PERSPECTIVES

8. T. Jung et al., Langmuir 20, 8587 (2004).

stable interfaces. The rheology of the incorpo-
rated biopolymers, not molecular recognition,
may very well be the critical factor in deter-
mining which internal facets are expressed in
single crystals of bioinorganic composites.
Given the dire consequences of ocean
acidification (15) and the enormous problem
of CO2 sequestration, the carbonates (e.g.,
CaCO3) may be the most ecologically impor-
tant minerals of the century. Approaches
such as the ones described by Li et al. will
be essential for a deeper understanding of
CaCO3 crystallization (and its dissolution) in
biological systems.
References and Notes
1. B. L. Smith et al., Nature 399, 761 (1999).
2. H. Li, H. L. Xin, D. A. Muller, L. A. Estroff, Science 326,
1244 (2009).
3. A. Berman et al., Science 259, 776 (1993).
4. J. Aizenberg, J. Hanson, T. F. Koetzle, S. Weiner, L. Addadi,
J. Am. Chem. Soc. 119, 881 (1997).
5. K. Henriksen, S. L. S. Stipp, J. R. Young, M. E. Marsh, Am.
Mineral. 89, 1709 (2004).
6. L. Addadi, S. Weiner, Nature 411, 753 (2001).
7. B. Kahr, R. W. Gurney, Chem. Rev. 101, 893 (2001).
9. C. A. Orme et al., Nature 411, 775 (2001).
10. P. A. Midgley, R. E. Dunin-Borkowski, Nat. Mater. 8, 271
(2009).
11. P. A. Midgley, M. Weyland, Ultramicroscopy 96, 413
(2003).
12. J. M. Thomas et al., Angew. Chem. Int. Ed. 43, 6745
(2004).
13. H. Li, L. A. Estroff, Adv. Mater. 21, 470 (2009).
14. J. Aizenberg, A. J. Black, G. M. Whitesides, Nature 398,
495 (1999).
15. J. C. Orr et al., Nature 437, 681 (2005).
16. This work was supported by the NSF (grant
CHE-0809845).
10.1126/science.1183122

CELL BIOLOGY
The machinery that exports small RNAs from

Nuclear Export of Small RNAs the cell nucleus is fine-tuned to recognize

when the processing of each class of RNA
Murray Stewart has been completed.

I
n eukaryotic organisms, RNAs are gen-
erated in the cell nucleus but many per-
form their functions in the cytoplasm and
so need to be exported through pores in the
nuclear envelope. Because many RNAs must
be processed or modified in the nucleus, it
is important that only the fully mature RNA
is exported. Stringent molecular recogni-
tion is required to identify the class of RNA
and ensure that its processing has been com-
pleted. On page 1275 of this issue, Okada et
al. provide remarkable insight into this export
process for pre-microRNAs (1), which—after
further digestion in the cytoplasm—function
to regulate specific mRNAs. Unexpectedly,
pre-microRNAs are recognized for export in
a quite different way to that recently reported
by Cook et al. for transfer RNA (tRNA) (2).
Large mRNAs and a number of
smaller RNAs—including tRNA and pre-
microRNA—are transported to the cyto-
plasm through nuclear pores by nuclear
export factors, or exportins, that are spe-
cific for each class of RNA (3). Both pre-
microRNA and tRNA are exported by
exportins of the β-karyopherin superfamily,
in conjunction with the guanosine triphos-
bound Ran (RanGTP) in the nucleus. Weak
interactions between nuclear pore proteins
(nucleoporins) and the exportins mediate
equilibration between the nucleus and cyto-
plasm though nuclear pores. When the export
complex reaches the cytoplasm, it dissoci-
ates, releasing the cargo. The RanGTP is then
hydrolyzed (providing the energy to power the
process), and the Ran and exportins are recy-
cled to the nucleus to participate in another
export cycle.
Both pre-microRNA and tRNA are gen-
erated from larger precursor RNAs that
undergo considerable nuclear processing. The
exported forms are both based on stem loops
with 3′ overhangs that are crucial for recog-
nition by the export machinery. After tran-
scription, capping, and polyadenylation, pri-
mary microRNAs are cleaved by the Drosha
ribonuclease to generate pre-microRNA stem
loops containing about 65 nucleotides that
have a two-nucleotide 3′ overhang. tRNAs
-t 6 7
Xp
o
4
5 Exp
are also cleaved from a larger precursor that
generates a three-nucleotide (CCA) 3′ over-
hang, located on the “acceptor helix” of the
L-shaped tRNA molecules, where amino
acids are attached in aminoacyl-tRNAs.
Probably for quality control, export gener-
ally occurs only after this step has been com-
pleted. Because nuclear processing of both
tRNA and microRNA needs to be completed
before they are exported, an effective molec-
ular recognition is required to distinguish
between the mature molecules and their vari-
ous precursor forms.
Exp-5 and Xpo-t are horseshoe-shaped
molecules constructed from HEAT repeats
(see the figure). HEAT repeats are based on
two antiparallel α helices that generate mol-
ecules resembling helical springs (4, 5). Like
other karyopherins (5), these springlike mol-
ecules are intrinsically flexible, which is cru-
cially important for both cargo binding and
release (2). In the cargo-bound state, RanGTP
-5
13 12

3'
phatase (GTPase) Ran. Pre-microRNA is 1 1
N N
exported primarily by exportin-5 (Exp-5) 3'
and tRNA by exportin-t (Xpo-t).
14

The export of pre-microRNA and tRNA 15

an
R

employs the general Brownian ratchet model an 19 GT

R

seen with other nuclear trafficking pathways
(4). This model is based on the nucleotide
state of Ran, which binds guanosine triphos-
phate (GTP) in the nucleus but guanosine
diphosphate (GDP) in the cytoplasm. Expor-
tins bind their cargoes together with GTP-
MRC Laboratory of Molecular Biology, Hills Road, Cam-
bridge CB2 0QH, UK. E-mail: ms@mrc-lmb.cam.ac.uk
GT C P 20
P C
tRNA export Pre-microRNA export
How tRNA and pre-microRNA bind to their exportins. The exportins are constructed from HEAT repeats
(yellow and red) and adopt a roughly U-shaped conformation. An RNA double helix (black) is bound along
most of the exportin’s length, interacting mainly with positively charged residues on the exportin (for clarity,
only the acceptor helix of tRNA is shown). RanGTP (blue) binds to HEAT repeats at either end of the exportin,
helping to maintain the U-shaped conformation. In both exportins, the 3′ end is buried in a cavity formed
by a group of HEAT repeats (red), yet the orientation of the tRNA helix (2) is opposite to that seen with pre-
microRNA (1).

www.sciencemag.org SCIENCE VOL 326 27 NOVEMBER 2009 1195

Published by AAAS
 PERSPECTIVES
interacts with regions near the N and C termini
of Xpo-t and Exp-5 to clamp them around a
double-stranded RNA helix (the tRNA accep-
tor helix or the pre-microRNA stem, respec-
tively). This configuration facilitates interac-
tions between positively charged residues on
the inner surface of the exportin horseshoe
and negatively charged RNA backbone phos-
phates. This binding is independent of the
sequence of the RNA, enabling the exportins
to bind a range of different RNAs.
Recognition of 3′ overhangs is crucial for
the function of both Exp-5 and Xpo-t, but
this is achieved in quite different ways, and
remarkably, the RNA helix that is gripped by
the karyopherin runs in opposite directions
(see the figure). In Exp-5, the two-nucleotide
3′ overhang is recognized by a deep tunnel-
like cavity formed by HEAT repeats 12 to 15
(1); in contrast, the tRNA 3′ CCA overhang is
recognized by a groove formed by the inter-
repeat sequences between HEAT repeats 4
and 7 of Xpo-t (2). In both pre-microRNA
and tRNA, steric clashes involving the 5′ end
VIROLOGY
Moving Forward in HIV Vaccine
Development
Norman L. Letvin
E
arly in the AIDS epidemic, efforts
to develop a vaccine to prevent HIV
infection were focused on two vaccine
strategies. It was hoped that the HIV envelope
glycoprotein (gp120) would generate an anti-
body response that would block the initiation
of HIV infections, and that a recombinant
canary pox construct expressing HIV genes
would elicit cellular immune responses that
would inhibit HIV replication. However, the
nature of the immune responses elicited by
each of these vaccine candidates in monkeys
and human subjects proved disappointing (1–
3). Despite these results, the U.S. Department
of Defense (DOD) proceeded with plans to
initiate the RV144 trial in Thailand to test the
efficacy of a vaccine regimen that included
both agents. At that time, the U.S. National
Institutes of Health assumed responsibil-
ity for a major component of the DOD HIV/
AIDS program, and provided a substantial
proportion of the funding for the Thai trial.
Department of Medicine, Beth Israel Deaconess Medical
Center, Harvard Medical School, Boston, MA, 02115, USA.
E-mail: nletvin@bidmc.harvard.edu
1196 27 NOVEMBER 2009 VOL 326 SCIENCE www.sciencemag.org
seem to be important in recognizing the cor-
rect length of the 3′ overhang.
Both tRNAs and pre-microRNAs are rec-
ognized primarily by the 3′ overhang, which
poses the question of why a separate export
pathway has evolved for pre-microRNA. One
possible explanation relates to the stability of
these different classes of RNA. tRNAs have
a compact, tightly folded structure and so
are comparatively stable. By contrast, while
they remain in the nucleus, pre-microRNAs
are susceptible to digestion by ribonucleases
that digest RNAs from either end. Many other
RNAs are protected by modifications at their
ends (such as adding a 5′ cap and 3′ polyA),
but these are removed when pre-microRNAs
are generated from primary microRNAs.
Exp-5 grasps its cargoes as in a mitt, resulting
in an intimate contact that may offer greater
protection from digestion prior to export than
would be afforded by binding to Xpo-t.
This protective feature may be important
in a wider context because, in addition to pre-
microRNAs, Exp-5 also exports other small
The decision to commit resources to carry
out the RV144 trial in the face of the limited
immunogenicity of both vaccine prototypes
created considerable concern within the HIV
vaccine research community (4). Opponents
of the trial argued that the resources being
dedicated to the vaccine study would be bet-
ter spent on basic research that might inform
HIV vaccine development. As a consequence
of discussions among HIV vaccine inves-
tigators, a viral load set point—the amount
of virus present in the blood during chronic
infection—was added as one of the primary
endpoints (an outcome that can be measured
objectively to determine whether the vacci-
nation was beneficial) in the RV144 trial a
year after its initiation. I shared the concerns
of my colleagues about the wisdom of pro-
ceeding with this clinical trial, but my oppo-
sition was tempered by an appreciation for
the commitment made by the DOD and the
government of Thailand to test this vaccine.
It is unfortunate that there was a partial
release of data before the formal announce-
ment and publication of the trial findings,
but we can now digest the complete results
Published by AAAS
RNAs, such as human Y1 RNA and adenovi-
rus VA1 RNA. It seems likely that all these car-
goes are recognized in similar ways, although
the length of the 3′ overhang appears to influ-
ence the strength of each interaction (1).
Overall, the structures of both Exp-5 and
Xpo-t show analogous recognition strate-
gies to ensure that nuclear processing of their
cargoes has been completed before they are
exported. However, although burying the 3′
overhang is central to both, the precise way
in which Exp-5 and Xpo-t achieve this dif-
fers in ways that facilitate protection of pre-
microRNAs and accommodation of tRNA
aminoacylation.
References and Notes
1. C. Okada et al., Science 326, 1275 (2009).
2. A. G. Cook et al., Nature 461, 60 (2009).
3. A. Köhler, E. Hurt, Nat. Rev. Mol. Cell Biol. 8, 761
(2007).
4. M. Stewart, Nat. Rev. Mol. Cell Biol. 8, 195 (2007).
5. E. Conti et al., Curr. Opin. Struct. Biol. 16, 237 (2006).
6. Supported in part by a Wellcome Trust Programme Grant.
10.1126/science.1183273
Clinical trial results of a vaccine regimen to
prevent HIV infection show modest protection
and suggest that changes are needed in the
direction of future research.
in the recently published report of the trial
(5). The basic message is that the trial results
show modest vaccine efficacy at 3 years after
initiating vaccination, with a lowered rate
of HIV infection by 31.2% (P = 0.04)—51
infections in the 8197 vaccine recipients and
74 infections in the 8198 placebo recipients.
This evaluation was done using a modified
Intent-to-Treat analysis, in which all unin-
fected participants enrolled in the trial were
considered, including those who missed a
shot or did not receive one at the appropriate
time. The modest statistical significance of
the finding of protection is lost, however, if a
different strategy—a Per Protocol analysis—
is employed for determining which study
volunteers to analyze. In the Per Protocol
analysis, only a subset of trial participants
are considered—those who had followed the
study design precisely and who also did not
become infected during the 6-month period
of vaccination. Both of these commonly
employed analytic strategies were formally
designated as the criteria for evaluating the
study data before the trial was initiated, and
therefore it is appropriate to evaluate the trial