGGA-3′) and (5′-GACCACCATTCTTGTTTGTAGGCA-3′) generating a 343 base pair probe. The procedure for in situ hybridization was carried out according to . A sense probe was used as a negative control.
Generation of antibodies and immunohistochemistry
A NH2-terminal peptide CNFDATLDAKAEEFFKLFREKWNM comprising aa 46-69 of Nbr was used to immunize a rabbit. Crude serum from the 2nd bleed was used in all experiments. For Drosophila whole-mount staining, a Nbr monoclonal antibody  was used at 1:100 and detected using 2nd antibodies coupled to Alexa 555. For all immunofluorescence pictures, a Zeiss LSM 710 was used. For super-resolution recording, an Airy-Scan (Zeiss™) assembly was used in combination with a 63× lens. For Western analysis, embryos from the cross of the paired (prd)-GAL4 > UAS-nbr strain (Bloomington stock #16587) were used for 4-8 h extracts which, together with a 4-8 h wild-type extract of similar protein concentration were separated on a 10% PAGE and probed with the Nbr antiserum at a 1:2000 dilution.
RNA interference in cells
S2 cells were propagated in 1× Schneider’s Drosophila medium (GIBCO), supplemented with 10% FBS, 50 units/ml penicillin, and 50 μg/ml streptomycin at 27 °C. dsRNAs were produced from amplified DNA templates using a MEGASCRIPT T7 transcription kit (Ambion) following the protocol of . DNA templates were amplified with primers containing a 5′ T7 RNA polymerase binding site (5′-TAATACGACTCACTATAGGGAGACCAC-3) followed by sequences specific for the targeted genes. The following primers were used: nbr (5′- TAATACGACTCACTATAGGGAGACCACaggagtgcgtcatatacctg-3′) and (5′- TAATACGACTCACTATAGGGAGACCACgcgttcaatgagcgtgttg-3′); GFP (5′- TAATACGACTCACTATAGGGAgaccaccctgacctacggc-3′) and (5′- TAATACGACTCACTATAGGGagaccacgaactccagcaggacc-3′) and mock-lacZ (5′- TAATACGACTCACTATAGG-3′) and (5′- TAATACGACTCACTATAGGGAGACCAccgaactgagatacctacagc-3′), amplifying the 838 bp sequence of the vector pBluescript SK downstream of the T7 RNA polymerase binding site that includes the lacZ alpha gene. dsRNA products were DNase- treated, ethanol-precipitated and resuspended in DEPC water. The dsRNAs were analyzed by agarose gel electrophoresis to ensure that single bands of the expected size were present. S2 cells were transfected using FuGENE 6 (Roche) in 3 cm dishes at 50%–70% confluence, following the manufacturer’s recommendation. The standard transfection reaction contained 2 μg of plasmid expressing GFP (pAC-EGFP), 2 μg of dsRNA targeting GFP and 2 μg of either nbr dsRNA or mock dsRNA.
Transgenic RNA interference construct
A 408 bp fragment at the 5′ of the nbr gene (Fig. 1c) was amplified from cDNA with primers 5′-TGGTACCAGTGATCTCAGTGTATTGCAG-3′ and 5′-CGGATCCTCAATCACTTAACATGGGCA -3′. After digestion with Kpn I/Bam HI, the fragment was subcloned both into pBluescript II (Stratagene) forming pKS-nbr and into pEGFP-N1 (Clontech), respectively. Inversion of the sequence was achieved by excision of the Nhe I/Bam HI fragment of the pEGFP-N1 construct and subsequent ligation with a 148 bp Sau3A linker, derived from digestion of pEGFP-N1 with Sau3A, into pKS-nbr, cut with Spe I/Bam HI. The 964 bp Kpn I fragment containing the inverse sequence separated by the linker was inserted into pUAST. Prior to transformation, the construct was verified by restriction analysis and sequencing.
Drosophila Nibbler belongs to the exonuclease D family
In search for 3′-5′ exonuclease domain-containing proteins within the Drosophila genome, we came across a transcript, originally termed CG9247 by the Drosophila sequencing consortium (BDGP; , which contained a typical 3′-5′ exonuclease domain found in many proteins from worms to humans (Fig. 1a), subsequently termed nibbler (nbr) [23, 32]. Analysis of the nbr open reading frame (ORF) revealed a protein of 625 amino acids with two domains shared by other proteins in the animal kingdom. At the amino terminus, there is no indication of a signal peptide indicative of a secreted or a transmembrane protein, suggesting that it is an intracellular protein. The first third of the protein (amino acids 1-181) does not contain any homology to any known protein, while the second third (amino acids 182-409) contains low homology to human protein FLJ20433 and C. elegans nuclease ZK1098.3. The last third of the protein contains the 3′-5′ exonuclease domain which was the initial searching bait. This domain is a widespread domain found in diverse proteins such as human Werner syndrome protein  or E. coli RNaseD . Due to the fact that human FLJ20433 and C. elegans ZK1098.3 contain a similar domain arrangement as CG9247 and because lengths of all three proteins are similar, we presume that these three proteins represent the respective nbr orthologues.
Analysis of the nbr 3′-5′ exonuclease domain revealed that the sequence homology is fairly good (Fig. 1b), in particular the absolutely preserved amino acids that are the typical characteristics of an exonuclease domain. This domain is part of a large DEDD subfamily of exoribonucleases , owing to the fact that they contain invariant acidic amino acids at certain position such as the aspartic acid D and the glutamic acid E within domain I (Fig. 1b) and two aspartic acids D in domain II and III, respectively (Fig. 1b). This DEDD subfamily includes the proof-reading domains of many DNA polymerases as well as other DNA exonucleases and shares a common catalytic mechanism characterized by the involvement of two metal ions . Of the different members of the DEDD subfamily, the Nbr protein resembles most the RNaseD proteins which further subdivide the DEDD subfamily into the DEDDy sub-subfamily , due to the presence of an invariant tyrosine Y within the catalytic domain III (Fig. 1b).
nbr exhibits nuclease activity
Localization of the nbr mRNA and protein
We employed in situ hybridization to detect the spatial transcript pattern of nbr in Drosophila whole mount embryos. nbr is expressed ubiquitously in the early developing embryo (Fig. 2a), suggesting a maternal deposition, also confirmed by FlyBase . At stage 5, i. e. at cellular blastoderm stage, nbr expression drops considerably, and transcripts are only detected ubiquitously at low level during the remaining embryonic stages (FlyBase, ; data not shown). To ease analysis of the localization and function of nbr, we constructed EGFP-Nbr flies under control of the GAL/UAS system that allows ectopic expression of any protein . To test the functionality of the EGFP-nbr-N transgene, we monitored the expression of the ectopic transcript driven by the paired (prd)-GAL4 driver using in situ hybridization. As is evident from Fig. 2b, the EGFP-nbr-N transgene is faithfully expressed in 7 stripes, compared to the low-level ubiquitous expression of the endogenous nbr transcripts.
To investigate the subcellular localization of the Nbr protein, we first analyzed the EGFP-Nbr wild-type fusion protein in salivary gland cells from third instar larvae using a tubulin (tub)-GAL4 driver line. As evident in Fig. 2c, fluorescence was detected at low levels in the cytoplasm and at a perinuclear localization, similar to the localization of Nbr in the nuage, as described by .
To monitor the subcellular expression, we stained Drosophila tissues using a monoclonal antibody against Nbr  along with a nuclear stain, DAPI. We first focused on oogenesis, since nbr was reported to play a critical role during this stage [15, 24]. We detected the protein during the earliest stages in the germarium and in stage 2 oocytes only (Fig. 3a), while all subsequent stages were devoid of Nbr. Using super-resolution microscopy aimed to detect Nbr localization subcellularly and in the nuage , the Nbr protein was detected uniformly in the cytoplasm of stage 2 oocytes (Fig. 3b). This localization data are inconsistent with the data of ) who reported staining in the nuage surrounding the early nurse cells, but otherwise devoid of any other cellular localization. Moreover, our subcellular Nbr localization data do not entirely fit the localization pattern of EGFP-Nrb (Fig. 2c), however, the data was recorded in two different tissues, the salivary gland vs. the oocyte. Moreover, both the data by  and the data of Fig. 2c were based on the use of GFP-Nbr constructs rather than true antibody detection. Hence, we propose that the Nbr-EGFP fusion proteins tend to accumulate in the nuage and thus lead to a misinterpretation of the location where Nbr acts. On the other hand, our antibody data fit another report that revealed Nbr ubiquitous expression in the cytoplasm of oocytes  using a Nbr-HA fusion protein and anti HA staining, hence the localization does not include an EGFP-tag, but rather a small and binding-neutral HA-tag. In summary, there are marked differences between the EGFP-Nbr localization and the true antibody staining which question the nuage staining by .
Later during oogenesis, i. e. during stage, Nrb was detected at low levels at the cortex of the oocyte (Fig. 3e) which persisted during later stages of oogenesis (data not shown). In freshly-laid embryos, the cortical pattern was particularly pronounced (Fig. 3h), due to the strong maternal loading of nbr mRNA (Flybase; . The cortical pattern persisted during early nuclear stages, including nc 11 embryos (Fig. 3i) when the majority of the nuclei have reached the periphery of the blastoderm. At cellular blastoderm, this cortical staining persisted (Fig. 3j), and Nrb was detected on the basal as well as on the apical side of the nuclei, however, at low levels, leaving the nuclei free of Nbr staining.
Down-regulation of the nbr nuclease activity can lead to tumor formation.
Knockdown of nbr reduces the effect of RNAi
Next, we pondered if nbr might be involved in mechanisms of the RNAi machinery. For this reason, we set up two parallel assay systems, a cell-culture based system and a transgenic fly approach to test if nbr is involved in RNAi. Drosophila Schneider cells S2 were transfected with a reporter plasmid driving EGFP by an actin promoter. In parallel, two RNA i , one against the RNA of EGFP and another one against the RNA of a mock gene, LacZ, were applied simultaneously.
Occurrence of posterior cross vein (PCV) in Dg knockdown flies, assayed together with 3 different genetic backgrounds
PCV < 5%
PCV < 50%
PCV > 50%
MS1096-GAL4 > Dg i /Y; ftl i /+
MS1096-GAL4 > Dg i /Y; 3505-2a i /+
MS1096-GAL4 > Dg i /Y; nbr i 37/+
nbr affects the levels of transposon RNA intermediates
Using in vivo studies and by exploiting the GAL4/UAS system in Drosophila, we have analyzed the function of the Nibbler protein for development and for cellular mechanisms. We have undisclosed novel functions of this protein which suggest more wide-spread functions than hitherto anticipated.
Data from Fig. 2 indicate that Nibbler possesses a general nuclease activity and is probably more widely involved in cellular activities than only involved in trimming small RNA ends [23, 32]. This result is not surprising, as the protein possesses an exonuclease domain, however, this report shows for the first time that Nbr shows a broader involvement in trimming mRNAs. Discussed as a possibility by  that Nbr affects the length of not only miRNAs, but also that of piRNAs, it was speculated that Nbr could potentially trim the 3′ ends of a much broader species of RNA substrates, including other long and short noncoding RNAs and mRNAs. However, presumed to be instructive for the piRNA pathway, nbr has received little attention in the context of general function of RNA trimming.
Nbr appears to control the expression levels of TEs, as exemplified by copia in Fig. 6. As noted by , copia expression is also increased in nbr mutants which is in line with our observation (Fig. 6) that nbr controls the activity of TEs. Whether the regulation is direct or indirect via the piwi pathway which is involved in regulating the levels of TEs in germ cells, in currently unknown, but we favor an involvement of the piwi pathway.
Our data on Fig. 4 indicated that knock-down of nbr provokes the formation of tumors. Our current hypothesis is that ablation of nbr increases the rate of transposition. In these cases, the piRNA pathway is probably not involved, as the pathway is restricted to germ cells and extremely little piwi expression was observed during larval stages (Flybase; . Instead, we envision that nbr is involved in the miRNA pathway, by controlling any of the multi-isoform miRNAs that are expressed during larval and pupal stage [23, 32]. These are then thought to control genes regulating cell-cycles or cell-cycle check points.
Since a while, it is known that miRNAs are involved in tumorigenesis, where the focus is mainly in humans [25, 30, 45]. To date, in Drosophila, only a handful of miRNA genes are known to be involved in the formation of cancer. One of them is the bantam gene, identified by a conventional gain of function screen which constitutes a miRNA gene that positively regulates cell proliferation and suppresses apoptosis – two features typical of oncogenes [5, 26]. However, with the advent of the availability of systematic studies by applying inducible Drosophila miRNA transgenes, scores of uncovered of surprisingly specific, dominant phenotypes were discovered [2, 16]. These surveys suggest that miRNA gain of function may generate diseases much more frequently than miRNA loss of function.
Our sensitive assay on Dg-RNAi-mediated depletion of the posterior cross vein (PCV) of Fig. 5c-e and Table 1 confirms a direct involvement of nbr in RNAi-mediated gene silencing. If nbr is reduced, the activity of the RNAi machinery is weakened and depletion of PCV structures is reduced substantially. Likewise, we could confirm the mechanistic action of nbr in cell culture assays which revealed that effect of RNAi was weakened when nbr activity was compromised (Fig. 5b). Hence, for the first time, we can demonstrate that nbr is involved in patterning processes involving whole tissues. Moreover, our data demonstrate that nbr is part of a general RNAi machinery and not just involved in trimming selected miRNAs [23, 32].
In the past, there has been considerable disagreement as to the localization of Nbr [15, 24]. The nuage-based localization of Nbr , based on its involvement in the piwi-pathway was born by the necessity to reveal colocalization of Nbr with Aub/Ago3 in the nuage, and to adapt its localization to fit the model. Arguably, for localization studies, it is not recommended to use a fusion protein involving EGFP as in , as it can lead to substantial localization artefacts due to oligomerization [35, 41]. Consistent with this observation was the fact that our EGFP-Nbr fusion protein, apart from general cytoplasmic localization, also showed perinuclear localization in 3rd instar salivary glands (Fig. 2c), similar to the EGFP-Nbr localization reported in the nuage . In fact, there is not an immediate necessity to describe Nbr enrichment in the nuage as claimed by . Instead, it would have sufficed to imply ubiquitous Nbr localization which also includes localization in the nuage, in order to fulfill the model. This argument was put forward by  who observed ubiquitous Nbr localization within oocytes as well, however, their expression profile differed slightly from ours and Nbr was reported to be ubiquitously expressed beyond oocyte stage 2. Ubiquitous Nbr expression rather than accumulation in the nuage also makes sense from another perspective: Given the wide-spread involvement [15, 49] (this report), the function of Nbr is needed in the whole cytoplasm and not just in the nuage.
We have shown that nbr is a tumor suppressor gene, and that the protein is involved in the RNAi machinery and controls the levels of transposons. Nbr is expressed only during a short time window during oogenesis and is not enriched in the nuage. Hence, we have described novel functions of nbr that go beyond from what was expected from previous knowledge on the mode of action of nbr. The ubiquitous localization of Nrb in oocytes necessitates a further careful analysis as to the mode of action of this protein. While it is not excluded that indeed it localizes to the nuage, it is not the sole subcellular location where Nbr resides which asks for further functions of this protein in other areas of the cytoplasm. Moreover, our data will encourage studies to show that Nbr is involved in many cellular processes.
S. B. thanks the Swedish Research Council, the Swedish Cancer Foundation and the Medical Faculty of Lund for support. C. C. L. thanks the “Crafoorddska Stiftelsen”, “Läkaresällskåpet i Lund” and the “Nilsson-Ehle Fonden” for support. We also thank Udo Häcker for providing reagents and Sol Da Rocha for excellent technical assistance.
Vetenskapsrådet. Award number 2010-4358.
Stiftelsen Olle Engkvist Byggmästare.
Erik Philipp Sörenssson Stiftelse.
Availability of data and materials
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
CCL designed, analyzed and interpreted the data and was a major contributor in writing the manuscript. XC was responsible for data of Fig. 3. KF was involved in experiments related to Fig. 5. SB was responsible for the data of Fig. 3 and writing of parts of the manuscript. All authors read and approved the final manuscript.
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