to stoichiometric assembly of proteins into macromolecular machines. Key to this mechanism is a
nuclear import adaptor, Syo1, that specifically
recruits the two functionally and topologically
linked r-proteins Rpl5 and Rpl11. It guarantees
that this cargo pair remains bound together from
the time of synthesis in the cytoplasm until delivery to the nascent 5S rRNA in the nucleus (see
Fig. 4E). In the broader sense, synchronous nuclear transport of topologically linked and/or functionally related cargo may represent a general
strategy to streamline downstream nuclear processes that depend on temporally or spatially controlled assembly steps.
References and Notes
1. F. M. Boisvert, S. van Koningsbruggen, J. Navascués,
A. I. Lamond, Nat. Rev. Mol. Cell Biol. 8, 574
2. D. L. Lafontaine, D. Tollervey, Nat. Rev. Mol. Cell Biol. 2,
3. T. Pederson, R. Y. Tsai, J. Cell Biol. 184, 771 (2009).
4. J. R. Warner, Trends Biochem. Sci. 24, 437 (1999).
5. A. K. Henras et al., Cell. Mol. Life Sci. 65, 2334
6. D. Kressler, E. Hurt, J. Bassler, Biochim. Biophys. Acta
1803, 673 (2010).
7. J. P. Staley, J. L. Woolford Jr., Curr. Opin. Cell Biol. 21,
8. H. Tschochner, E. Hurt, Trends Cell Biol. 13, 255
9. Y. M. Chook, K. E. Süel, Biochim. Biophys. Acta 1813,
10. M. P. Rout, G. Blobel, J. D. Aitchison, Cell 89, 715
11. D. Görlich, U. Kutay, Annu. Rev. Cell Dev. Biol. 15, 607
12. M. Stewart, Nat. Rev. Mol. Cell Biol. 8, 195 (2007).
13. A. Ben-Shem et al., Science 334, 1524 (2011).
14. S. Ferreira-Cerca et al., Mol. Cell 28, 446 (2007).
15. G. Pöll et al., PLoS ONE 4, e8249 (2009).
16. M. Ciganda, N. Williams, Wiley Interdiscip. Rev. RNA 2,
17. M. Claussen, F. Rudt, T. Pieler, J. Biol. Chem. 274, 33951
18. J. Zhang et al., Genes Dev. 21, 2580 (2007).
19. S. Amlacher et al., Cell 146, 277 (2011).
20. M. A. Andrade, C. Petosa, S. I. O’Donoghue, C. W. Müller,
P. Bork, J. Mol. Biol. 309, 1 (2001).
21. D. Xu, A. Farmer, Y. M. Chook, Curr. Opin. Struct. Biol.
20, 782 (2010).
22. K. E. Süel, A. E. Cansizoglu, Y. M. Chook, Methods 39,
23. M. Marfori et al., Biochim. Biophys. Acta 1813, 1562
24. Y. Matsuura, M. Stewart, EMBO J. 24, 3681 (2005).
25. R. Bayliss, T. Littlewood, L. A. Strawn, S. R. Wente,
M. Stewart, J. Biol. Chem. 277, 50597 (2002).
26. S. A. Adam, R. S. Marr, L. Gerace, J. Cell Biol. 111, 807
27. B. Bradatsch et al., Mol. Cell 27, 767 (2007).
28. F. R. Bischoff, C. Klebe, J. Kretschmer, A. Wittinghofer,
H. Ponstingl, Proc. Natl. Acad. Sci. U.S.A. 91, 2587
29. B. J. Lee et al., Cell 126, 543 (2006).
30. K. E. Süel, H. Gu, Y. M. Chook, PLoS Biol. 6, e137 (2008).
31. J. D. Aitchison, G. Blobel, M. P. Rout, Science 274, 624
Acknowledgments: We thank A. Hendricks and C. Déforel
for technical assistance, J. Kopp and C. Siegmann from the
BZH Cluster of Excellence:CellNetworks crystallization platform,
J. Lechner and his team for mass spectrometry, T. Ruppert and
M. Mayer (Zentrum für Molekulare Biologie Heidelberg) for
access to the core facility for mass spectrometry for the HX-MS
experiments, J. Woolford for sharing published rpl5 mutant
plasmids, and L. Dimitrova (Hurt laboratory) for providing the
full-length ctKAP104 clone. Data collection was performed at
the European Synchrotron Radiation Facility, Grenoble. This
work was funded by the German Research Council (Hu363/9-4
to E.H. and SFB 638 to I.S.), the Swiss National Science
Foundation (PP00P3_123341 to D.K.), and the Japan Society
for the Promotion of Science (no. 21570195 and no.
21247032 to J.K. and Y. Y., respectively). I.S. and E.H. are
investigators of the Cluster of Excellence:CellNetworks, and
G.B. is a fellow of the Peter and Traudl Engelhorn Foundation.
D.K., G.B., I.S., and E.H. conceived the experiments. D.K., G.B.,
J.K., I.S., and E.H. analyzed the data. D.K., G.B., I.S., and E.H.
wrote the paper. D.K. and D.S. constructed yeast strains and
carried out all yeast genetic experiments. D.K., G.B., and S.A.
constructed plasmids. D.K. carried out tandem-affinity
purifications of yeast proteins. G.B. and D.K. performed
binding assays. G.B. determined crystal structures. G.S. carried
out the HX-MS experiments. D.P. and D.S. performed
fluorescence microscopy of live yeast cells. B.B. performed
in vitro FG-repeat binding assays. Y.O. performed in vitro import
assays with permeabilized HeLa cells under the supervision of
J.K. in the laboratory of Y. Y. J.K. measured binding constants
by BIAcore. D.K. and G.B. contributed equally to this study.
All authors commented on the manuscript. Atomic coordinates
and structure factors for the reported crystal structures have
been deposited with the Protein Data Bank (PDB) under accession
codes 4GMNO (ctSyo1) and 4GMN (ctSyo1/ctL5-N). The authors
declare no competing financial interests. Correspondence and
requests for materials should be addressed to D.K. (dieter.
firstname.lastname@example.org), I.S. ( email@example.com),
or E.H. ( firstname.lastname@example.org).
4 July 2012; accepted 6 September 2012
Materials and Methods
Figs. S1 to S12
Tables S1 to S5
Gene Loops Enhance
Sue Mei Tan-Wong,1* Judith B. Zaugg,2* Jurgi Camblong,1* Zhenyu Xu,3
David W. Zhang,4 Hannah E. Mischo,1 Aseem Z. Ansari,4 Nicholas M. Luscombe,2,5,6
Lars M. Steinmetz,3† Nick J. Proudfoot1†
Eukaryotic genomes are extensively transcribed, forming both messenger RNAs (mRNAs)
and noncoding RNAs (ncRNAs). ncRNAs made by RNA polymerase II often initiate from
bidirectional promoters (nucleosome-depleted chromatin) that synthesize mRNA and ncRNA
in opposite directions. We demonstrate that, by adopting a gene-loop conformation, actively
transcribed mRNA encoding genes restrict divergent transcription of ncRNAs. Because
gene-loop formation depends on a protein factor (Ssu72) that coassociates with both the
promoter and the terminator, the inactivation of Ssu72 leads to increased synthesis of
promoter-associated divergent ncRNAs, referred to as Ssu72-restricted transcripts (SRTs).
Similarly, inactivation of individual gene loops by gene mutation enhances SRT synthesis.
We demonstrate that gene-loop conformation enforces transcriptional directionality on
otherwise bidirectional promoters.
Ssu72, localized at the 5′ and 3′ ends of genes
(12, 13). On the basis of quantitative 3C analysis,
we initially confirmed that mutation of Ssu72
(ssu72-2) prevents gene-loop formation across
FMP27 (Fig. 1A). We also detected an increase
in promoter-associated antisense ncRNA and in-
creased Pol II density over the FMP27 promoter
region in ssu72-2 (Fig. 1, B and C). Furthermore,
we observed unanticipated genetic interactions
between either Ssu72- or pAC-associated Pta1
and the nuclear exosome component Rrp6, which
is responsible for the degradation of many ncRNAs,
especially cryptic unstable transcripts (CUTs)
(fig. S1) (6, 7). Taken together, our initial results
indicate that the loss of gene-loop formation by
inactivation of Ssu72 results in the production of
Eukaryotic genomes are ubiquitously tran- scribed, generating an extensive network ofnoncoding RNAs(ncRNAs) (1, 2). Most
ncRNAs are made by RNA polymerase II (Pol II),
which can initiate transcription nonspecifically
and bidirectionally on nucleosome-depleted chro-
matin (3–5). Although this promiscuous tran-
scription is partly restricted by rapid transcript
degradation (6, 7), we demonstrate that actively
transcribed genes adopt a gene-loop conforma-
tion that reduces aberrant transcription by fo-
cusing Pol II into productive mRNA synthesis
(see the supplementary materials and methods).
Gene-loop formation depends on both promoter-
associated transcription factors and polyade-
nylation complex (pAC) factors (8–11) such as
1Sir William Dunn School of Pathology, University of Oxford,
South Parks Road, Oxford OX1 3RE, UK. 2European Molecular
Biology Laboratory, European Bioinformatics Institute, Cambridge CB10 1SD, UK. 3Genome Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg,
Germany. 4Department of Biochemistry, University of Wisconsin–
Madison, 433 Babcock Drive, Madison, WI 53706, USA. 5Uni-
versity College London Genetics Institute, Gower Street, London
WC1E 6BT, UK. 6Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail:
email@example.com (L.M.S.); firstname.lastname@example.org.