Male Germ Cells Express Abundant Endogenous Sirnas

Male germ cells express abundant endogenous siRNAs

Rui Song, Grant W. Hennig, Qiuxia Wu, Charlie Jose, Huili Zheng, and Wei Yan1

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557

Edited by Ryuzo Yanagimachi, The Institute for Biogenesis Research, University of Hawaii, Honolulu, HI, and approved July 7, 2011 (received for review May 27, 2011)

In mammals, endogenous siRNAs (endo-siRNAs) have only been window (31, 32). Those studies suggest that endo-siRNAs play a reported in murine oocytes and embryonic stem cells. Here, we show critical role in normal development of oocytes and embryonic that murine spermatogenic cells express numerous endo-siRNAs, stem cells. Previous data appear to support the notion that both which are likely to be derived from naturally occurring double- oocytes and ES cells, unlike most of the somatic cell types, lack or stranded RNA (dsRNA) precursors. The biogenesis of these testicular are insensitive to the IFN response triggered by dsRNAs because introduction of dsRNAs into these cells do not activate the OAS– endo-siRNAs is DROSHA independent, but DICER dependent. These ’ – male germ cell endo-siRNAs can potentially target hundreds of RNaseL pathway, which usually leads to the cell s demise (33 35). transcripts or thousands of DNA regions in the genome. Overall, our Given that the testis is an immune-privileged organ that has been shown to tolerate antigen introduction without eliciting immune work has unveiled another hidden layer of regulation imposed by responses (36, 37) and to respond poorly to RNA virus stimula- small noncoding RNAs during male germ cell development. tion (38, 39), testicular cells, at least the developing male germ cells, may lack IFN response and thus can produce endo-siRNAs RNA interference | spermatogenesis | testis | interferon response using naturally occurring long dsRNAs. Here, we show that the mouse testis, indeed, expresses numerous endo-siRNAs, which NA interference (RNAi) is a highly conserved gene silencing are mostly derived from trans-nat-dsRNAs and hairpin dsRNAs. Rmechanism by which double-stranded RNAs (dsRNAs) are These testicular endo-siRNAs could potentially target hundreds processed into single-stranded RNAs (ssRNAs) followed by loading of transcripts expressed during all phases of spermatogenesis. onto effector complexes to modulate gene expression (1, 2). Small Alternatively, they can recognize thousands of DNA sites across interfering RNAs (siRNAs) represent one of several distinct classes the genome, where they display complementary sequences to, and of small noncoding RNAs identified so far. Previously, siRNAs thus may have an epigenetic role as reported in yeasts, plants, mainly referred to small ssRNAs processed in the host cells C. elegans, and flies (13, 14, 30). from exogenous dsRNAs (e.g., hairpin dsRNAs, synthetic short dsRNAs, etc.), and these artificial siRNAs have been widely used to Results and Discussion suppress target gene expression both in vitro and in vivo (3–5). The Murine Testis Expresses Numerous Endo-siRNAs. The size of Endogenous siRNAs (endo-siRNAs) were initially identified in endo-siRNAs is ∼21 nt, which is distinct from that of piwi-inter- yeasts, plants, and Caenorhabditis elegans (6–9), and biogenesis of acting RNAs (piRNAs) at ∼31 nt. The sequence of an endo- endo-siRNAs in these organisms depends on the activity of RNA- siRNA is completely complementary to its target transcripts, dependent RNA polymerase (RdRP), which catalyzes the replica- whereas a miRNA is usually partially complementary to their tion of RNA from an RNA template (8–11). Double-stranded targets. Mature miRNAs are derived from precursor miRNAs, RNAs (dsRNAs) produced by RdRP are then cleaved by the RNase which possess the short stem-loop structures, whereas endo-siR- III DICER to generate single-stranded mature endo-siRNAs. By NAs are processed from long dsRNAs without the short stem- associating with Argonaute (AGO) proteins, siRNAs negatively loop structures (24–26). These major characteristics distinguish regulate the expression of targeting genes at posttranscriptional endo-siRNAs from other two well-known small RNA species: levels by inducing mRNA degradation and/or translational sup- piRNAs and miRNAs. Using these characteristics as criteria, we pression (10–12). Alternatively, these endo-siRNAs can function as searched for endo-siRNAs in a small RNA library of the adult guidance molecules to direct associated protein factors to target mouse testes sequenced using the 454 platforms (SI Appendix, specific genomic regions by DNA cytosine methylation or pro- Fig. S1). A total of 73 testicular endo-siRNAs were identified moting the formation of heterochromatin (13, 14). from approximately half a million reads, which were named Although RdRP has not been found in flies or mice, certain cell endo-siRNA-T1 to -T73 (SI Appendix, Table S1). These murine types of these two species appear to be able to generate endo- testicular endo-siRNAs rarely displayed unique chromosome siRNAs by processing the naturally occurring dsRNAs (15–23). hits. Instead, the majority of endo-siRNAs were matched to These dsRNA precursors include hairpin-dsRNAs, trans-natural hundreds of different sites on multiple chromosomes (Fig. 1 and antisense transcript-derived dsRNAs (trans-nat-dsRNAs), and cis- SI Appendix, Table S2). For example, endo-siRNA-T19 was natural antisense transcript-derived dsRNAs (cis-nat-dsRNAs) matched to every single chromosome, and on each chromo- (24–26). Endo-siRNAs differ from microRNAs (miRNAs) in that some except Y there were ∼850 copies of this endo-siRNA (SI the former are processed from long dsRNA precursors, whereas Appendix, Fig. S2 and Table S2). Therefore, endo-siRNAs are the later are mainly derived from precursors containing the short remarkably different from miRNAs in that miRNAs usually come stem-loop structure, although production of both requires DICER from a unique locus or very few loci (40), whereas endo-siRNAs activity in the cytoplasm. On the other hand, the microprocessor can have hundreds or even thousands of chromosome hits. A total complex (DROSHA–DGCR8) is required for cleaving precursor of 57 out of the 73 testicular endo-siRNAs possessed fewer than miRNAs out of the primary miRNA transcripts in the nucleus (27– 10 target transcripts predicted on the basis of sequence comple-

29). miRNAs are thus dependent upon DROSHA activity in the mentarity, whereas the remaining 16 displayed numerous target BIOLOGY

nucleus, whereas endo-siRNAs do not need DROSHA in their DEVELOPMENTAL biogenesis. In ﬂies, endo-siRNAs have also been found to be involved in heterochromatin formation in addition to their roles in Author contributions: R.S. and W.Y. designed research; R.S., G.W.H., Q.W., C.J., H.Z., and posttranscriptional regulation (30). W.Y. performed research; R.S., G.W.H., Q.W., H.Z., and W.Y. analyzed data; and R.S. and In mammals, endo-siRNAs have only been reported in murine W.Y. wrote the paper. oocytes and embryonic stem cells (21–23). Two recent in- The authors declare no conﬂict of interest. dependent studies revealed that the function of miRNAs is vir- This article is a PNAS Direct Submission. tually silenced during oocyte maturation and preimplantation 1To whom correspondence should be addressed. E-mail: [email protected]. embryonic development, whereas endo-siRNAs appear to be This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. required for the cellular events occurring during the same time 1073/pnas.1108567108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1108567108 PNAS | August 9, 2011 | vol. 108 | no. 32 | 13159–13164 Downloaded by guest on September 26, 2021 Fig. 1. Chromosome mapping of mouse testicular endo-siRNAs and their predicted targets. Each shade from solid blue to solid red in the spectrum represents each endo-siRNA from T1 to T73. The number and type of endo-siRNAs located in bins of ∼140 kbp (corresponding to one vertical pixel) were drawn on the Right sides of chromosomes, with each horizontal pixel representing one occurrence. The locations of predicted target transcripts were drawn on the Left sides of chromosomes indicated with a green line.

transcripts ranging from 10 up to 140 (Figs. 1 and 2A), suggesting dsRNA is formed by the partial sequences of the 3′-UTRs of these these endo-siRNAs can target a greater number of transcripts. two mRNAs and seeds three endo-siRNAs (endo-siRNAs-T26, Among all of the predicted target transcripts, the majority -T32, and -T71) (Fig. 3A). Hsd3b2 locates on Chr. 3 and Zfp488 (∼92%) were mRNAs and the remaining belonged to transcripts locates on Chr. 14. The partial 3′-UTR sequences of these two of pseudogenes (∼3%), retrotransposons (∼1%), and noncoding mRNAs are complementary to each other; these 3′-UTRs could, RNAs (∼4%) (Fig. 2B). Among all of the mRNA targets, an therefore, form a trans-nat-dsRNA, which contains two endo- average of 93% had endo-siRNA targeting sites in their 3′-UTRs, siRNAs (endo-siRNAs-T27 and -T45) (Fig. 3B). The Zfp353 tran- 4% in 5′-UTRs, and 3% in the coding regions (Fig. 2C). These script possesses extensive 5′-UTR and 3′-UTR, and their sequences results suggest that endo-siRNAs, like miRNAs, can target 3′- are partially complementary to each other. Therefore, a large UTRs of mRNAs and thus may regulate gene expression at hairpin-dsRNA can be formed by the noncoding sequences of this posttranscriptional levels. mRNA, which seeds one endo-siRNA, endo-siRNA-T50 (Fig. 3C). It is noteworthy that each of these endo-siRNAs appears to Although our endo-siRNAs could be mapped to multiple have many more hits on DNA (thousands) than on RNA (hun- chromosome sites, we found dsRNA precursors for only 42 of the dreds) (Fig. 1 and SI Appendix, Tables S1 and S2). If these endo- 73 endo-siRNAs. This was likely due to the incomplete collection siRNAs indeed interact with genomic DNA, their effects would of transcripts in the currently available databases. A total of 60 be much greater at DNA levels than at RNA levels on the basis intermolecular dsRNA precursors were studied here, which could of the number of potential targets. potentially generate 42 unique endo-siRNAs. We further analyzed these precursor dsRNAs for length, percentage of com- Mouse Testicular Endo-siRNAs Are Mainly Derived from Naturally plementarity, and transcript sources. The length of these dsRNAs Occurring dsRNAs. dsRNAs formed by two complementary tran- ranged from 118 to 1,400 bp, with an average of 337 bp. The scripts derived from different loci are called intermolecular percentage of complementarity between the two strands ranged dsRNAs. These include trans-natural antisense transcript- from 80 to 100%, with an average of 90% (Fig. 3D and SI Ap- dsRNAs (trans-nat-dsRNAs) in which two transcripts come from pendix, Dataset 1A). Among all 60 intermolecular dsRNAs, 58 different loci and cis-natural antisense transcript-dsRNAs (cis- were trans-nat-dsRNAs whereas only two appeared to be cis-nat- nat-dsRNAs) in which two transcripts come from the bidi- dsRNAs (Fig. 3D and SI Appendix, Dataset 1A). By studying the rectional transcription of the same chromosome locus. Hairpin- transcript sources for dsRNA precursors, we found that 40 out of dsRNAs, which result from complementary sequences within the 60 predicted intermolecular dsRNA precursors formed by a single transcript, are called intramolecular dsRNAs (24–26). All paring between two 3′-UTRs (Fig. 3E and SI Appendix, Dataset three types of long dsRNAs could serve as precursors for endo- 1A). In addition, we found 17 intramolecular dsRNA precursors siRNA production. For example, Tmod1 and Tstd2 are two neigh- that could potentially produce 12 endo-siRNAs. The average boring genes on chromosome (Chr.) 4 with the opposite orien- length of these hairpin-dsRNAs was 176 bp, and the average tations, which partially overlap in their last exons. This overlapping percentage of complementarity between the two strands was 83% region can be transcribed in a bidirectional manner, which can re- (Fig. 3F and SI Appendix, Dataset 1B). The 5′-UTR and the 3′- sult in the potential formation of a cis-nat-dsRNA. This cis-nat- UTR of the Zfp353 transcript could form a hairpin dsRNA,

13160 | www.pnas.org/cgi/doi/10.1073/pnas.1108567108 Song et al. Downloaded by guest on September 26, 2021 requires the use of sequences of small RNAs as the forward primers (41), whereas the sequences of some endo-siRNAs were not qualified as workable primers. Four general expression patterns were observed among the 58 endo-siRNAs examined (Fig. 4): (i) Eleven endo-siRNAs displayed an onset of expression at approximately postnatal day 14 (P14), followed by increasing levels from P17 to P28 and decreasing levels from P35 to adulthood. These endo-siRNAs are likely to be expressed in pachytene spermatocytes and/or round spermatids because the developmental timing coincides with the first appearance and accumu- lation of these spermatogenic cells in the testis (i.e., pachytene spermatocytes first appear at P14 and round spermatids first appear at P20). (ii) Fifteen endo-siRNAs were first detected at ∼P14 and their levels kept increasing thereafter until adulthood, suggesting these endo-siRNAs are mainly expressed in pachytene spermatocytes, round and elongated spermatids. (iii) Four endo- siRNAs (T10, T9, T64, and T12) showed the highest levels of expression between birth and P14, and levels diminished drasti- cally thereafter, suggesting that these endo-siRNAs are either expressed in the somatic cell types (Sertoli or Leydig cells) or spermatogonia because these cells are proportionally diluted significantly by the increasing number of meiotic and haploid germ cells. (iv) The remaining 28 endo-siRNAs displayed rela- tively constant levels in the developing testes, suggesting these endo-siRNAs may represent those housekeeping ones. These dynamic expression patterns imply that these endo-siRNAs are expressed in specific stages during male germ cell development. Moreover, these endo-siRNAs are mainly expressed in developing germ cells because levels should have decreased if they are solely expressed in somatic cell components of the testis due to the dilution effects of the increasing number of spermatogenic cells during testicular development (42). Indeed, all except four endo-siRNAs with early expressions (pattern 3 described above) were detected in purified pachytene spermatocytes (see below). Fig. 2. Transcripts predicted to be targeted by the 73 mouse testicular Moreover, their levels were all significantly down-regulated in the endo-siRNAs. (A) Number of predicted transcripts targeted by all 73 testic- Dicer-null, but not in Drosha-null spermatogenic cells (see below), ular endo-siRNAs. (B) Category and proportion of all predicted transcripts. supporting the notion that these endo-siRNAs are expressed in Note that the majority of the predicted transcripts (92.3%) are mRNAs. (C) germ cells rather than somatic cell types in the testis. Targeting sites in all predicted mRNA targets. Endo-siRNAs tend to target ′ the 3 -UTRs of mRNAs. Biogenesis of Testicular Endo-siRNAs Is DROSHA Independent, but DICER Dependent. Given that endo-siRNAs are mainly derived whereas the other 16 hairpin-dsRNAs were derived from hairpin from DICER-mediated processing of dsRNAs formed in the cy- folding of single 3′-UTRs (SI Appendix, Dataset 1B). toplasm, the true endo-siRNAs should be independent of the It is quite remarkable that almost all of the dsRNA precursors microprocessor (DROSHA–DGCR8 complex) activity, which is are formed between noncoding regions of the mRNAs (either 5′- required for precursor miRNA production in the nucleus. To test UTRs or 3′-UTRs) and/or pseudogene or transposon transcripts. whether the endo-siRNAs that we identified were affected by This finding suggests that endo-siRNAs possess a lower degree of either Dicer or Drosha inactivation, we generated two conditional fi sequence specificity toward their target transcripts because they knockout mouse lines with postnatal germ cell-speci c in- appear to mainly come from 3′-UTRs and predominantly target activation of Dicer or Drosha, respectively, by crossing a postnatal fi 3′-UTRs of transcripts. This may allow endo-siRNAs to target male germ cell-speci c Cre line, Stra8-iCre, with Dicer-loxp or a greater number of genes in the developing male germ cells. Drosha-loxp line. The Stra8-iCre male mice start to express Cre Because the formation of intermolecular dsRNA precursors exclusively in spermatogonia starting at postnatal day 3 (43). To visualize the Cre-expressing cells, we further crossed the Stra8- requires concurrent expression of two transcripts, we examined +/lox tg/tg lox/lox the spatiotemporal expression profiles of these predicted pre- iCre-Dicer mice with Rosa26mTmG -Dicer mice and fi Dicer cursor transcripts during the testicular development using semi- generated compound, male germ cell-speci c knockout mice (Stra8-iCre-Dicerlox/lox-Rosa26mTmG+/tg), in which quantitative PCR and/or by analyzing the Gene Expression all Cre-expressing (true knockout) cells were green due to Cre- Omnibus (GEO) database (SI Appendix, Fig. S3). Transcript pairs mediated activation of eGFP expression (44). Similarly, Stra8- predicted to form intermolecular dsRNA precursors appeared to iCre-Droshalox/lox-Rosa26mTmg+/tg male mice were generated for display similar or at least partially overlapping spatiotemporal the purification of Drosha-null pachytene spermatocytes. Both expression patterns during testicular development, suggesting Stra8-iCre-Dicerlox/lox and Stra8-iCre-Droshalox/lox males were in- that these predicted dsRNAs indeed could be formed in vivo. The fertile due to oligozoospermia or azoospermia caused by constant BIOLOGY

same analyses were also performed for the transcripts that were depletion of pachytene spermatocytes and spermatids in the adult DEVELOPMENTAL predicted to form intramolecular dsRNA precursors, and results mouse testes. However, the germ cell depletion was progressive confirmed that they did express during testicular development. with age, and at P25, a portion of Dicer-null or Drosha-null spermatocytes and round spermatids were being depleted, but the Stage-Specific Expression of Testicular Endo-siRNAs in Developing majority of spermatogenic cells remained normal looking within Male Germ Cells. To validate the expression of these cloned endo- the seminiferous tubules (SI Appendix, Fig. S4A), which allowed siRNAs in vivo, we examined their expression levels in developing us to purify pachytene spermatocytes using the STA-PUT method testes (Fig. 4) and purified spermatogenic cells (Fig. 5 and SI (45) for molecular analyses reported below. Appendix, Fig. S7). We determined the expression profiles for 58 We purified pachytene spermatocytes from the control (Stra8- of the 73 testicular endo-siRNAs because the PCR method used iCre-Rosa26mTmG+/tg), Drosha KO (Stra8-iCre-Droshalox/lox-Rosa

Song et al. PNAS | August 9, 2011 | vol. 108 | no. 32 | 13161 Downloaded by guest on September 26, 2021 Fig. 3. Double-stranded RNA (dsRNA) precursors for mouse testicular endo-siRNAs. (A) Representative cis-natural antisense transcript-derived dsRNA (cis-nat- dsRNA) precursor for three testicular endo-siRNAs (T26, T32, and T71). (B) Representative trans-natural antisense transcript-derived dsRNA (trans-nat-dsRNA) precursor that seeds two testicular endo-siRNAs (T27 and T45). (C) Representative hairpin dsRNA precursor that can produce testicular endo-siRNA-T50. (D) Length and complementarity of 60 predicted intermolecular dsRNA precursors (58 trans-nat-dsRNAs and 2 cis-nat-dsRNAs), which could potentially produce 42 testicular endo-siRNAs. (E) Sources of the two strands in each of the 60 predicted intermolecular dsRNA precursors. (F) Length and complementarity of 17 predicted intramolecular/hairpin dsRNA precursors, which could potentially generate 12 testicular endo-siRNAs. The longest hairpin-dsRNA precursor can be formed by pairing between 5′-UTR and 3′-UTR of Zfp353 mRNA, and the remaining 16 hairpin-dsRNAs are formed by two complementary sequences within mRNA 3′-UTRs.

26mTmg+/tg), and Dicer KO (Stra8-iCre-Dicerlox/lox-Rosa26m by down-regulation of global transcription or transcript stability in TmG+/tg) testes and the purified pachytene spermatocytes dis- cells that were undergoing apoptosis. Overall, these data demon- played a purity ranging from 85 to 95% on the basis of microscopic strate that biogenesis of these testicular endo-siRNAs requires examination (green vs. red cells) (SI Appendix,Fig.S4B)and DICER, but not DROSHA. This is consistent with the findings quantitative real-time PCR analyses of marker genes for different on endo-siRNAs identified in mouse oocytes and embryonic types of testicular cells (SI Appendix, Fig. S5). Levels of Dicer and stem cells (23, 31). The expression pattern of these endo-siRNAs Drosha mRNAs, compared with those of the controls, were dras- (SI Appendix,Fig.S7) was different from that of miRNAs (SI fi tically reduced in pachytene spermatocytes puri ed from Dicer Appendix,Fig.S6C) in the same sets of samples, further demon- KO (Stra8-iCre-Dicerlox/lox-Rosa26mTmG+/tg) and Drosha KO lox/lox +/tg strating that miRNA production is dependent upon both DRO- (Stra8-iCre-Drosha -Rosa26mTmg ) testes, respectively SHA and DICER activities, whereas biogenesis of endo-siRNAs (SI Appendix,Fig.S6A and B). Furthermore, levels of 12 miRNAs requires DICER, but not DROSHA. Expression profiles of four known to be expressed in pachytene spermatocytes (46) were all fi fi endo-siRNAs (T10, T9, T64, and T12) during testicular de- signi cantly lowered in both puri ed Dicer-null and Drosha-null velopment (Fig. 4) suggested that they were expressed mainly in pachytene spermatocytes than in the controls (SI Appendix,Fig. fi spermatogonia or in testicular somatic cell types (Sertoli and S6C). Together, these data con rmed the effective inactivation fi of Dicer and Drosha in the pachytene spermatocytes of Stra8- Leydig cells). No signi cant changes in levels of these four endo- iCre-Dicerlox/lox-Rosa26mTmG+/tg and Stra8-iCre-Droshalox/lox- siRNAs in the control, Dicer-null, or Drosha-null pachytene Rosa26mTmg+/tg mice, respectively. spermatocytes further support that these endo-siRNAs are mainly In purified pachytene spermatocytes, 54 out of the 58 endo- expressed in spermatogonia and/or somatic cells within the testis. siRNAs examined showed a similar pattern of changes with sig- We also examined the expressions of these 58 endo-siRNAs in nificantly lowered levels in Dicer-null cells (P < 0.001, n = 54), but human testes. Although primers for these endo-siRNAs were not in Drosha-null cells (Fig. 5 and SI Appendix,Fig.S7). Average designed on the basis of the mouse sequences, PCR products with levels of these endo-siRNAs in Dicer-null pachytene spermato- the expected sizes were observed in both mouse and human testes cytes were reduced by ∼75% compared with those in the controls samples (SI Appendix, Fig. S8). We then sequenced the PCR (Fig. 5). In contrast, these endo-siRNAs displayed an average of products and the sequencing results confirmed that sequences of ∼27% decrease in levels in the Drosha-null pachytene spermato- these testicular endo-siRNAs were exactly the same between mice cytes compared with the controls (Fig. 5). Given that the depletion and humans (sequencing data available upon request). These data of those Drosha-null pachytene spermatocytes was ongoing at P25 suggest that endo-siRNAs are conserved in male germ cells be- (SI Appendix,Fig.S4A), this degree of decrease was likely caused tween mice and humans.

13162 | www.pnas.org/cgi/doi/10.1073/pnas.1108567108 Song et al. Downloaded by guest on September 26, 2021 could not distinguish these two types of small RNAs, resulting in amplification of endo-siRNAs in the testis and those larger small RNAs in other tissues. The larger small RNAs appear to be similar to those piRNA-like RNAs identified in tissues other than the testis previously (47). Further investigation on this small RNA species is underway. These findings suggest that these testicular endo-siRNAs are exclusively expressed in the testis, more specifi- cally in the testicular germ cells. None of the 73 testicular endo- siRNAs was found among the mouse oocyte- or ES cell-expressed endo-siRNAs previously reported (21–23). Although there might be more endo-siRNAs that are yet to be identified in both male and female germ cells, two unique sets of endo-siRNAs identified so far in the male and female gametes, respectively, suggest that male germ cell-expressed endo-siRNAs have functions that are specific to the male germ cell developmental program and vice versa.

Endo-siRNAs Effectively Induce Target mRNA Degradation in Vitro. Endo-siRNA-T6 and its two predicted targets—Frmpd1 and Lrrc2—displayed similar expression patterns during testicular development (Fig. 4 and SI Appendix, Fig. S9A). Similar expression patterns were also observed between endo-siRNA-T40 and its predicted target Kif17 and between endo-siRNA-T19 and its predicted targets Spata1 and Syt11 (Fig. 4 and SI Appendix, Fig. S9A). Similar spatiotemporal expression patterns between endo- siRNAs and their predicted targets support a true targeting relationship in vivo To further test the target relationship between

Fig. 4. Expression proﬁles of testicular endo-siRNAs during development. Semiquantitative PCR was performed to determine levels of 58 testicular endo-siRNAs in the testes at the ages of newborn, postnatal day 7 (P7), P10, P14, P17, P21, P28, P35, and adult. Relative expression levels were then converted into a color-coded heat map.

We also examined the tissue distribution of these testicular endo-siRNAs by PCR using multiple mouse organs including brain, heart, liver, spleen, lung, kidney, small intestine, stomach, ovary, and uterus. We detected PCR products of slightly larger sizes, and sequencing analyses revealed that these small RNAs ampliﬁed from tissues other than the testis were ∼31 nt long and their sequences overlapped with those of the testicular endo-siR- NAs, which are 21 nt long. Because partial sequences of these longer small RNAs are the same as those testicular endo-siRNAs, the PCR primers designed on the basis of endo-siRNA sequences BIOLOGY DEVELOPMENTAL

Fig. 6. Endo-siRNAs can induce degradation of their target mRNAs in vitro. Fig. 5. Levels of 54 endo-siRNAs in Drosha-orDicer-null pachytene sper- In a luciferase-based reporter assay, NIH 3T3 cells were cotransfected with matocytes. TaqMan-based quantitative real-time PCR was used to quantify firefly luciferase expression plasmids bearing the 3′-UTR of Frmpd1, Lrrc2, levels of endo-siRNAs in pachytene spermatocytes purified from the testes of Kif17, Spata1,orSyt11, and its corresponding targeting endo-siRNAs (T6, T40, the control (Stra8-iCre-Rosa26mTmG+/tg), Drosha knockout (Stra8-iCre- and T19). Relative levels of the firefly vs. Renilla luciferase mRNAs are shown. Droshalox/lox-Rosa26mTmG+/tg), and Dicer knockout (Stra8-iCre-Dicerlox/lox- Three types of controls included transfection without corresponding endo- Rosa26mTmG+/tg) mice at postnatal day 25. Levels of endo-siRNAs in Drosha- siRNAs (open bars), the native firefly luciferase plasmid (none), and the firefly or Dicer-null cells relative to those in the control cells were plotted, and the luciferase plasmid bearing Klhl10 3′-UTR, which did not contain any targeting average levels are shown. sites for three endo-siRNAs tested. Experiments were performed in triplicate.

Song et al. PNAS | August 9, 2011 | vol. 108 | no. 32 | 13163 Downloaded by guest on September 26, 2021 endo-siRNAs and predicted target transcripts, we performed direct their associated epigenetic factors [e.g., site-specificDNA in vitro luciferase assays using National Institutes of Health methyltransferase (MET1) or histone methyltransferases (HMTs)] (NIH) 3T3 cells. Three endo-siRNAs and five predicted target to target specific genomic regions through DNA cytosine methyl- transcripts were not expressed in NIH 3T3 cells (SI Appendix, Fig. ation or promoting the formation of heterochromatin, respec- S9 B and C), which eliminated the endogenous influences on tively. Interestingly, like yeast and plants, endo-siRNAs appeared the results. to be involved in chromatin modifications in flies (30). Given that The native firefly luciferase plasmid and the plasmid bearing ′ the testicular endo-siRNAs display numerous hits on multiple Klhl10 3 -UTR were used as controls because neither contained chromosomes (Fig. 1), it is intriguing to further explore whether any targeting sites for the three endo-siRNAs tested. No signifi- fi fl these murine endo-siRNAs have any nuclear effects on methyla- cant changes in levels of re y mRNAs were detected in the fi control groups, whereas highly reduced mRNA levels were con- tion and/or chromatin modi cation in addition to their well- sistently observed in experimental groups (Fig. 6). Moreover, the established cytoplasmic roles as posttranscriptional regulators. efficiency for each of the three endo-siRNAs tested to degrade Materials and Methods the target mRNAs was >50% (Fig. 6). Accordingly, protein production was significantly suppressed in all three sets of experi- Quantitative analyses of endo-siRNAs, miRNAs, and other transcripts were ments (SI Appendix,Fig.S9D). These data demonstrated that conducted as described in refs. 41 and 46. Spermatogenic cells were purified endo-siRNAs could effectively induce degradation of their target as described in refs. 42 and 46. Bioinformatic analyses were performed using mRNAs in vitro. It is conceivable that these testicular endo-siR- an in-house computer program. For an extensive description of the materials NAs may exert similar effects in vivo, causing degradation of their and methods, see SI Appendix, Materials and Methods. target mRNAs. In addition to their role as a posttranscriptional regulator, endo- ACKNOWLEDGMENTS. The Drosha-loxp mouse line was provided by Dr. Dan siRNAs of plants and yeasts have been found to function as an R. Littman, Skirball Institute of Biomolecular Medicine, New York University epigenetic regulator and thus affect transcriptional activities of School of Medicine. This work was supported by Grants HD050281 and HD060858 from the National Institutes of Health (NIH) (to W.Y.). The soft- genes through either RNA-directed DNA methylation (RdDM) ware for bioinformatic analyses was developed in the Imaging Core (Core D), mechanism or RNA-mediated heterochromatin formation (13, with support by Centers of Biomedical Research Excellence Grant P20- 14). In both cases, endo-siRNAs may act as guide molecules to RR18751 from the NIH.

1. Hannon GJ (2002) RNA interference. Nature 418:244–251. 25. Golden DE, Gerbasi VR, Sontheimer EJ (2008) An inside job for siRNAs. Mol Cell 31: 2. Tomari Y, Zamore PD (2005) Perspective: Machines for RNAi. Genes Dev 19:517–529. 309–312. 3. Elbashir SM, et al. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference 26. Okamura K, Lai EC (2008) Endogenous small interfering RNAs in animals. Nat Rev Mol in cultured mammalian cells. Nature 411:494–498. Cell Biol 9:673–678. 4. Paddison PJ, Caudy AA, Hannon GJ (2002) Stable suppression of gene expression by 27. Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ (2004) Processing of primary – RNAi in mammalian cells. Proc Natl Acad Sci USA 99:1443 1448. microRNAs by the Microprocessor complex. Nature 432:231–235. 5. Lu PY, Xie F, Woodle MC (2005) In vivo application of RNA interference: From func- 28. Gregory RI, et al. (2004) The Microprocessor complex mediates the genesis of mi- – tional genomics to therapeutics. Adv Genet 54:117 142. croRNAs. Nature 432:235–240. 6. Cogoni C, Macino G (2000) Post-transcriptional gene silencing across kingdoms. Curr 29. Han J, et al. (2004) The Drosha-DGCR8 complex in primary microRNA processing. Opin Genet Dev 10:638–643. Genes Dev 18:3016–3027. 7. Ruby JG, et al. (2006) Large-scale sequencing reveals 21U-RNAs and additional mi- 30. Fagegaltier D, et al. (2009) The endogenous siRNA pathway is involved in hetero- croRNAs and endogenous siRNAs in C. elegans. Cell 127:1193–1207. chromatin formation in Drosophila. Proc Natl Acad Sci USA 106:21258–21263. 8. Pak J, Fire A (2007) Distinct populations of primary and secondary effectors during 31. Suh N, et al. (2010) MicroRNA function is globally suppressed in mouse oocytes and RNAi in C. elegans. Science 315:241–244. early embryos. Curr Biol 20:271–277. 9. Sijen T, Steiner FA, Thijssen KL, Plasterk RH (2007) Secondary siRNAs result from un- 32. Ma J, et al. (2010) MicroRNA activity is suppressed in mouse oocytes. Curr Biol 20: primed RNA synthesis and form a distinct class. Science 315:244–247. – 10. Vaucheret H (2006) Post-transcriptional small RNA pathways in plants: Mechanisms 265 270. fi and regulations. Genes Dev 20:759–771. 33. Wianny F, Zernicka-Goetz M (2000) Speci c interference with gene function by – 11. Martienssen RA, Zaratiegui M, Goto DB (2005) RNA interference and heterochro- double-stranded RNA in early mouse development. Nat Cell Biol 2:70 75. fi matin in the fission yeast Schizosaccharomyces pombe. Trends Genet 21:450–456. 34. Yang S, Tutton S, Pierce E, Yoon K (2001) Speci c double-stranded RNA interference 12. Yigit E, et al. (2006) Analysis of the C. elegans Argonaute family reveals that distinct in undifferentiated mouse embryonic stem cells. Mol Cell Biol 21:7807–7816. Argonautes act sequentially during RNAi. Cell 127:747–757. 35. Stein P, Zeng F, Pan H, Schultz RM (2005) Absence of non-specific effects of RNA 13. Verdel A, Vavasseur A, Le Gorrec M, Touat-Todeschini L (2009) Common themes in interference triggered by long double-stranded RNA in mouse oocytes. Dev Biol 286: siRNA-mediated epigenetic silencing pathways. Int J Dev Biol 53:245–257. 464–471. 14. Lejeune E, Allshire RC (2011) Common ground: Small RNA programming and chro- 36. Fijak M, Bhushan S, Meinhardt A (2011) Immunoprivileged sites: The testis. Methods matin modifications. Curr Opin Cell Biol 23:258–265. Mol Biol 677:459–470. 15. Ghildiyal M, et al. (2008) Endogenous siRNAs derived from transposons and mRNAs in 37. Fijak M, Meinhardt A (2006) The testis in immune privilege. Immunol Rev 213:66–81. Drosophila somatic cells. Science 320:1077–1081. 38. Dejucq N, Liénard MO, Jégou B (1998) Interferons and interferon-induced antiviral 16. Okamura K, et al. (2008) The Drosophila hairpin RNA pathway generates endogenous proteins in the testis. J Reprod Immunol 41:291–300. – short interfering RNAs. Nature 453:803 806. 39. Melaine N, et al. (2003) Production of the antiviral proteins 2’5’oligoadenylate syn- 17. Czech B, et al. (2008) An endogenous small interfering RNA pathway in Drosophila. thetase, PKR and Mx in interstitial cells and spermatogonia. J Reprod Immunol 59:53–60. – Nature 453:798 802. 40. He L, Hannon GJ (2004) MicroRNAs: Small RNAs with a big role in gene regulation. 18. Kawamura Y, et al. (2008) Drosophila endogenous small RNAs bind to Argonaute 2 in Nat Rev Genet 5:522–531. somatic cells. Nature 453:793–797. 41. Ro S, Park C, Jin J, Sanders KM, Yan W (2006) A PCR-based method for detection and 19. Okamura K, Balla S, Martin R, Liu N, Lai EC (2008) Two distinct mechanisms generate quantification of small RNAs. Biochem Biophys Res Commun 351:756–763. endogenous siRNAs from bidirectional transcription in Drosophila melanogaster. Nat 42. Bellvé AR, et al. (1977) Spermatogenic cells of the prepuberal mouse. Isolation and Struct Mol Biol 15:581–590. morphological characterization. J Cell Biol 74:68–85. 20. Chung WJ, Okamura K, Martin R, Lai EC (2008) Endogenous RNA interference pro- 43. Sadate-Ngatchou PI, Payne CJ, Dearth AT, Braun RE (2008) Cre recombinase activity vides a somatic defense against Drosophila transposons. Curr Biol 18:795–802. fi – 21. Watanabe T, et al. (2008) Endogenous siRNAs from naturally formed dsRNAs regulate speci c to postnatal, premeiotic male germ cells in transgenic mice. Genesis 46:738 742. fl transcripts in mouse oocytes. Nature 453:539–543. 44. Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L (2007) A global double- uorescent – 22. Tam OH, et al. (2008) Pseudogene-derived small interfering RNAs regulate gene ex- Cre reporter mouse. Genesis 45:593 605. fi pression in mouse oocytes. Nature 453:534–538. 45. Bellvé AR (1993) Puri cation, culture, and fractionation of spermatogenic cells. 23. Babiarz JE, Ruby JG, Wang Y, Bartel DP, Blelloch R (2008) Mouse ES cells express en- Methods Enzymol 225:84–113. dogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent 46. Song R, et al. (2009) Many X-linked microRNAs escape meiotic sex chromosome in- small RNAs. Genes Dev 22:2773–2785. activation. Nat Genet 41:488–493. 24. Nilsen TW (2008) Endo-siRNAs: Yet another layer of complexity in RNA silencing. Nat 47. Ro S, et al. (2007) Cloning and expression profiling of testis-expressed piRNA-like Struct Mol Biol 15:546–548. RNAs. RNA 13:1693–1702.

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