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Eukaryotic ribosome assembly, transport and quality control
Cohue Peña1,2, Ed Hurt3 & Vikram Govind Panse2
Eukaryotic ribosome synthesis is a complex, energy-consuming process that takes place across the nucleolus, nucleoplasm
and cytoplasm and requires more than 200 conserved assembly factors. Here, we discuss mechanisms by which the ribosome
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
assembly and nucleocytoplasmic transport machineries collaborate to produce functional ribosomes. We also highlight recent
cryo-EM studies that provided unprecedented snapshots of ribosomes during assembly and quality control.
The ribosome translates the genetic code into proteins. In the
eukaryotic model organism Saccharomyces cerevisiae, this universal
two-subunit machine is composed of a large 60S subunit, consisting
of three rRNAs (25S, 5.8S, 5S) and 46 different ribosomal proteins
(r proteins), and a small 40S subunit, consisting of 18S rRNA and
33 r proteins1,2. In contrast to that of their bacterial and archaeal
counterparts, assembly of eukaryotic ribosomes requires more than
200 conserved assembly factors, including diverse RNA-binding
proteins, endo- and exonucleases, RNA helicases, GTPases and
ATPases. These assembly factors promote pre-rRNA folding and
processing, remodeling of protein–protein and RNA–protein
networks, nuclear export and quality control3.
Eukaryotic ribosome assembly is a complex process spanning
various cellular compartments (Box 1). It starts with transcription
of a single RNA polymerase (Pol) I transcript, the 35S pre-rRNA, in
the nucleolus. This transcript undergoes several processing steps to
eventually give rise to mature 18S, 5.8S and 25S rRNAs (Fig. 1). In
the 35S pre-rRNA, 18S rRNA is preceded by a 5′ external transcribed
spacer sequence (5′ ETS) and followed by the internal transcribed
spacer sequence ITS1 which, together with ITS2, separates 5.8S rRNA
from the downstream 25S rRNA. Cotranscriptional cleavage at the A1
and A2 sites releases the 20S pre-rRNA, associated assembly factors
and r proteins as the earliest 40S preribosome. The remaining 27S
A2 pre-rRNA recruits 60S-specific r proteins and assembly factors
to form the earliest pre-60S ribosome. 60S pre-ribosomes undergo
sequential pre-rRNA processing to yield mature 25S and 6S rRNA
before nuclear export. Exported 40S pre-ribosomes containing 20S
pre-rRNA undergo final RNA processing to yield mature 18S rRNA,
and 6S pre-rRNA within exported 60S pre-ribosomes gets trimmed
to mature 5.8S rRNA.
A critical task for the ribosome assembly machinery is to precisely
incorporate r proteins onto dynamically folding pre-rRNA. This is a
logistical challenge given that r proteins, which are synthesized in the
cytoplasm, need to be targeted to the nucleus and then handed over
to the ribosome assembly machinery. Recent studies have provided
1Instituteof Biochemistry, ETH Zurich, Zurich, Switzerland. 2Institute of Medical
Microbiology, University of Zurich, Zurich, Switzerland. 3Biochemie Zentrum
Heidelberg, University of Heidelberg, Heidelberg, Germany. Correspondence
should be addressed to V.G.P. (vpanse@imm.uzh.ch).
Received 6 June; accepted 27 July; published online 7 September 2017;
doi:10.1038/nsmb.3454
nature structural & molecular biology  VOLUME 24  NUMBER 9  SEPTEMBER 2017 689
mechanistic insights into how r proteins are guided to their rRNA-
binding site on the maturing preribosome4,5. Moreover, advances in
cryo-EM revealed structural snapshots of different stages of eukaryo-
tic ribosome assembly, enabling further hypothesis-driven functional
studies. In this Review, we discuss the current view of ribosome
assembly in the context of recent structural insights and focus on how
structure-guided functional studies have advanced the understanding
of ribosome assembly and nucleocytoplasmic transport.
Targeting r proteins to assembling preribosomes
During a single 90-min generation, a yeast nucleus imports ~14
million r proteins for assembly and at the same time exports 200,000
preribosomes to the cytoplasm6. Rapid transport of r proteins,
assembly factors, and preribosomes through nuclear pore complexes
(NPCs) is essential for ribosome production7. In yeast, Kap104, Pse1
and Kap123 are the primary importins that target r proteins to the
nuclear compartment8. Unlike typical import cargos, r proteins are
disordered, which makes them prone to nonspecific interactions with
nucleic acids, aggregation and degradation in their nonassembled
state2. Thus, nuclear import of r proteins and their subsequent trans-
fer to their rRNA-binding site on the assembling preribosome poses
a challenge. Another challenge for the ribosome assembly machinery
is to ensure that stoichiometric levels of r proteins are incorporated
into every preribosome before nuclear export.
Previous studies have revealed that the ribosome-anchored NAC
and SSB–RAC chaperones stabilize aggregation-prone r proteins in
the cytoplasm before their nuclear import9. Simultaneous inactiva-
tion of NAC and SSB–RAC induces aggregation of r proteins of both
ribosomal subunits. Apart from these chaperone systems, importins,
in addition to their role in mediating their nuclear import, also sta-
bilize r proteins10. Owing to the high demand for r proteins during
ribosome formation, however, these factors are not sufficient, and
specialized mechanisms have evolved to protect r proteins during
their journey to the nucleolus.
This is achieved by a functional class of dedicated chaperones that
interact with newly synthesized r proteins and facilitate their nuclear
import and/or escort them to preribosomes4. At least nine r pro-
teins, uS3 (Rps3), uS11 (Rps14), eS26 (Rps26), uL3 (Rpl3), uL4 (Rpl4),
uL14 (Rpl23), uL16 (Rpl10), uL18 (Rpl5) and uL5 (Rpl11) (r protein
nomenclature according to Ban et al.11), are bound by dedicated
chaperones in vivo. Affinity purification of several of these chaper-
ones selectively enriches the mRNA encoding their r-protein client,
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Box 1  State of the art of eukaryotic ribosome assembly

Eukaryotic ribosome assembly begins with RNA Pol I–driven transcription of rDNA repeats in the nucleolus to produce 35S pre-rRNA, the precursor to mature
18S, 5.8S and 25S rRNAs40. The emerging 35S pre-rRNA associates with U3, several small nucleolar RNAs (snoRNAs), 40S-specific r proteins and assembly
factors to form the 90S preribosome. Cotranscriptional cleavage at site A2 then separates the small subunit from the emerging large pre-60S subunit, both of
which undergo independent maturation pathways. Transiently associating assembly factors drive maturation of the preribosomal subunits as they travel across
the nucleoplasm and eventually translocate to the cytoplasm. This process is aided by multiple transport receptors that facilitate translocation of preribosomes
through NPCs into the cytoplasm. Prior to achieving translation competence, pre-ribosomes undergo functional proofreading, which is intimately linked to the
release of remaining assembly factors and transport factors.

Cytoplasm
Nucleolus Nucleoplasm

5S RNP 60S

Pol III
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

Pre-60S

Pol I

A2 cleavage
40S
rDNA

90S Pre-40S

Pol I

Emerging
35S rRNA r proteins

Assembly factors

suggesting that r proteins are captured as they emerge from the
translating ribosome12. However, it is still unclear how dedicated
chaperones identify their specific target r proteins at translating
ribosomes. Below, we review several of these dedicated chaperones
and the mechanisms by which they target r proteins to assembling
ribosomes (Fig. 2).
Yar1, an ankyrin-repeat protein, binds to the N-terminal domain
of uS3 in the cytoplasm and accompanies the r protein into the
nucleus13–15 (Fig. 2a). Surprisingly, uS3 contains a nuclear locali-
zation signal (NLS) in its N domain that interferes with the Yar1-
binding site and recruits the classical importin dimer Kap60–Kap95.
However, when associated with Yar1, the C-terminal domain of uS3
interacts with a second copy of uS3. This allows binding of Yar1 to
one N-terminal domain and Kap60 to the second N-terminal domain
of dimerized uS3, thereby orchestrating simultaneous nuclear import
and protection of this r protein.
Rrb1 and Sqt1 are both WD-repeat β-propeller proteins that bind
to uL3 and uL16, respectively12,16 (Fig. 2b,c). Rrb1 localizes to the
nucleolus, and its overexpression leads to nuclear accumulation of
uL3. Translation inhibition mislocalizes Rrb1 to the cytoplasm17. This
suggests that Rrb1 accompanies uL3 into the nucleus, but it remains
unclear how their nuclear import is mediated. In contrast, uL16 is
incorporated into the late cytoplasmic pre-60S subunit, and therefore
the Sqt1–uL16 pair is not imported into the nucleus18. Structural
analysis of Sqt1 bound to the N-terminal extension of uL16 suggests
that Sqt1 protects the N terminus of uL16 during its incorporation
into the preribosome12. Release of Sqt1 might then allow the
N-terminal extension of uL16 to stably incorporate into its cognate
binding site helix H89 of the 25S rRNA.
Syo1 adopts an elongated α-solenoid fold through an unusual com-
bination of four armadillo repeats and six HEAT repeats19. It is unique
in that it functions as import adaptor for two different r proteins,
uL18 (Rpl5) and uL5 (Rpl11), via two different binding sites (Fig. 2d).
An N-terminal proline-tyrosine-NLS in Syo1 recruits the importin
Kap104 and facilitates simultaneous coimport of the r-protein clients.
Upon arrival in the nucleus, the trimeric Syo1–uL18–uL5 cargo com-
plex is released in a RanGTP-dependent manner. Whether coimport
of uL18 or uL5 into the nucleus is affected in Syo1 mutants that are
selectively impaired in binding these r proteins, however, remains
unclear19. uL18 and uL5 are functionally related and, together with
5S rRNA, they are incorporated into early pre-60S subunits as part of
the 5S ribonucleoprotein (RNP) complex. The Syo1–uL18–uL5 trimer
therefore serves as an assembly platform to allow 5S rRNA recruit-
ment to form a pre-5S RNP. In this complex, Syo1 binds to the same
surface on uL5 that interacts with helix H84 of 25S rRNA, suggesting
that Syo1 protects this binding site before being replaced by helix H84
(ref. 20). It is unclear, however, if Syo1 remains associated with the 5S
RNP during its incorporation into pre-60S subunits.
Acl4 is a tetratricopeptide-repeat-containing protein that, unlike
the aforementioned dedicated chaperones, does not bind to the
N-terminal region of uL4 (refs. 21,22) (Fig. 2e). Instead, one copy of
Acl4 binds to a long internal loop in uL4, while a second copy of Acl4

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B1L eS26 is extracted in the nucleus from the importin by an unloading

A0 A1 D A2 A3 B1S E C2 C1 B2 B0
35S 18S 5.8S 25S
Nucleolus
5S
factor, the escortin Tsr2, without the aid of RanGTP. Once bound
5′ ETS ITS1 ITS2 3′ ETS
to Tsr2, eS26 is shielded from proteolysis, enabling its safe transfer
Utp24 cleavage A0/A1 Rnt1/Rex1 cleavage B0/B2 Rex1, Rex2, Rex3
B1L exonuclease to the 90S preribosome. Intriguingly, in mature ribosomes, eS26 is
D A2 A3 B1S E C2 C1 located between the r protein uS11 and the very 3 end of 18S rRNA2.
32S
Although uS11 could be readily assigned in recent cryo-EM struc-
? cleavage A2
B1L tures of 90S pre-ribosomes, eS26 could not be visualized in these
D A3 B1S E C2 C1
structures29–31, even though mass spectrometric analysis indicates
20S 27S
Las1 cleavage C2
its presence on the yeast 90S (refs. 30,31). It is conceivable that eS26
Mrp cleavage A3
? processing B1L has not achieved its native state, thus precluding the reliable modeling
Rat1 exonuclease B1S
of its native structure on the 90S. Alternatively, missing densities at
E C1
the very 3′ end of 18S rRNA, following ITS1 on the 90S, suggest that
7S 25.5S
eS26 might be present in a flexible region, making it refractory to
Rrp6 (exosome)
exonuclease E′
Rat1 exonuclease C1
structural studies.
D E Recently, Bcp1 was shown to function as an escortin for the r pro-
20S 6S 25S 5S tein uL14 (Rpl23) by extracting uL14 from its importins Kap121 and
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

Nucleoplasm Kap123 in a RanGTP-dependent manner32 (Fig. 2g), but how eS26

D E
20S 6S
Nob1 cleavage D
Rex1, Rex2, Ngl2
exonuclease E
18S 5.8SL/S 25S
Figure 1  Pre-rRNA processing pathway. The Pol I–transcribed 35S pre-
rRNA contains sequences for 18S, 5.8S and 25S rRNA, flanked and
separated by external transcribed spacers (ETS) and internal transcribed
spacers (ITS) (indicated in red). Pre-5S rRNA is transcribed by RNA
Pol III. A series of co- and post-transcriptional endo- and exonucleolytic
events removes the spacer sequences to yield mature rRNA (pre-
rRNA intermediates are indicated in blue, enzymes and corresponding
processing sites in green). pre-rRNA processing is reviewed in detail in
ref. 40. Figure adapted from ref. 3.
binds to its eukaryotic-specific C-terminal extension23. Reminiscent
of Yar1–uS3 nuclear import, the C-terminally bound copy of Acl4
can be displaced by the importin Kap104 to form an import-
competent heterotrimeric complex. Upon arrival in the nucleus,
Acl4–uL4 is released from Kap104 in a Ran-dependent manner,
thereby permitting its incorporation into the 60S preribosome. Thus,
like Yar1, Acl4 has a dual function to facilitate nuclear import and
protect unassembled uL4.
How the release of an r protein from its dedicated chaperone is
coupled to its incorporation into the assembling preribosome remains
unknown. In case of Acl4–uL4, an interaction of the uL4 eukaryotic-
specific C-terminal extension with eL18 on the 60S preribosomal
surface is thought to trigger energy-independent disassembly of
the Acl4–uL4 complex and allow uL4 incorporation21. In contrast,
incorporation of uS11 is catalyzed by the essential chaperone Fap7 in
an ATP-dependent manner. Fap7 binds to and stabilizes uS11 both
in vivo and in vitro24–27. The ATPase activity triggers uS11 release onto
helix H23 within 18S rRNA26,27. Structural analysis of the Fap7–uS11
complex revealed that Fap7 serves as an RNA mimic for helix H23,
the binding site of uS11 in mature 40S subunits25,26. RNA mimicry
may provide a surrogate platform to fold an r protein and prepare
it for loading on the preribosome. In addition, complex formation
with a dedicated chaperone may prevent nonproductive interactions
between the r protein and other cellular RNAs.
In contrast to r proteins described above, a different mechanism
is employed by the r protein eS26 to reach the 90S preribosome28
(Fig. 2f). eS26, like a typical import cargo, directly binds to importins
for targeting to the nucleus. However, unlike a typical import cargo,
and uL14 are released from their escortins remains unclear.
Cytoplasm
How does the ribosome assembly machinery guarantee stoichio-
metric integration of r proteins into pre-ribosomes? At least two dif-
ferent mechanisms seem to ensure that correct levels of r proteins
are delivered to the preribosome. As discussed previously, the sym-
5S
portin Syo1 has been proposed to coordinate the transfer of spa-
tially proximal r proteins uL18 and uL5 onto 5S rRNA19 (Fig. 2d).
Here, Syo1 employs separate binding sites to directly bind r proteins
uL18 and uL5 and synchronize their import for 5S RNP assembly.
The second mechanism involves the prefabrication of ribosomal
protein complexes, as exemplified by the ATPase Fap7 that assem-
bles a native-like uS11–eS26 ribosomal protein complex before its
stable incorporation into the 90S preribosome27 (Fig. 2f). uS11, when
bound to Fap7, directly recruits eS26 through tertiary contacts found
between these r proteins on the mature 40S subunit. Mutations in
these conserved contacts preclude stoichiometric prefabrication
of the uS11–eS26 complex. Fap7 ATPase activity then unloads the
uS11–eS26 complex onto rRNA helix H23. In addition, several r proteins
cluster on the surface of the ribosome through a network of intricate
tertiary contacts33. Prefabricating these r-protein subcomplexes
through tertiary contacts before their assembly also ensures accuracy
of ribosome production.
In the absence of dedicated chaperones, orphaned r proteins are
efficiently recognized and degraded by the cellular protein quality-
control machinery. Recent work has implicated the HECT-domain E3
ubiquitin ligase Tom1 in clearing excess r proteins34,35. Interestingly,
Tom1 ubiquitinates unassembled r proteins through residues that are
inaccessible in mature ribosomes and targets them for proteasome-
dependent degradation. This degradation pathway, termed ERISQ,
for excess ribosomal protein quality control, may be one mechanism
employed to maintain cellular homeostasis of r proteins.
Small subunit assembly and acquisition of export competence
Early nucleolar preribosome assembly occurs dichotomously:
production of the pre-40S subunit is followed by pre-60S subunit
synthesis36,37. Initially, a large RNP complex, referred to as 90S or
the small subunit (SSU) processome, assembles on the emerging
RNA Pol I–transcribed 5′ ETS and 18S rRNA to form the character-
istic terminal knobs seen on Miller spreads38,39. The 90S assembly
process is aided by ~70 assembly factors and small nucleolar RNAs
(snoRNAs), most prominently the U3 snoRNP. The 90S complex con-
sists of structurally autonomous subcomplexes and proteins that join
the nascent 5′ ETS and 18S and ITS1 rRNA in a sequential manner40.
Recent work has elucidated in more detail the temporal events

nature structural & molecular biology  VOLUME 24  NUMBER 9  SEPTEMBER 2017 691
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a Cytoplasm Nucleus
Importin
Yar1 Importin Pre-40S
Ran Yar1

Nascent uS3 uS3 uS3

uS3 uS3
80S uS3
Yar1 Yar1

AAAAA

b Importin
Ran Rrb1 Pre-60S
Rrb1
Importin
Nascent uL3 uL3 uL3
Rrb1 uL3
80S Rrb1

AAAAA

c
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

Sqt1
uL16 uL16
Nascent uL16 Pre-60S
Sqt1
80S
Sqt1

AAAAA

d Syo1 Kap104
Nascent uL18 Ran Syo1
Kap104
uL5 uL18
Syo1 Syo1 uL18 Syo1 uL18
80S uL18
uL5 uL5 uL5

AAAAA 5S rRNA Pre-60S

e Kap104
Acl4 Acl4
Ran Pre-60S
Acl4 Acl4 Kap104 Acl4

uL4 uL4 uL4

80S Nascent uL4 Acl4 Acl4 Acl4 uL4 uL4
Kap104 Acl4
Acl4
AAAAA

f eS26 90S
Importin Tsr2

Importin
N Tsr2 Fap7
80S
eS26
eS26 eS26 eS26
uS11 uS11
Tsr2 Fap7
AAAAA
uS11

g Importin
uL14
Bcp1 Pre-60S
Ran
Bcp1
Importin
N Bcp1
80S
uL14
uL14 uL14
AAAAA

Figure 2  Targeting r proteins to assembling preribosomes. (a) Yar1 captures the N terminus of uS3 (Rps3), which dimerizes with a second uS3 molecule
to allow importin recruitment. After nuclear import, the uS3–uS3–Yar1 complex is dissociated to incorporate uS3 into the pre-40S subunit. (b) Rrb1
cotranslationally captures uL3 (Rpl3) and accompanies the r protein into the nucleus to its assembly site on the pre-60S. (c) Sqt1 binds the nascent
N terminus of uL16 (Rpl10) in a cotranslational manner and assists uL16 incorporation into pre-60S subunits in the cytoplasm. (d) Syo1 captures uL18
(Rpl5) cotranslationally and binds uL5 (Rpl11) post-translationally. Kap104 then binds to Syo1 to mediate synchronized nuclear import of the
r proteins. After recruitment of the 5S rRNA to the Syo1–uL18–uL5 complex in the nucleus, Syo1 is released and the 5S RNP is incorporated into pre-
60S subunits. (e) Acl4 cotranslationally binds to a long internal loop in uL4 (Rpl4). A second copy of Acl4 recognizes the C terminus of uL4 before being
displaced by Kap104 to mediate nuclear import of Acl4–uL4. In the nucleus, Kap104 dissociates in a Ran-dependent manner to allow rebinding of the
second Acl4 copy. Acl4 dissociates upon incorporation of uL4 into pre-60S subunits. (f) eS26 (Rps26) is imported into the nucleus and then picked up
from its importin in a Ran-independent manner by the escortin Tsr2. Fap7 and uS11 (Rps14) then join eS26 to prefabricate a ribosomal subcomplex
that allows stoichiometric incorporation of uS11 and eS26 into the 90S. (g) uL14 (Rpl23), bound to its importin, is imported into the nucleus and then
released in a Ran-dependent manner. Bcp1 then picks up uL14 to escort the r protein to its assembly site on the pre-60S. Figure adapted from ref. 4.

692 VOLUME 24  NUMBER 9  SEPTEMBER 2017  nature structural & molecular biology
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a Kre33
Kre33
Utp22 r proteins
Utp20
Enp2 Rrp5
Utp20 Utp22
18S Rrp5
rRNA
18S
Rrp7
Rcl1 rRNA
Bms1 r proteins
Utp13 Utp13
Sof1
Krr1
Fcf1
Utp12 Rrp9 Utp12
Rrp9
Mpp10 U3 Pno1
Imp4 snoRNA Imp3
180° Snu13 Utp7
Emg1 U3
Enp1 Nop56 Utp1

Utp30 Nop58 Utp21

Utp15 Utp18
Nop1
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

Utp17
Utp10 Utp10

Utp5 Utp8
Utp8
Utp5 Utp4 Utp17
Utp9
5′ ETS 5′ ETS

b Central 3′ major 3′ minor

Mpp10
UtpB 5′ domain
Imp3 Sof1
Imp4 Fcf1
5′ ETS U3

UtpA

5′ domain Central 3′ major 3′ min

5′ ETS 18S ITS1

Figure 3  90S pre-ribosome assembly. (a) Cryo-EM structure of the S. cerevisiae 90S preribosome. 18S rRNA is colored in gray; all yellow regions
depict r proteins (model shows a composite structure based on PDB 5TZS and PDB 5WYK). (b) The 90S complex consists of structurally autonomous
subcomplexes and proteins that join the nascent 5′ ETS and following 18S and ITS1 rRNA in a sequential manner. The order of events (top) is
represented by highlighting the subcomplexes in distinct colors based on their association with the emerging pre-rRNA (depicted at the bottom). min,
minor. Figure adapted from refs. 30,31.

leading to the pre-40S subunit formation41,42. Further, three inde-
pendent cryo-EM studies have provided insights into the earliest steps
of pre-40S subunit assembly29–31 (Fig. 3).
First, the UtpA subcomplex interacts with several RNA helices
formed by the 5′ ETS and primes it to recruit U3 snoRNP and the
UtpB modules41–43. UtpA and UtpB are evolutionarily related mul-
tiprotein subcomplexes that scaffold the SSU processome to sub-
sequently assemble the 18S rRNA-containing pre-40S ribosome.
UtpA is composed of seven subunits, of which two contain tandem
β-propellers (Utp4 and Utp17) and four contain one β-propeller and
an α-helical C-terminal domain (Utp5, Utp8, Utp9 and Utp15), which
induces oligomerization. Utp10, the last subunit, is a large helical
repeat protein that wraps around the UTP modules. In contrast, UtpB
is composed of six subunits, with Utp1, Utp12, Utp13 and Utp21
forming a tetramer of tandem β-propeller proteins containing an
α-helical C-terminal domain. The remaining subunits, Utp6 and
Utp18, are located in proximity to the U3 snoRNP, which undergoes
extensive base-pairing interactions with regions of the 5′ ETS as well
as the beginning of 18S rRNA. Together with the Mpp10–Imp3–Imp4
complex and additional large helical proteins, these 5′ ETS compo-
nents create an assembly platform that hugs the emerging 18S rRNA
domains and spatially segregates them to facilitate access of enzymes
and assembly factors to the 90S core.
Chaperoned by the UtpC complex, the emerging 5′ domain of
18S rRNA allows recruitment of a first set of 40S r proteins41,42.
Subsequently, the core body of the 90S is formed by recruiting a subset
of assembly factors including the GTPase Bms1, the methyltransferase
Emg1, the homodimeric acetyltransferase Kre33 and the large 288-
kDa α-helical Utp20. Bms1 forms a complex with Rcl1 and has been
proposed to catalyze cleavage at the A2 site44–46. However, within the
90S, Bms1–Rcl1 bridges distant 5′ and 3′ domains of 18S rRNA, which
does not support a direct role in cleavage. Emg1 methylates 18S rRNA
at nucleotide position 1,191 (refs. 47,48), and Kre33 acetylates 18S
rRNA nucleotides 1,280 and 1,773 (ref. 49). Whereas Emg1 is readily
positioned toward nucleotide 1,191, Kre33 is located far away from
its substrates. It is therefore possible that the activities of Bms1–Rcl1
and Kre33 might require global pre-rRNA rearrangements. Utp20 and
additional unresolved helical-repeat proteins may act as mediators of

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 RE V IE W

long-range interactions to stabilize the 90S. In its final form, the SSU
processome contains the PIN-domain nuclease Utp24 that has been
suggested to catalyze cleavage at the A0 and A1 site and release the 5′
ETS particle from the assembled pre-40S (ref. 50). In yeast, cleavage
at site A2 predominantly occurs cotranscriptionally and releases the
early pre-40S particle containing 20S pre-rRNA into its independent
nuclear assembly pathway51. Under unfavorable growth conditions,
however, the pre-rRNA is cleaved at site A3 to generate 23S pre-rRNA.
This process is regulated by the TOR1 pathway52,53. How exactly
the 90S transitions into the pre-40S stage remains unclear from a
structural viewpoint. A comparison between the 90S-associated 18S
rRNA and its mature fold suggests that only the 5′ domain of 18S

a Solvent-exposed interface Subunit interface

Pre-60S (Nog2 particle)

5S rRNA (premature) Rsa4

Nsa2
Rsa4 Nog2
Rpf2 Rrs1
Mrt4 Nsa2 Rpf2 Nog1
Rrs1
ITS2 180° ITS2
Mrt4
Nop15
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

Cic1 Tif6
25S Rlp7
rRNA
Nop53
Nop7
r proteins 5.8S rRNA Bud20
Rlp24
Arx1 Arx1

b
Pre-60S (Rix1 particle)

Rea1
Rea1

Rsa4 Rsa4

5S rRNA (premature)
Nog1
Mrt4
Sda1
Mrt4
180°

25S Tif6
rRNA

Rlp24
r proteins

Arx1 Arx1

c Rea1

ATP Rsa4
5S rRNA ~180° rotation
Rix1 5S
NA
5S

rR Rrs1 complex
rR

5S Rsa4 Sda1 Rsa4

NA

Rpf2
Nog2 Rix1 complex Sda1 Nog2 Nmd3
Nsa2 Nsa2 Nmd3 Nsa2
GTP Rea1 GTP Mex67–Mtr2
Nog1 Nog1 Nog1
Mrt4 Mrt4 Mrt4

Mex67
Tif6 Tif6 Tif6
Bud20 Bud20 Bud20

Rlp24 Rlp24 Rsa4 Rlp24

ITS2 ITS2
Rpf2 Rea1ADP
Rrs1 Nog2GDP
Rix1 complex
Arx1 Arx1 Sda1 Arx1

Nog2 particle Rix1 particle Export-competent pre-60S

Figure 4  Nuclear pre-60S assembly. Cryo-EM structures of (a) Nog2 and (b) Rix1 particles representing two late nuclear pre-60S intermediates. The
Nog2 particle represents an earlier assembly intermediate in which the ITS2 spacer has not yet been removed and the 5S rRNA is still in a premature
conformation. In the Rix1 particle, ITS2 is removed and the 5S rRNA has rotated into its mature position upon recruitment of the Rix1 subcomplex and
Rea1. 25S rRNA is depicted in gray, 5.8S and 5S in black, ITS2 in brown and r proteins in yellow. (Nog2 PDB 3JCT, ref. 62; Rix1 PDB 5Fl8, EMDB-3199,
ref. 64.) (c) Scheme for acquisition of export competence for the large pre-60S subunit. Only assembly factors with known binding sites are indicated.

694 VOLUME 24  NUMBER 9  SEPTEMBER 2017  nature structural & molecular biology

a 60S–Rei1–Arx1–Alb1 b 60S–Nmd3–Lsg1–Tif6
solvent-exposed interface
CP
P stalk 5S rRNA
Nmd3
Lsg1
5.8S rRNA
Alb1
Rei1 Arx1
d
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
Lsg1GTP
Nm Nm
d3 d3
Tif6 Lsg1
Tif6
GTP
Jjj1ATP
PET
Arx1
Ssa1/2
Alb1
Alb1
Rei1 Rei1
Arx1
Figure 5  Cytoplasmic quality control of the pre-60S subunit. Cryo-EM structures of S. cerevisiae 60S subunits reconstituted with (a) Rei1, Arx1, Alb1
(PDB 5APN, ref. 88), (b) Nmd3, Lsg1, Tif6 (PDB-5T62, ref. 70) and (c) D. discoideum 60S subunits reconstituted with human EFL1, human Sdo1 (SBDS)
and endogenous eIF6 (Tif6) (EMDB-3146, ref. 91). (d) Scheme for cytoplasmic quality control of the pre-60S subunit. Only assembly factors resolved in
a–c are shown. The C terminus of Rei1 is inserted into the polypeptide exit tunnel (PET). PTC, peptidyl transferase center; CP, central protuberance.
rRNA has adopted its final conformation. It is possible that the central
and 3′ domains fold into their mature configuration after release of
the 5′-ETS particle. In general, the structural landmarks as seen in
the mature 40S subunit have yet to achieve a compacted state in the
90S subunit. It appears that the 90S has a perforated structure with
numerous cavities and channels in its inner core.
Nuclear pre-40S particles acquire export competence through the
recruitment of a distinct set of assembly factors, Enp1, Dim1 and
Pno1, which join during the 90S stage, and Tsr1, Rio2, Ltv1, Hrr25 and
Nob1, probably joining after A2 cleavage54. Prior to nuclear export,
pre-40S subunits undergo an essential maturation step during which
the binding of r protein uS3 (Rps3) to the ‘beak’, a structural landmark
in the head domain of mature 40S subunits, is reorganized. In these
pre-40S particles, a subcomplex composed of the assembly factors
Enp1, Ltv1 and uS3 is bound in proximity to the beak structure 55.
Phosphorylation of Enp1 and uS3 by the kinase Hrr25 weakens their
affinity for the pre-40S and increases the conformational flexibility of
the head. It has been proposed that beak flexibility might be critical
for nuclear pre-40S particles to acquire export competence. One of the
earliest cryo-EM studies on ribosome assembly allowed mapping of
Enp1, Ltv1, Tsr1, Rio2, Dim1, Pno1 and Nob1 on a late 40S pre-ribos-
ome56, but the inherent structural flexibility of this particle appears to
be a major hurdle to performing high-resolution analyses57.
Large subunit assembly and acquisition of export competence
Release of the pre-40S particle permits the remaining pre-rRNA
to assemble into a pre-60S subunit. Functional studies suggest that
early assembly of the pre-60S follows principles similar to those of
the pre-40S. Using plasmid-encoded pre-rRNA fragments of increas-
ing lengths, a stepwise association of assembly factors and ribosomal
proteins with emerging 27S pre-rRNA of the pre-60S subunit was
nature structural & molecular biology  VOLUME 24  NUMBER 9  SEPTEMBER 2017 695
RE V IE W
c 60S–EFL1–SBDS–eIF6
subunit interface subunit interface
CP CP
5S rRNA
P stalk
L1 stalk
EFL1
SBDS
eIF6
Tif6
Sdo1
Efl1GTP
Efl1
Sdo1
GTP
PTC Tif6
GDP
Lsg1 Efl1GDP
Nmd3 Sdo1
Mature 60S
Tif6
demonstrated. This supports the idea that, like the 90S, the early pre-60S
is assembled cotranscriptionally rather than post-transcriptionally58.
Obtaining a structural snapshot of such a pre-60S ribosome, analogous
to the 90S, remains a future challenge. A myriad of assembly factors
are implicated in nuclear pre-60S maturation, including snoRNPs,
RNA helicases and RNA-modifying or RNA-processing enzymes.
Together, they are involved in further cleavage and trimming of 27S
rRNA to remove most of the ITS1, ITS2 and 3′-ETS sequences. In
addition, the remaining 5S RNP is recruited, and the pre-60S is com-
pacted into its characteristic structure (reviewed in ref. 59).
In contrast to early pre-60S assembly, subsequent steps leading to acqui-
sition of export competence are amongst the best characterized. One of the
first cryo-EM reconstructions representing a nuclear pre-60S intermediate,
the ‘Arx1 particle’, revealed regions of extra densities attributed to associ-
ated biogenesis factors60. Named after its association with the assembly fac-
tor Arx1, this pre-60S particle exhibits characteristic structural landmarks,
such as a large ‘foot’ structure originating from the unprocessed ITS2-
containing 7S pre-rRNA and, importantly, a 5S RNP and a central protu-
berance that are rotated ~180° compared to a mature 60S particle61.
The Woolford and Gao labs were able to assign densities to sev-
eral assembly factors on a nucleoplasmic particle isolated using the
conserved GTPase Nog2 (Nug2) as bait62 (Fig. 4a). The structure of
this particle, which partially overlaps with the Arx1 particle, revealed
assembly factors involved in building the ITS2-containing foot struc-
ture. The foot structure is established through an intricate interaction
network involving the assembly factors Nop15, Cic1, Rlp7, Nop7 and
Nop53. Processing of 7S pre-rRNA, and thus removal of the foot struc-
ture, is carried out by the exosome and requires prior release of Nop15,
Cic1 (also known as Nsa3) and Rlp7. Nop53 initiates processing of
the 7S pre-rRNA by recruiting the exosome-associated helicase Mtr4
(ref. 63); however, how these events are coordinated remains unclear.
 RE V IE W

a Subunit interface Solvent-exposed interface

Pre-40S (Rio2 particle)

Head Head

Platform

Rio2
Nob1
Ltv1
Pno1
Beak Dim1 ~180° Beak
Enp1
Tsr1
18S
rRNA
helix 44
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

Foot Foot

b
Ltv1
Rio1 60S
Enp1 Nob1 Nob1
eIF5B
Rio2 Pno1 Rio1 Pno1 20S pre-rRNA
Tsr1 processing
Dim1 eIF5B

Ltv1 ITS1
Enp1 Nob1
ITS1 Rio2 ITS1 Pno1
60S
Tsr1 Rio1
Dim1 80S-like eIF5B
Pre-40S Mature 40S

Figure 6  Cytoplasmic quality control of the pre-40S subunit. (a) Cryo-EM structure of the Rio2 particle representing a cytoplasmic pre-40S ribosome.
Assembly factors and 18S rRNA helix 44 are depicted in colors, and remaining rRNA and r proteins are shown in gray (EMDB-1927, ref. 56).
(b) Scheme for cytoplasmic quality control of the pre-40S subunit. Only assembly factors with known binding sites are indicated.

The structure of the Nog2 particle also revealed how the rotated
5S RNP is docked on the assembly factors Rpf2, Rrs1 and Rsa4. To
acquire export competence, the 5S RNP has to rotate ~180° into its
final position. This dramatic remodeling step is likely carried out
through recruitment of the Rix1 subcomplex and its interactor Rea1,
a dynein-related 550-kDa AAA-ATPase. A cryo-EM study using Rix1
as bait revealed the structural landscape of how these two factors form
a checkpoint for acquisition of export competence64 (Fig. 4b). In the
Rix1 particle, the 5S RNP is rotated into its mature position where it
contacts Rix1. Thus, recruitment of Rix1 is thought to destabilize the
immature 5S RNP conformation, thereby inducing its rotation. Only
after accurate 5S RNP rotation can the huge Rix1–Rea1 machinery
be stably anchored through multiple contact points on the pre-60S.
Correct positioning of Rea1 has been suggested to trigger its ATPase
activity, which motors the removal of Rsa4 by an ATP-hydrolysis-
driven “power stroke”65. Intriguingly, the Rea1 ATPase activity is also
coupled to the release of Nog2, a K+-dependent GTPase whose enzy-
matic activity is required for its own release66. Nog2 is a placeholder
for Nmd3, an essential assembly factor required for nuclear export of
pre-60S subunits67. Thus, the Rix1–Rea1 machinery and the coupled
enzymatic activities of Rea1 and Nog2 may serve as a checkpoint to
render pre-60S subunits competent for nuclear export (Fig. 4c).
Nuclear export of pre-ribosomes
In growing yeast cells, every minute, ~25 preribosomal particles are
exported into the cytoplasm6, with the exportin Crm1 (yeast Xpo1)
playing an essential role the transport7. To initiate export, Crm1, in
the presence of RanGTP, needs to recognize a nuclear export signal
(NES) on adaptor proteins bound to preribosomes and cooperatively
form a Crm1-export complex.
Nmd3 is an essential export adaptor protein for Crm1-dependent
export of the 60S preribosome67,68. It contains a bipartite leucine-
rich NES in its C-terminal domain that is essential for yeast viability.
Overexpression of nmd3∆NES leads to dominant-negative effects
and impairs pre-60S-subunit export. Two recent cryo-EM struc-
tures of pre-60S subunits have revealed how Nmd3 is bound to a
ribosome69,70. Purification of native Nmd3-TAP from yeast led to a
structural snapshot of a cytoplasmic pre-60S containing Nmd3 and
the assembly factors Lsg1 (Kre35), Tif6 and Reh1 (ref. 69). Using a dif-
ferent approach, Malyutin et al. reconstituted mature 60S with recom-
binant Nmd3, Lsg1 and Tif6 and were able to solve most of the Nmd3
structure70. Interestingly, the NES-containing C terminus of Nmd3
was not resolved in either study, supporting the idea that recruitment
of Crm1 occurs via a flexible region. In contrast to Nmd3-dependent
pre-60S export, no essential NES-containing export adaptor for pre-
40S export has been identified. However, the assembly factors Ltv1
and the atypical kinase Rio2 carry a leucine-rich NES at their C ter-
mini that recruits Crm1 in the presence of RanGTP, suggesting that
there are redundant mechanisms for 40S preribosome export71,72.
A common feature of NESs is their low affinity toward Crm1, even
in the presence of RanGTP. Given the rate of preribosome export,
eukaryotes must have evolved mechanisms to efficiently assemble
Crm1-export complexes to boost preribosome export. For pre-60S
export, Nmd3 may employ a bipartite NES and enhance recruitment

696 VOLUME 24  NUMBER 9  SEPTEMBER 2017  nature structural & molecular biology

of Crm1. It is plausible that a Crm1 dimer is loaded to the NESs, as
has been reported for the Rev oligomer that functions in HIV RNA
export73. Pre-40S subunits appear to employ two RanGTP-binding
proteins, Slx9 and Yrb2, to promote assembly of the Crm1-export
complex. Slx9 was identified as a RanGTP-binding protein that
facilitates recruitment of Crm1 to Rio2 (ref. 74). In vitro, Slx9 binds
Rio2 and RanGTP to form a ternary complex, which then directly
recruits Crm1. Slx9 seems to prime the Rio2 NES and possibly orient
RanGTP in such a manner as to enhance Crm1 loading and boost pre-
40S export. Yrb2 is another RanGTP-binding protein that enhances
assembly of Crm1-export complexes75. Yrb2 uses its two hydrophobic
FG-repeat domains to bind Crm1 and its C terminus to bind RanGTP,
thus forming a Yrb2–RanGTP–Crm1 complex 76. In this ternary
complex, the NES-binding cleft of Crm1 is in a closed conformation.
Loading of the NES onto the NES-binding cleft readily displaces the
C terminus of Yrb2 from the complex through an allosteric mecha-
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
nism. FRET analyses of this Yrb2–RanGTP–Crm1–NES complex
further showed that binding of the NES to Crm1 is accelerated in
the presence of Yrb2. Unlike Slx9, Yrb2 primes RanGTP and Crm1
for rapid loading onto NES-containing cargos. Curiously, yrb2∆ cells
are impaired in pre-40S-subunit export but not pre-60S export75,77.
However, the precise targets of Yrb2 to boost 40S preribosome export
remain unknown.
In addition to Crm1-dependent export adaptors, preribosomes
recruit auxiliary factors that directly interact with the NPC. The
Mex67–Mtr2 heterodimer and Rrp12 have been identified as com-
mon factors that promote the nuclear export of pre-60S and pre-40S
particles. UV crosslinking and analysis of cDNA demonstrated that
Mex67 interacts in vivo with the 20S pre-rRNA and 5.8S rRNAs78.
Reconstitution of a late 60S preribosome with Mex67–Mtr2 in vitro
and identification of additional crosslinks to H42-43 of the 25S
rRNA79 suggests two separate binding sites for this transport receptor
on the 60S preribosome. Additional factors Arx1, Ecm1, Bud20 and
Gle2 also facilitate efficient export of the 60S preribosome. Although
these export factors are nonessential, their deletions are syntheti-
cally lethal when combined with each other or additional export
mutants80,81. These genetic interactions suggest redundant roles of
export factors and support the idea that the large preribosomal cargos
require multiple cooperative interactions for translocation through
the NPC channel3.
Cytoplasmic quality control of the ribosome
Upon arrival in the cytoplasm, preribosomal particles undergo late
maturation before achieving translation competence. These final steps
involve further processing of pre-RNA, incorporation of remaining
r proteins and release of associated assembly and transport factors.
Cytoplasmic maturation also provides a time window for the quality-
control machinery to functionally proofread preribosomes3,59.
In the case of pre-60S subunits, energy-consuming enzymes trig-
ger these steps in a stepwise fashion. The AAA-ATPase Drg1 initi-
ates cytoplasmic maturation of the 60S preribosome by specifically
binding and releasing the ribosomal-like protein Rlp24, a place-
holder for the r protein eL24 (refs. 82,83). Expression of a dominant-
negative allele of Drg1 traps cytoplasmic pre-60S subunits by blocking
the recycling of not only Rlp24 but further assembly factors
including Nog1, Mrt4, Tif6, Bud20, Nmd3, Mex67 and Nsa2 (ref. 81).
This Drg1-dependent event is therefore critical for ribosomal tunnel
maturation and stalk assembly.
The presence of r protein eL24 on the 60S preribosome triggers Rei1
and Jjj1 recruitment to the ribosomal exit tunnel, which is sealed by
Arx1 (refs. 84–86). Jjj1 is a J-domain protein that binds in the immediate
nature structural & molecular biology  VOLUME 24  NUMBER 9  SEPTEMBER 2017 697
RE V IE W
vicinity of the Arx1–Rei1 interaction site and recruits and activates
the Hsp70-type ATPase Ssa1 or its paralog Ssa2, inducing Arx1
release. Structural analyses of a reconstituted 60S subunit with Arx1,
Rei1 and Jjj1 revealed how Rei1 probes the ribosome polypeptide
tunnel87,88 (Fig. 5). The C-terminal tail of Rei1 is deeply inserted into
the ribosomal tunnel, where it forms several contacts along its entire
length, thereby assessing tunnel integrity. Failure to insert the C-
terminal tail of Rei1 into the tunnel inhibits Arx1 release and blocks
further cytoplasmic maturation of the 60S preribosome, including
downstream events that lead to the release of Tif6, the last step in
cytoplasmic maturation. However, the molecular basis of this block
remains unresolved.
The final events also involve the release of the export adaptor
Nmd3. Recent cryo-EM analyses of Nmd3-containing pre-60S par-
ticles showed that Nmd3 is positioned across the peptidyl transferase
center (PTC), where it contacts Tif6 and Lsg1 (Kre35), a GTPase
whose enzymatic activity is required to release Nmd3 (refs. 69,70).
The structure of the in vitro–reconstituted 60S–Nmd3–Lsg1–Tif6
complex supports a model in which 25S rRNA helix H69 triggers the
GTPase activity of Lsg1, which in turn would release Nmd3 from the
pre-60S ribosome70 (Fig. 5).
Release of Nmd3 seems to be tightly coupled with release of Tif6,
an assembly factor that prevents pre-60S ribosomes from interact-
ing with mature 40S subunits. It has been proposed that recruitment
of the assembly factors Efl1 and Sdo1 disengages Nmd3 from Tif6
(ref. 70). This allows Efl1 and Sdo1 to promote the release of Tif6
from pre-60S ribosomes86,89,90. A cryo-EM study in which human
Sdo1 (SBDS) and Efl1 were bound to Dictyostelium discoideum 60S
subunits revealed that upon binding of Efl1, Sdo1 is repositioned
around helix H69, thus facilitating a conformational switch in Efl1
that displaces Tif6 (ref. 91) (Fig. 5). Efl1 shares sequence similarity
with the GTPase elongation factor Ef-2. Thus, Efl1 could check the
integrity of the GTPase-activating center, while Sdo1 proofreads the
PTC of the ribosome92. In this way, cytoplasmic release factors may
couple the recycling of shuttling assembly factors and simultaneously
check ribosome function.
Similar to the pre-60S export factors, recycling of pre-40S export
factors Ltv1 and Rio2 is energy dependent. Ltv1 is released through
phosphorylation by Hrr25, the same kinase that is also involved in
conferring export competence for the pre-40S in the nucleus93. In
contrast, Rio2 uses its intrinsic ATPase activity to dissociate from
the pre-40S94. Release of Rio2 allows recruitment of Rio1, a related
ATPase implicated in final quality control of the 40S subunit95.
Cryo-EM studies of a late 40S preribosome assigned binding sites
for multiple assembly factors including Enp1, Ltv1, Nob1, Pno1,
Dim1, Rio2 and Tsr1 (refs. 56,57) (Fig. 6). These analyses suggest
that these assembly factors prevent premature translation initiation
by inhibiting access of translation factors. Ltv1 and Enp1 directly bind
uS3 (Rps3) on its solvent-exposed side, thereby blocking the opening
of the mRNA channel. Nob1 and Pno1 inhibit the binding of eIF3 and
thereby interfere with translation initiation. Dim1, Rio2 and Tsr1 are
localized at the subunit interface, thus preventing premature inter-
actions with the 60S subunit and translation initiation factor eIF1A.
Moreover, Tsr1 acts as an inactive structural mimic of eIF5B (Fun12),
a GTPase involved in the final steps of pre-40S maturation96. The
location of Tsr1 is further predicted to interfere with binding of Rio1,
strongly indicating that removal of both Rio2 and Tsr1 is essential for
40S maturation.
Release of Rio2 and Tsr1 then allows the recruitment of Rio1 and
Fun12. The resulting pre-40S intermediate appears to mimic the
translation-initiation surface of a 40S subunit, because it allows the
 RE V IE W
interaction with a mature 60S subunit to form an 80S-like particle97,98
(Fig. 6). Curiously, formation of the 80S-like particle containing
active Rio1 and Fun12 stimulates release of Pno1 and triggers the
endonuclease activity of Nob1 (refs. 95,97–99), suggesting that 20S
pre-rRNA processing may be a quality-control mechanism triggered
only on translation-competent 40S pre-ribosomes.
Perspectives
During the last decade, biochemical purification and MS permitted
the isolation and compositional analyses of preribosomes, providing
‘biochemical snapshots’ of the eukaryotic ribosome assembly pathway.
Cryo-EM studies are now beginning to offer ‘structural snapshots’
of assembling ribosomes. Thus, the ribosome-studying community
is equipped with a powerful toolbox to uncover the functional envi-
ronment of assembly factors, a key step toward mechanistic and
in vitro reconstitution studies. Genetic trapping of preribosomes using
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
dominant-negative alleles followed by biochemical purifications and
structural studies will be instrumental in uncovering the roles of
the >50 steps along the assembly pathway. Such analyses may reveal
mechanisms that structurally proofread ribosome quality during
assembly. Further, understanding how preribosomes dock at the NPC
for productive translocation through the NPC may reveal general
mechanisms that govern transport of large cargos between the nucleus
and cytoplasm. Exciting developments in cryo-electron tomography
and super-resolution microscopy are likely to trigger breakthroughs
in visualizing ribosome formation and transport in vivo.
Acknowledgments
We apologize to those authors whose work has not been cited due to space
limitations. We thank S. Gerhardy and P. Nerurkar for help with figure preparation.
V.G.P. is supported by grants from the Swiss National Science Foundation, NCCR
RNA & Disease, Novartis Foundation, and Olga Mayenfisch Stiftung and a Starting
Grant Award from the European Research Council (EURIBIO260676). E.H. is
supported by grants from the German Research Council (DFG; HU363/15-1,
HU363/12-1).
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html. Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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