Herpesvirus Saimiri Noncoding RNA Demián Cazalla, Et Al

Down-Regulation of a Host MicroRNA by a Herpesvirus saimiri Noncoding RNA Demián Cazalla, et al. Science 328, 1563 (2010); DOI: 10.1126/science.1187197

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To analyze the cell cycles of germ cells in the To confirm that the nos2-expressing Gs cells 6. K. Eggan, S. Jurga, R. Gosden, I. M. Min, A. J. Wagers, germinal cradles, we assessed the S-phase index, a generated fertile eggs, F1 embryos from heat- Nature 441, 1109 (2006). 7. R. R. Tokarz, in The Vertebrate Ovary, R. E. Jones, Ed. measure of the percentage of nuclei in the S phase. treated transgenic medaka were genotyped (fig. S8 (Plenum, New York, 1978), pp. 145–179. Adult female medaka were exposed to BrdU for and table S2). Some embryos possessed an un- 8. R. A. Wallace, K. Selman, J. Electron Microsc. Tech. 16, various periods of time (fig. S6A). After 24 hours, cleaved loxP region, whereas others exhibited the 175 (1990). nearly 60% of the Gs cells were BrdU positive, characteristic pattern of successful loxP-mediated 9. C. Morinaga et al., Proc. Natl. Acad. Sci. U.S.A. 104, 9691 (2007). and the rate of newly stained cells slowed over the recombination (Fig. 2E). Embryos with the cleaved 10. V. P. I. Vidal, M. C. Chaboissier, D. G. de Rooij, A. Schedl, following week such that about 75% of the Gs loxP region were produced for 3 months after Nat. Genet. 28, 216 (2001). cells were BrdU positive. Thus, Gs cells appear heat treatment (table S2), indicating that the pop- 11. T. Wagner et al., Cell 79, 1111 (1994). to be heterogeneous. Piecewise linear regression ulation of nos2-expressing Gs cells was capable 12. N. Klüver, M. Kondo, A. Herpin, H. Mitani, M. Schartl, analysis revealed two distinct populations: fast- of continuously generating fertile eggs. Dev. Genes Evol. 215, 297 (2005). 13. S. Nakamura et al., Mol. Reprod. Dev. 75, 472 (2008). dividing (Gsf) and slow-dividing (Gss) germ cells. We identified and characterized ovarian cords 14. H. Kurokawa et al., Dev. Growth Differ. 48, 209 (2006). BrdU pulse-chase experiments also identified within the germinal epithelia of medaka ovaries. 15. M. Tanaka, M. Kinoshita, D. Kobayashi, Y. Nagahama, label-retaining germ cells among the Gs cells (fig. These cords were composed of sox9b-expressing Proc. Natl. Acad. Sci. U.S.A. 98, 2544 (2001). S6, B to D). These experiments suggest that at least cells and contained mitotic nos2-expressing Gs 16. Y. Aoki et al., Dev. Dyn. 237, 800 (2008). 17. Y. Aoki, S. Nakamura, Y. Ishikawa, M. Tanaka, Zoolog. 60% of Gs cells are Gsf cells. In contrast, the per- cells in discrete structures referred to as germinal Sci. 26, 112 (2009). centage of BrdU-positive Gcys nuclei increased cradles. These mitotic oogonia continually gave rise 18. T. Iwai et al., Exp. Cell Res. 312, 2528 (2006). from about 35% after 1 hour of BrdU labeling to to germ cells that developed in the ovary, finally 19. E. Passegué, A. J. Wagers, S. Giuriato, W. C. Anderson, nearly 100% after 24 hours. Thus, no quiescent resulting in fertile eggs. Thus, we conclude that the I. L. Weissman, J. Exp. Med. 202, 1599 (2005). 20. Y. Stahl, R. Simon, Int. J. Dev. Biol. 49, 479 (2005). Gcys cells were present in the germinal cradles. ovarian cord harbors the histological niche within 21. J. W. Zhang et al., Nature 425, 836 (2003). We next addressed the potential functions of the ovary where germinal cradles are formed 22. We thank M. Yamashita and T. Iwai for anti-medaka SYCP the Gs cells. We conducted experiments to de- (Fig. 3). Moreover, these cradles contain oogonia antibodies, T. Czerny for plasmids containing the hs termine whether Gs cells facilitate the recovery characteristic of germline stem cells that contrib- promoter region, and S. Yoshida for helpful discussions. We are grateful to Y. Ichikawa and C. Kinoshita for of Gcys cells after busulfan treatment. Three- to ute to the production of fertile eggs (Fig. 3). maintaining the fish colony. This work was supported in 4-month-old female medaka were treated with The germinal cradle in the ovarian cord is rem- part by Grants-in-Aid for Scientific Research on 10 ng/ml busulfan for 1 week to eliminate mitot- iniscent of the germarium from the Drosophila Innovative Areas, “Gamete Stem Cells” (grant 21116509) ically active Gcys cells. The number of germinal ovary (1); both promote the development of germ and for Young Scientists (B) (21770072) (to S.N.), and on December 1, 2010 cradles with Gcys cells was markedly reduced cells from germline stem cells to very early diplo- for Scientific Research on Priority Areas (B) (21370101); the National BioResource Project Medaka; and the Daiko 1 month after treatment, before recovering to the tene oocytes in a unique histological compartment Foundation and the Center for the Promotion of numbers observed in untreated control samples within the ovary. These similarities might reflect Integrated Science (CPIS) of SOKENDAI (to M.T.). 3 months after the treatment (fig. S7). These re- a fundamental process governing oogenesis across Supporting Online Material sults suggest that the Gs cells present 1 month animal species. www.sciencemag.org/cgi/content/full/science.1185473/DC1 after oogenesis was disrupted are capable of re- Materials and Methods generating Gcys cells in the germinal cradles. References and Notes SOM Text Figs. S1 to S9 To confirm that Gs cells in the ovary were stem 1. M. D. Wong, Z. Jin, T. Xie, Annu. Rev. Genet. 39, 173 Tables S1 and S2 cell–like germ cells, we conducted clonal analysis (2005).

References www.sciencemag.org 2. T. C. A. Kumar, Proc. R. Soc. London Ser. B 169, 167 by using transgenic medaka harboring two dis- Movies S1 to S3 hsp (1968). tinct fluorescent constructs { -cre:mCherry/ 3. J. Johnson, J. Canning, T. Kaneko, J. K. Pru, J. L. Tilly, 3 December 2009; accepted 5 May 2010 nos2p-loxP[DsRed]-EGFP-olvas3′ untranslated Nature 428, 145 (2004). Published online 20 May 2010; region (UTR)} (fig. S8). In these medaka, heat 4. K. Zou et al., Nat. Cell Biol. 11, 631 (2009). 10.1126/science.1185473 treatment transiently induces EGFP expression 5. J. Pacchiarotti et al., Differentiation 79, 159 (2010). Include this information when citing this paper. only in nos2-expressing Gs cells, and EGFP transcripts are stabilized in all germ cells through the ′ olvas 14 15

action of the 3 UTR of ( , ). Downloaded from Adult transgenic fish (n = 21) were heated to Down-Regulation of a Host 39°C for 2 hours, and immunohistochemistry was performed by using antibodies specific for MicroRNA by a Herpesvirus saimiri GFP and the pan–germ cell marker tdrd1 (16). Several different combinations of EGFP fluores- Noncoding RNA cent cells were found in the germinal cradles, including cradles in which the only EGFP-labeled Demián Cazalla, Therese Yario, Joan Steitz* germ cells were Gs cells (Gs clone type) (Fig. 2A and fig. S9, E and F), cradles containing fluores- T cells transformed by Herpesvirus saimiri express seven viral U-rich noncoding RNAs of unknown function cent Gs cells as well as Gcys cells and/or Gdip called HSURs. We noted that conserved sequences in HSURs 1 and 2 constitute potential binding sites oocytes (mosaic clone type) (Fig. 2B and fig. S9, for three host-cell microRNAs (miRNAs). Coimmunoprecipitation experiments confirmed that HSURs 1 G and H), and cradles entirely occupied by flu- and 2 interact with the predicted miRNAs in virally transformed T cells. The abundance of one of these orescent germ cells (full clone type) (Fig. 2C and miRNAs, miR-27, is dramatically lowered in transformed cells, with consequent effects on the expression of fig. S9, I and J). Forty-eight hours after heat treat- miR-27 target genes. Transient knockdown and ectopic expression of HSUR 1 demonstrate that it ment, most germinal cradles were the Gs clone directs degradation of mature miR-27 in a sequence-specific and binding-dependent manner. This viral type (97.0 T 2.0%, mean T SEM, n = 7), whereas 3 strategy illustrates use of a ncRNA to manipulate host-cell gene expression via the miRNA pathway. weeks later, the full clone type was the most pre- valent germinal cradle structure (77.8 T 3.3%, erpesvirus saimiri (HVS) infects T cells HVS transcripts are seven small noncoding RNAs mean T SEM, n = 8) (Fig. 2D). Thus, all of the and causes aggressive leukemias and lym- (ncRNAs) called HSURs (H. saimiri U-rich RNAs) H 1 2–4 germ cells in the germinal cradles were derived phomas in New World primates ( ). In ( ). HSURs exhibit structural but little sequence from the population of nos2-expressing Gs cells. transformed marmoset T cells, the most abundant similarity to cellular small nuclear RNAs (snRNAs).

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HSURs are encoded by all HVS subgroups; HSURs Coimmunoprecipitation experiments on ex- formed T cells lacking HSURs 1 and 2 (Fig. 2A). 1and2(Fig.1A)arethemosthighlyconservedand tracts of virally transformed marmoset T cells using Levels of miR-23a and miR-24, two miRNAs the only snRNAs expressed by the closely related antibodies to Ago2 showed that HSURs 1 and 2 contained in the same primary transcript as miR-27a Herpesvirus ateles (5). Because HSURs are dis- were selectively present as compared with control (14), were unchanged (Fig. 2A and fig. S5), which pensable for transformation in vitro (6, 7), their immunoprecipitates, whereas all other HSURs suggested posttranscriptional differences in the strong conservation suggests an in vivo role in in- (Fig. 1B, lanes 3 and 5) and cellular small nuclear expression of miR-27 between these two cell lines. fected monkeys. HSURs 1 and 2 contain highly ribonucleoproteins (snRNPs) (fig. S3) did not de- The abundance of the precursor miRNAs (pre- conserved AU-rich 5′-end sequences (Fig. 1A and tectably associate with micro-ribonucleoproteins miRNAs) for miR-27a and miR-27b (fig. S6) did figs. S1 and S2) that are similar to AU-rich ele- (miRNPs). Immunoprecipitation was then per- not differ between the two cell lines, nor did that ments (AREs) found in the 3′ untranslated regions formed on extracts from T-cell lines transformed of the passenger strand of the miR-27a duplex (3′UTRs)ofshort-livedmRNAs(8–10). HSURs with either the wild-type HVS strain A11 or a mu- (Fig. 2A), which suggests that miRNA processing 1 and 2 are responsible for the up-regulation of tantdeletedforHSURs1and2(7) by using anti- by Drosha and Dicer (15) is not altered. a handful of host proteins that are hallmarks of bodies to Sm proteins, which recognize both cellular To determine whether the difference in the T-cell activation (11) and may contribute to an and viral snRNPs (3). Figure 1C (lanes 5 and 10) abundance of miR-27 is due to a change in the enhanced growth rate (7) of transformed cells. reveals the association of miR-16, miR-27, and stability of the mature miRNA, we designed a pulse- Comparisons of HSUR 1 (fig. S1) and HSUR miR-142-3p, and not of the control miR-20, but chase strategy using synthetic miRNA duplexes 2 (fig. S2) between HVS strains identified stretches only when HSURs 1 and 2 are present. Psoralen (16) in which only the guide strand was radio- of perfectly or highly conserved sequences (Fig. [aminomethyltrioxsalen (AMT)] crosslinking ex- actively labeled. After nucleofection (the “pulse”), 1A, bold nucleotides). Bioinformatic searches periments (fig. S4) (13) confirmed the existence we monitored the miRNA remaining over time then revealed complementarity between these of in vivo interactions between miR-27 and HSURs. (the “chase”; Fig. 2B) in marmoset T cell lines trans- HSUR sequences and three microRNAs (miRNAs) We noticed a distinct difference in the over- formed by either wild-type HVS or mutant HVS expressed in T cells: miR-142-3p, miR-27, and all level of miR-27 in the marmoset T-cell line lacking HSURs 1 and 2. miR-27a was degraded miR-16 (Fig. 1A) (12). transformed by wild-type HVS as compared with more rapidly in the wild-type transformed cells, that of the mutant lacking HSURs 1 and 2 (Fig. 1C whereas no difference was observed for either and fig. S5). The miR-27 family includes miR-27a miR-16, which is predicted to bind HSUR 2 (Fig. Department of Molecular Biophysics and Biochemistry, and miR-27b, which are transcribed from differ- 1A), or for the control miR-20a (fig. S7). Howard Hughes Medical Institute, Yale University School of ent chromosomes and differ by only one nucleotide HSURs 1 and 2 do not affect the steady-state

Medicine, Boyer Center for Molecular Medicine, 295 Congress on December 1, 2010 Avenue, New Haven, CT 06536, USA. near the 3′ end. Quantitative real-time polymerase levels of host mRNAs in virally transformed *To whom correspondence should be addressed. E-mail: chain reaction (PCR) confirmed the higher abun- marmoset T cells, except for eight genes (8, 11) [email protected] dance of both miR-27a and miR-27b in trans- that are not predicted targets of HSUR-bound

Fig. 1. HSURs 1 and 2 bind host miRNAs in virally transformed T cells. (A) Sequences and predicted secondary structures of HSURs 1 and 2. Bold nucleotides are perfectly conserved in all available genome

sequences from independent isolates of www.sciencemag.org HVSA,B,andCstrainsandalsoinH. ateles (figs. S1 and S2). Complementarity between HSURs and miRNAs is represented by dots; miRNA seed regions are in yellow. (B) Coimmunoprecipitation of HSURs from extracts of virally transformed marmoset T cells with antibody to Flag (lane 3) or

antibody to Ago2 (lane 5). I, input (5%); S, Downloaded from supernatant (5%); P, pellet (100%). (C) Coimmunoprecipitation of miRNAs from extracts of virally transformed marmoset T cell lines expressing (Wt, lanes 1 to 5) or lacking HSURs 1 and 2 (Mut, lanes 6 to 10) with Y12 antibody (aSm, lanes 4, 5 and 9, 10) or nonimmune serum (C, lanes 2, 3 and 7, 8). I, input (2%); S, supernatant (2%); P, pellet (100%). Northern blots in (B) and (C) were probed for HSURs, miRNAs, or U4atac, as an aSm immunoprecipitation control.

1564 18 JUNE 2010 VOL 328 SCIENCE www.sciencemag.org REPORTS miRNAs (17). We analyzed the levels of fork- higher levels of miR-27 (Fig. 3, A and B) and with stably transfected with a plasmid deleted for the head box 1 (FOXO1) protein, whose mRNA is a lower levels of the miR-27 target protein, FOXO1 HSUR 1 gene (Fig. 4B, lanes 6 to 10). Likewise, validated target of miR-27 (18). The difference in (Fig. 3C), which suggests that HSUR 1 is mutation of the conserved miR-27 binding site in miR-27 abundance correlates with up-regulation specifically involved in regulating miR-27. HSUR 1 [Fig. 4A, HSUR 1 mutant (H1Mt)] abol- of the FOXO1 protein (Fig. 2C) in the presence Direct base-pairing between HSUR 1 and ished the immunoprecipitation of miR-27 (Fig. 4B, of HSURs 1 and 2, which suggests that these miR-27 is required to control miRNA abundance. lanes 11 to 15). Furthermore, mutations in HSUR HVS ncRNAs perturb host gene expression via Human Jurkat T cells were stably transfected with 1 that were designed to produce complementarity the miRNA pathway. a plasmid containing HVS DNA that encodes all to miR-20a (Fig. 4A, H1m20) enabled a previ- To confirm that the difference in miR-27 levels seven HSURs, including their endogenous tran- ously unknown interaction with miR-20 (Fig. 4B, does not result from accumulated mutations in the scription and processing signals (7). Precipitation lanes16to20). two HVS-transformed T cell lines, we treated cells of HSUR snRNPs with antibodies to Sm proteins Expression of wild-type HSUR 1 alone (fig. that contained wild-type HVS with chimeric oligo- confirmedtheirassociationwithmiR-27inextracts S8) in Jurkat T cells (fig. S9) is sufficient to down- nucleotides that effectively induce degradation of of this cell line (Fig. 4B, lanes 1 to 5). In contrast, regulate the level of miR-27a as compared with complementary nuclear RNAs (19). Knockdown antibodies to Sm proteins did not coimmunopre- transfection with the empty vector [Fig. 4C; green of HSUR 1 but not of HSUR 2 correlated with cipitate miR-27 from extracts of Jurkat T cells fluorescent protein (GFP)]. Direct interaction between HSUR 1 and miR-27 is required because cells transfected with a H1Mt that is unable to bind miR-27 (Fig. 4B) have levels of miR-27 comparable with those of cells transfected with the empty vector. Moreover, the miR-20a level was substantially lower after transfection of the HSUR 1 mutant (H1m20) that binds this miRNA (Fig. 4B). Together, these results indicate that base-pairing to an internal site in HSUR 1 is both necessary and sufficient to direct a mature miRNA into a cellular degradation pathway. The ARE-like sequence in HSUR 1 is known on December 1, 2010 to induce in vivo decay of HSUR 1 itself (9), suggesting that the ARE could be involved in the HSUR 1–dependent decay of miR-27. We transfected Jurkat T cells with a mutant HSUR 1 containing two U→G substitutions in the ARE (H1M1) that were previously shown to stabilize and raise cellular levels of HSUR 1 (9). This mutation re- sulted in higher levels of HSUR 1 (fig. S9) and did Fig. 2. ThepresenceofHSURs1and2affectsmiR-27aabundance, decay, and target expression. (A) not alleviate but produced a more pronounced Relative levels of different mature miRNAs in virally transformed marmoset T cells expressing (Wt) or lacking www.sciencemag.org down-regulation of the abundance of miR-27 as (Mut) HSURs 1 and 2 were determined by means of quantitative real-time PCR. (B) Pulse-chase assay compared with wild-type HSUR 1 (Fig. 4C), in- assessing the decay of radioactively labeled synthetic miR-27a and miR-16. (C) Western blot analysis of FOXO1 in marmoset T cells transformed by HVS expressing (Wt) or lacking (Mut) HSURs 1 and 2. dicating that HSUR 1 directs the degradation of miRNAs by an ARE-independent mechanism. We have demonstrated that HSUR 1 and 2 snRNPs directly bind specific host miRNPs in virally transformed T cells. Whereas the interac-

tion of miR-27 with an internal site in HSUR Downloaded from 1 results in the degradation of this miRNA, the binding of miR-142-3p and miR-16 to HSURs 1 and 2 does not result in their lowered levels (Fig.2,AandB,andfig.S5).Nonetheless,mu- tational alteration of its binding site in HSUR 2 indicates that the interaction with miR-16 also occurs via base-pairing (fig. S10), and it is con- ceivable that if this base-pairing were stronger, decay would be induced. Because HSURs are comparable in abundance with the bound miRNAs in virally transformed T cells (table S1), it seems Fig. 3. HSUR 1 regulates the unlikely that they could effectively compete with abundance of miR-27 in virally mRNA targets and act as miRNA sponges (20) transformed T cells. (A)Northern even though down-regulating the activity of these blot analyses of miRNAs and miRNAs might be advantageous for the virus. HSURs after nucleofection with For instance, miR-16 is reported to target cell-cycle chimeric oligonucleotides anti- and apoptosis regulators such as Bcl-2 and cyclins sensetoGFP(lane1),HSUR1(lane D1 and E1 (21, 22), but we do not observe differ- 2), or HSUR 2 (lane 3). (B)Quan- ences in levels of miR-16 target proteins in the tification of miRNAs from three independent experiments performed as in (A). (C) Western blot of FOXO1 presence versus absence of HSURs 1 and 2 (fig. in HVS-transformed marmoset T cells nucleofected as described in (A). S11). The functional importance of the interaction

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Fig. 4. HSUR 1 down-regulates mature miRNAs in a sequence-specific and binding- dependent manner. (A) Partial sequences of HSUR 1 (positions 40 to 62) and its mutants (in red) H1Mt and H1m20. Bold nucleotides are perfectly conserved (Fig. 1A). (B)Coim- munoprecipitation of miRNAs with aSm, as in Fig. 1C, from extracts of Jurkat T cells stably expressing HSURs 2 to 7 and either wild-type HSUR 1 (Wt, lanes 1 to 5), no HSUR 1 (DH1, lanes 6 to 10), mutant HSUR 1 H1Mt (lanes 11 to 15), or mutant HSUR 1 H1m20 (lanes 16 to 20). (C) miRNA levels in Jurkat T cells fluorescence- activated cell sorted for GFP after transient transfection with empty vector (GFP) or with plasmids expressing GFP and the following: HSUR 3 (GFP-H3),WtHSUR 1 (GFP-H1), H1Mt (GFP-H1Mt), H1m20 (GFP-H1m20), or H1M1 (GFP-H1M1).

betweenHSURs1and2andmiR-16andmiR- 18. I. K. Guttilla, B. A. White, J. Biol. Chem. 284, 23204 for technical assistance, K. Tycowski and K. Riley for critical (2009). commentary, and A. Miccinello for editorial assistance.

142-3p requires further investigation. on December 1, 2010 19. T. Ideue, K. Hino, S. Kitao, T. Yokoi, T. Hirose, RNA 15, This work was supported by grant CA16038 from the It is not yet clear how down-regulation of miR- 1578 (2009). NIH. The content is solely the responsibility of the authors 27 benefits HVS. Down-regulation of the same 20. M. S. Ebert, J. R. Neilson, P. A. Sharp, Nat. Methods 4, and does not necessarily represent the official views host miRNA has been reported for another herpes- 721 (2007). oftheNIH.J.S.isaninvestigatorattheHoward virus, murine cytomegalovirus, upon infection of 21. A. Cimmino et al., Proc. Natl. Acad. Sci. U.S.A. 102, Hughes Medical Institute. 13944 (2005). cell lines and primary macrophages apparently also 22. Q. Liu et al., Nucleic Acids Res. 36, 5391 (2008). Supporting Online Material www.sciencemag.org/cgi/content/full/328/5985/1563/DC1 at the posttranscriptional level (23). Only a few 23. A. H. Buck et al., RNA 16, 307 (2010). Materials and Methods targets of miRNA-27, including the transcription 24. O. Ben-Ami, N. Pencovich, J. Lotem, D. Levanon, Y. Groner, Proc. Natl. Acad. Sci. U.S.A. 106, 238 (2009). Figs. S1 to S11 factors FOXO1, RUNX1 and PAX3, have been Table S1 18 24 25 25. C. G. Crist et al., Proc. Natl. Acad. Sci. U.S.A. 106, 13383

validated ( , , ). Thus, identification of www.sciencemag.org (2009). References additional targets of miR-27 in T cells transformed 26. We thank A. Giraldez for initial bioinformatic searches, 18 January 2010; accepted 14 April 2010 with HVS is needed, as well as elucidation of the R. C. Desrosiers for plasmids and cell lines, R. Jobava 10.1126/science.1187197 molecular mechanism by which association with HSUR 1 leads to miR-27 decay. References and Notes MicroRNA-33 and the SREBP Host 1. A. Ensser, B. Fleckenstein, Adv. Cancer Res. 93,91(2005). 2. B. Biesinger, J. J. Trimble, R. C. Desrosiers, B. Fleckenstein,

Virology 176, 505 (1990). Genes Cooperate to Control Downloaded from 3. S. I. Lee, S. C. Murthy, J. J. Trimble, R. C. Desrosiers, J. A. Steitz, Cell 54, 599 (1988). 4. D. A. Wassarman, S. I. Lee, J. A. Steitz, Nucleic Acids Res. Cholesterol Homeostasis 17, 1258 (1989). 5. J. C. Albrecht, J. Virol. 74, 1033 (2000). S. Hani Najafi-Shoushtari,1,2 Fjoralba Kristo,3 Yingxia Li,4 Toshi Shioda,1 David E. Cohen,4 6. A. Ensser, A. Pfinder, I. Müller-Fleckenstein, B. Fleckenstein, 3,5 1,2 J. Virol. 73, 10551 (1999). Robert E. Gerszten, Anders M. Näär * 7. S. C. Murthy, J. J. Trimble, R. C. Desrosiers, J. Virol. 63, 3307 (1989). Proper coordination of cholesterol biosynthesis and trafficking is essential to human health. The sterol 8. H. L. Cook, H. E. Mischo, J. A. Steitz, Mol. Cell. Biol. 24, regulatory element–binding proteins (SREBPs) are key transcription regulators of genes involved in 4522 (2004). cholesterol biosynthesis and uptake. We show here that microRNAs (miR-33a/b) embedded within introns 9. X. C. Fan, V. E. Myer, J. A. Steitz, Genes Dev. 11, 2557 (1997). 10.V.E.Myer,S.I.Lee,J.A.Steitz,Proc. Natl. Acad. Sci. U.S.A. of the SREBP genes target the adenosine triphosphate–binding cassette transporter A1 (ABCA1), an 89, 1296 (1992). important regulator of high-density lipoprotein (HDL) synthesis and reverse cholesterol transport, for 11. H. L. Cook et al., Curr. Biol. 15, 974 (2005). posttranscriptional repression. Antisense inhibition of miR-33 in mouse and human cell lines causes 12. P. Landgraf et al., Cell 129, 1401 (2007). up-regulation of ABCA1 expression and increased cholesterol efflux, and injection of mice on a western- 13. G. D. Cimino, H. B. Gamper, S. T. Isaacs, J. E. Hearst, – Annu. Rev. Biochem. 54, 1151 (1985). type diet with locked nucleic acid antisense oligonucleotides results in elevated plasma HDL. Our findings 14. M. Lagos-Quintana, R. Rauhut, W. Lendeckel, T. Tuschl, indicate that miR-33 acts in concert with the SREBP host genes to control cholesterol homeostasis and Science 294, 853 (2001). suggest that miR-33 may represent a therapeutic target for ameliorating cardiometabolic diseases. 15. V. N. Kim, J. Han, M. C. Siomi, Nat. Rev. Mol. Cell Biol. 10, 126 (2009). holesterol and other lipids play key roles including atherosclerosis, metabolic syndrome, 16. H. W. Hwang, E. A. Wentzel, J. T. Mendell, Science 315, in many physiological processes in meta- and type II diabetes, underscoring the importance 97 (2007). 17. Materials and methods are available as supporting Czoans, and aberrant cholesterol/lipid ho- of understanding fully how cholesterol/lipid ho- material on Science Online. meostasis has been linked to a number of diseases, meostasis is regulated (1, 2).

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Battling the Paper Glut THE ACADEMIC COMMUNITY CONTINUES TO BELIEVE THAT THE FORMAL SCHOLARLY PUBLISHING vitally important way for researchers to con- process separates sound research from shoddy or biased counterparts. Unfortunately, scholarly tribute to scholarship, and should reward it as publishing may not be able to effectively fulﬁ ll its role as a gatekeeper much longer. such. One way to accomplish this would be As soon as the “publish or perish” concept (the imperative to publish work constantly to a new generation of review impact indexes, further or sustain an academic career) surfaced in the United States in the early 1950s, academ- based on information provided by publishers ics criticized it openly as a recipe for disaster (1, 2). Nevertheless, in the early to mid-1980s, (3, 5). Effectiveness in peer-reviewing should administrators in universities systematically began to use the number of articles published per be viewed as an essential skill to acquire for year by individual faculty members as a measure of their productivity. Ph.D. students, worldwide. Journals should on December 1, 2010 The shift transformed scholarly publishing. Researchers began “salami slicing” their manu- demand that for every paper submitted, an scripts in ever smaller “least publishable units” and began rushing manuscripts to publication author provide three reviews of other man- before proper replication or evaluation of results. Multi-authored manuscripts increased, regard- uscripts. Perhaps if authors knew that their less of true contribution to the work. Doc- reviewing workload would increase dramati- toral students began to write dissertations cally with the number of papers they submit, as a series of publishable chapters, some they would craft fewer and better papers, ulti- submitted even before the defense. As a mately beneﬁ ting all involved. result, the quantity of articles published DONALD SIEGEL1* AND PHILIPPE BAVEYE2

in scholarly journals increased on aver- 1Department of Earth Sciences, Syracuse University, Syra- www.sciencemag.org age by about 200 to 300% from the early cuse, NY 13244, USA. 2SIMBIOS Centre, Abertay Uni versity, 1980s to the late 1990s (3). Dundee DD1 1HG, UK. Researchers in countries such as *To whom correspondence should be addressed. E-mail: China and India are subjected to a num- [email protected] bers game similar to that in the West, References sometimes with the added incentive of 1. L. Hurt, Coll. Engl. 23, 5 (1961). monetary rewards for articles published 2. R. W. Beard, J. Higher Ed. 36, 455 (1965). 3. P. Baveye, J. Scholarly Pub. 41, 191 (2010). Downloaded from in “top” journals. In 2008, China passed 4. M. Angell, Ann. Int. Med. 104, 261 (1985). the United States to become the second 5. P. C. Baveye, J. T. Trevors, Water Air Soil Pollut., 10.1007/ scholarly producer (in total number of s11270-010-0355-7 (2010). articles) after Europe. Researchers have reacted to this publication glut by developing bibliometric indices, such as India’s Courteous Creativity the h- and g-indexes, based on citation counts, to evaluate a researcher’s impact in their disci- pline. Perhaps these indexes do evaluate impact better than counting annual number of articles. IN HIS EDITORIAL “IRREVERENCE AND INDIAN However, in various ways, they also encourage researchers to publish more articles to directly science” (30 April, p. 547), R. A. Mashelkar infl ate their own citations or to cite friends who then cite them in return. observes that Indian science lacks adventure The top journals now are fl ooded with numbers of manuscripts beyond most editors’ capac- and a spirit of questioning established ideas. ity to handle. Reviewers are solicited to scrutinize not just manuscripts but also research pro- He suggests that the situation has deep roots posals and governmental reports. Yet, peer-reviewing is rarely, if ever, valued by academic in Indian culture and tradition. institutions as a fruitful way for researchers to spend their time, so fi nding good reviewers has I disagree. Creativity can and does exist become more and more diffi cult. in a society that values decorum over irrev- Researchers need to fi ght to contain the current paper glut. The number of articles pub- erence, such as India. In fact, a healthy skep- lished per year should never be used, under any circumstance, as a criterion in tenure or promo- ticism, an ability to be introspective, and an tion decisions, or to rank academic institutions. As the medical community proposed 25 years urge to revisit and reexamine existing ideas ago (4), researchers should never be allowed to include more than three publications per year have always been part of India’s intellectual in activity reports; in research proposals, principal investigators should cite no more than 10 tradition. Take, for example, literary works

papers. University administrators should consider peer-reviewing as not only legitimate, but a known as bhashyas, which are commen- ANNE-LOUISE QUARFOTH/ISTOCKPHOTO.COM CREDIT:

1466 17 SEPTEMBER 2010 VOL 329 SCIENCE www.sciencemag.org Published by AAAS live, then more complex population dynam- Photons take Cognitive ics are indicated. Understanding the archaeo- a random walk certainty logical chronology behind the peopling of the Qinghai-Tibet Plateau is critical to evaluating the tempo of selection operating on contem- 1477 1478 porary human populations. P. JEFFREY BRANTINGHAM,1* DAVID RHODE,2 DAVID B. MADSEN3 1Department of Anthropology, University of California, Los taries or interpretations of classical works. gins up to 3300 m by ~15,000 yr B.P. (4). Angeles, Los Angeles, CA 90095, USA. 2Desert Research Institute, Reno, NV 89512, USA. 3Texas Archaeological Bhashyas contain interpretations from oppos- Directly dated sites documenting human Research Laboratory, University of Texas at Austin, Austin, ing perspectives by different commentators. presence above 4000 m are younger still, at TX 78758, USA. The body of work, collectively known as ~11,000 to 8000 yr B.P. (1). These early sites Upanishads, is considered by many to be a col- represent intermittent, seasonal occupations *To whom correspondence should be addressed. E-mail: [email protected] laborative exploration of knowledge by stu- by populations who most likely spent much dent and teacher. In a culture that usually gives of their time at lower elevations. Foragers may References 1. P. J. Brantingham et al., in Human Adaptaton to Climate an exalted position to the teacher, questioning have established more permanent occupa- Change in Arid China, D. B. Madsen, F. H. Chen, X. Gao, authority is permissible, albeit with decorum. tions on the Plateau margins as high as 3300 Eds. (Elsevier, Amsterdam, 2007), pp. 129–150. Ideally, education should strike a balance m after ~7000 yr B.P. (5–7), but these groups 2. C. M. Beall et al., Proc. Natl. Acad. Sci. U.S.A. 107, 11459 (2010). between sound training in the fundamentals interacted extensively with agricultural pop- 3. M. Zhao et al., Proc. Natl. Acad. Sci. U.S.A. 106, 21230 and motivation to critically question those ulations in low-elevation environments. (2009). fundamentals in order to advance knowledge. Year-round occupation above 4000 m likely 4. D. B. Madsen et al., J. Archaeol. Sci. 33, 1433 (2006). 5. M. S. Aldenderfer, in Human Adaptaton to Climate C. V. Raman (who discovered the Raman became possible only after 4000 yr B.P. with Change in Arid China, D. B. Madsen, F. H. Chen, X. Gao, effect), Megnad Saha (distinguished astro- the emergence of dedicated pastoralist adap- Eds. (Elsevier, Amsterdam, 2007), pp. 151–165. physicist), and Jagdish Chandra Bose and tations centered on domesticated yaks (6, 8). 6. D. Rhode et al., J. Archaeol. Sci. 34, 600 (2007). 7. D. Rhode et al., Quat. Int. 218, 29 (2010). Satyen Bose (both renowned physicists), who If the genetic traits suggested by Simonson et 8. R. K. Flad, J. Yuan, S. C. Li, in Human Adaptaton to on December 1, 2010 functioned during the Indian colonial rule, al. and Yi et al. evolved in response to selec- Climate Change in Arid China, D. B. Madsen, F. H. Chen, were not only well trained in the fundamen- tion on populations living exclusively above X. Gao, Eds. (Elsevier, Amsterdam, 2007), pp. 167–203. tals of science but also capable of questioning 4000 m, then the genetic divergence dates established ideas. It is not clear that the lack of ~2750 yr B.P. reasonably agrees with the Response of an adventurous spirit in modern Indian archaeological evidence. If selection for these WE THANK BRANTINGHAM ET AL. FOR THEIR science, or for that matter, in the humanities traits occurred among populations below interest in our study; we agree that both molec- and the social sciences, can be readily attrib- 4000 m (2), where most Tibetans currently ular and archaeological evidence should be uted to India’s cultural roots.

T. N. NARASIMHAN www.sciencemag.org CORRECTIONS AND CLARIFICATIONS Department of Materials Science and Engineering, Uni- versity of California at Berkeley, Berkeley, CA 94720–1760, News Focus: “From pigs to people: The emergence of a new superbug” by D. Ferber (27 August, p. 1010). Tara Smith’s afﬁ li- USA. E-mail: [email protected] ation is the University of Iowa in Iowa City. Reports: “Down-regulation of a host microRNA by a Herpesvirus saimiri noncoding RNA” by D. Cazalla et al. (18 June, Archaeology Augments p. 1563). The third author should have been listed as Joan A. Steitz. The correction has been made in the HTML version online. Reports: “Evolution of an expanded sex-determining locus in Volvox” by P. Ferris et al. (16 April, p. 351). The legend for

Tibet’s Genetic History fi gure S12 (Alternative splicing of female and male MAT3) in the Supporting Online Material should include the citation Downloaded from A. Kianianmomeni et al., Plant Cell 20, 2399 (2008), which reported two instances of unregulated intron retention (corre- T. S. SIMONSON ET AL. (“GENETIC EVIDENCE sponding to E1_3.3, E3_5.4) and one instance of unregulated alternative splice site usage (corresponding to E13_16.5) in for high-altitude adaptation in Tibet,” Reports, female MAT3. A revised Supporting Online Material has been posted online at www.sciencemag.org/cgi/content/full/328/5976/ 2 July, p. 72) and especially X. Yi et al. 351/DC1. (“Sequencing of 50 human exomes reveals Reports: “N-doping of graphene through electrothermal reactions with ammonia” by X. Wang et al. (8 May 2009, p. 768). adaptation to high altitude,” Reports, 2 July, After publication, the authors discovered that the graphene sheet (GS) sample used to take the x-ray photoelectron spec- p. 75) estimate that the genetic divergence troscopy (XPS) and nanometer-scale secondary ion mass spectroscopy data in Fig. 4 was unintentionally oxidized by air from a leak that had not been detected during the experiment. The NH3 annealing environment for the GS sample in Fig. 4 of Tibetan populations with unique high- should be corrected to ~800 mtorr of NH and an estimated partial pressure of oxygen of tens of millitorr. altitude adaptations occurred as late as ~2750 3 Later, the authors found that as-made GSs annealed in NH without any oxygen showed little n-doping within the detec- years ago. We have investigated this same 3 tion limit of XPS, which is much lower than the doping level for the gas-phase–oxidized GS in Fig. 4. In a systematic study, problem from an archaeological perspec- they used XPS to observe n-doping and covalent N incorporation into the lattice of pre-oxidized GSs upon annealing in tive. Our results partly support the genetic- NH3 [X. Li et al., J. Am. Chem. Soc. 131, 15939 (2009)]. They found that graphene oxide (with reduced oxidation and based scenario but suggest some contradic- defect densities by stepwise thermal treatment) showed reduced n-doping levels upon NH3 annealing [X. Li et al., J. Am. tions between the two data sets. We currently Chem. Soc. 131, 15939 (2009)], suggesting that the degree of n-doping scales with the degree of oxidation or concen- have no evidence of permanent occupations tration of defects in the graphene lattice. on the Qinghai-Tibet Plateau before the The above fi ndings are consistent with each other and do not change the main conclusions of the original publication— i.e., that annealing of graphene in NH affords n-doping most likely at the edges and defect sites. The sample in Fig. 4 with middle Holocene, ~7000 years before the 3 unintended oxidation showed higher N signals than later samples without oxidation after similar NH annealing, because 1 3 present (yr B.P.) ( ), contrary to claims of gas-phase oxidation generated more defects and oxygen groups in the GS and increased its reactivity, allowing for large occupations as old as 30,000 yr B.P. (2, 3). amounts of n-dopants to be incorporated into the GS. This fi nding is consistent with the authors’ original suggestion and also Mobile foragers did exploit the Plateau mar- shows that a higher defect density in graphene introduced by gas-phase oxidation allows for higher n-doping.

www.sciencemag.org SCIENCE VOL 329 17 SEPTEMBER 2010 1467 Published by AAAS LETTERS

used to understand the demographic history Late Paleolithic inhabitants on the plateau. If TANG,11 GUOYI GAO,12 YONG CHEN,5 ZHEN LUO,5 of the Tibetan people. Our Report focused this is the case, even if the EPAS1 variant was LAMU GUSANG,5 ZHENG CAO,1 QINGHUI ZHANG,1 not on the demographic history of the Tibetan present in the early inhabitants of Tibet, strong WEIHAN OUYANG,1 XIAOLI REN,1 HUIQING LIANG,1 population, but rather the selection acting on selection would be needed to increase its fre- HUISONG ZHENG,1 YEBO HUANG,1 JINGXIANG LI,1 speciﬁ c putatively adaptive mutations segre- quency in the modern Tibetan gene pool. The LARS BOLUND,1 KARSTEN KRISTIANSEN,1,7 YIN- 1 1 1 gating in the Tibetan population. We included understanding that the majority of the current GRUI LI, YONG ZHANG, XIUQING ZHANG, RUIQ- 1,7 1 1 some limited demographic analyses because population of the Tibetan plateau may trace IANG LI, SONGGANG LI, HUANMING YANG, 1,3,7 1,7 1 they helped illuminate our results regard- their genetic ancestry back to quite recent RASMUS NIELSEN, * JUN WANG, * JIAN WANG * ing natural selection. The real demographic immigrants into Tibet, even though humans 1BGI-Shenzhen, Shenzhen 518083, China. 2The Graduate University of Chinese Academy of Sciences, Beijing 100062, model is clearly likely to be more complex have lived in Tibet for a much longer time— China. 3Department of Integrative Biology and Department than the simple models of two populations possibly with some continuity of culture—is of Statistics, University of California Berkeley, Berkeley, CA diverging from each other. For example, Zhao important for understanding the difference 94820, USA. 4Innovative Program for Undergraduate Stu- et al 1 dents, School of Bioscience and Biotechnology, South China . ( ) used mitochondrial DNA to argue between inferences based on archaeology and University of Technology, Guangzhou 510641, China. 5The that late settlers of the Tibetan plateau may inferences based on genetics. People’s Hospital of the Tibet Autonomous Region, Lhasa not have entirely replaced the original popula- XIN YI,1,2 YU LIANG,1,2 EMILIA HUERTA-SANCHEZ,3 850000, China. 6Department of Evolution and Ecology, Uni- 7 tion but that a small proportion of them carry XIN JIN,1,4 ZHA XI PING CUO,2,5 JOHN E. POOL,3,6 versity of California Davis, Davis, CA 95616, USA. Depart- ment of Biology, University of Copenhagen, DK-1165 Copen- XUN XU,1 HUI JIANG,1 NICOLAS VINCKENBOSCH,3 mitochondrial DNA lineages tracing back to hagen, Denmark. 8Innovative Program for Undergraduate THORFINN SAND KORNELIUSSEN,7 HANCHENG Students, School of Science, South China University of Tech- Letters to the Editor ZHENG,1,4 TAO LIU,1 WEIMING HE,1,8 KUI LI,2,5 nology, Guangzhou 510641, China. 9Genome Research Insti- RUIBANG LUO,1,4 XIFANG NIE,1 HONGLONG WU,1,9 tute, Shenzhen University Medical School, Shenzhen 518060, China. 10The People’s Hospital of Lhasa, Lhasa, 850000, China. Letters (~300 words) discuss material published MEIRU ZHAO,1 HONGZHI CAO,1,9 JING ZOU,1 YING Science 11The Military General Hospital of Tibet, Lhasa, 850007, China. in in the previous 3 months or issues of 1,4 1 1 1,2 SHAN, SHUZHENG LI, QI YANG, ASAN, PEIXI- 12 general interest. They can be submitted through The Hospital of XiShuangBanNa Dai Nationalities, Autono- ANG NI,1 GENG TIAN,1,2 JUNMING XU,1 XIAO LIU,1 mous Jinghong 666100, Yunnan, China. the Web (www.submit2science.org) or by regular TAO JIANG,1,9 RENHUA WU,1 GUANGYU ZHOU,1 mail (1200 New York Ave., NW, Washington, DC *To whom correspondence should be addressed. E-mail: MEIFANG TANG,1 JUNJIE QIN,1 TONG WANG,1 SHUI- [email protected] ( Ji.W.); [email protected]. 20005, USA). Letters are not acknowledged upon 1 1 1 cn (Ju.W.); [email protected] (R.N.) receipt, nor are authors generally consulted before JIAN FENG, GUOHONG LI, HUASANG, JIANGBAI

1 1 1 on December 1, 2010 publication. Whether published in full or in part, LUOSANG, WEI WANG, FANG CHEN, YADING Reference 1 1,2 1 letters are subject to editing for clarity and space. WANG, XIAOGUANG ZHENG, ZHUO LI, ZHUOMA 1. M. Zhao et al., Proc. Natl. Acad. Sci. U.S.A. 106, 21230 BIANBA,10 GE YANG,10 XINPING WANG,11 SHUHUI (2009).

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