MOLECULAR AND CELLULAR BIOLOGY, May 2007, p. 3303–3312
Vol. 27, No. 9
Conservation of a Masked Nuclear Export Activity of La Proteins and
Its Effects on tRNA Maturation?
Mark A. Bayfield, Trish E. Kaiser,† Robert V. Intine,‡ and Richard J. Maraia*
Intramural Research Program, National Institute of Child Health and Human Development,
U.S. National Institutes of Health, Bethesda, Maryland
Received 5 January 2007/Returned for modification 1 February 2007/Accepted 14 February 2007
La is an RNA-processing-associated phosphoprotein so highly conserved that the human La protein (hLa)
can replace the tRNA-processing function of the fission yeast La protein (Sla1p) in vivo. La proteins contain
multiple trafficking elements that support interactions with RNAs in different subcellular locations. Prior data
indicate that deletion of a nuclear retention element (NRE) causes nuclear export of La and dysfunctional
processing of associated pre-tRNAs that are spliced but 5? and 3? unprocessed, with an accompanying decrease
in tRNA-mediated suppression, in fission yeast. To further pursue these observations, we first identified
conserved residues in the NREs of hLa and Sla1p that when substituted mimic the NRE deletion phenotype.
NRE-defective La proteins then deleted of other motifs indicated that RNA recognition motif 1 (RRM1) is
required for nuclear export. Mutations of conserved RRM1 residues restored nuclear accumulation of NRE-
defective La proteins. Some RRM1 mutations restored nuclear accumulation, prevented disordered pre-tRNA
processing, and restored suppression, indicating that the tRNA-related activity of RRM1 and its nuclear export
activity could be functionally separated. When mapped onto an hLa structure, the export-sensitive residues
comprised surfaces distinct from the RNA-binding surface of RRM1. The data indicate that the NRE has been
conserved to mask or functionally override an equally conserved nuclear export activity of RRM1. The data
suggest that conserved elements mediate nuclear retention, nuclear export, and RNA-binding activities of the
multifunctional La protein and that their interrelationship contributes to the ability of La to engage its
different classes of RNA ligands in different cellular locations.
La is an abundant protein whose capacity to bind a variety of
noncoding RNAs and mRNAs lends itself to numerous activ-
ities (26). La proteins from yeast to human have been impli-
cated in the production of tRNAs, rRNAs, ribosomal proteins,
and other components of the translational machinery (13, 16,
19, 20). In human cells, most La is phosphorylated on serine-
366 by protein kinase CK2, resides in the nucleoplasm, and is
associated with nascent pre-tRNAs (17, 32). Nonphosphory-
lated La is most concentrated in the nucleolus (15, 16) and was
independently found at tRNA and other RNA polymerase
III-transcribed genes (4), but it also resides in the cytoplasm
associated with 5?TOP mRNAs that encode ribosomal proteins
and translation factors (16). Trafficking signals in human La
include a nuclear localization signal (NLS), a nuclear retention
element (NRE) first identified by microinjection of Xenopus
oocytes and later confirmed in fission yeast (14, 35), and a
nucleolar localization signal (10, 15). Although trafficking may
be important for its different activities (19), knowledge of the
functional significance of alterations in La trafficking in specific
RNA pathways is limited.
Sequence-specific binding to 3? UUU-OH, the termination
motif found on nascent pre-tRNAs and other transcripts syn-
thesized by RNA polymerase III, is the activity responsible for
the best-established function of La proteins, protection of
RNA ligands from 3? exonucleolytic digestion (11, 12; reviewed
in references 27 and 41). Nascent pre-tRNAs require 5? and 3?
end processing, numerous modifications, CCA addition, ami-
noacylation, nuclear export, and splicing, if necessary, prior to
the appearance of a mature functional tRNA in the cytoplasm
(9). Recent findings have revealed that the tRNA production
pathway is highly complex in biochemistry, spatial organiza-
tion, and sequential order (9, 29). In what order are the 5?
leader, intron, and 3? trailer normally removed from a pre-
tRNA, and in which cellular compartments do these reactions
In yeast, removal of the 5? leader by RNase P appears to be
the earliest processing step for most pre-tRNAs, occurring at
or near tRNA transcription sites, in the nucleolus (2; reviewed
in references 6, 9, and 30). Consistent with this and the idea
that La is the first protein to interact with nascent pre-tRNAs,
La directs 5? processing to precede 3? processing, since this
order is reversed in its absence (17, 43). Removal of the 3?
trailer is believed to occur in the nucleus, as do some modifi-
cations, CCA addition, and aminoacylation (9, 24, 42). The
discovery that tRNA splicing occurs in the cytoplasm in yeast
(44) is consistent with splicing occurring after 5? end process-
ing (9, 29). An equally surprising discovery which revealed
further complexity was that tRNAs can move in a “retrograde”
manner, from the cytoplasm to the nucleus (29, 33, 36). Ret-
rograde tRNA transport may also occur in mammals (45).
Although more data are needed, this raises the possibility that
nuclear enzymes may have more than one chance to process
pre-tRNA, once prior to export and again following retrograde
* Corresponding author. Mailing address: 31 Center Drive, Rm.
2A25, Bethesda, MD 20892-2426. Phone: (301) 402-3567. Fax: (301)
480-6863. E-mail: email@example.com.
† Present address: Rosalind Franklin University of Medicine and
Science, Dept. of Cell Biology and Anatomy, 3333 Green Bay Rd.,
North Chicago, IL 60064.
‡ Present address: William M. Scholl College of Podiatric Medicine,
3333 Green Bay Rd., North Chicago, IL 60064.
?Published ahead of print on 16 February 2007.
import. The recent advances raise new questions as to the
order and transport aspects of tRNA processing.
In contrast to the disordering of 5? and 3? end processing that
either human La (hLa) or Schizosaccharomyces pombe La (Sla1p)
(hLa?NRE or Sla1p?NRE, respectively) leads to disorder in
pre-tRNA splicing relative to end processing (hLa?NRE ?
hLa?316-332 and Sla1p?NRE ? Sla1p?234–255) (14). In the
associates with pre-tRNAs but is inappropriately exported to the
cytoplasm without the pre-tRNAs undergoing end processing
(14). Thus, deletion of the NRE promotes the nuclear export of
La, which causes associated pre-tRNAs to bypass nuclear 5?
and 3? end processing and produce spliced pre-tRNAs that
retain their 5? and 3? extensions, which do not support tRNA-
mediated suppression (14). Since, as reviewed above, the cur-
rent understanding is that 5? end processing is an early step
followed by nuclear export and cytoplasmic splicing, we will
refer to the processing defect caused by NRE-deficient La
proteins as disordered because it causes splicing to occur prior
to end processing.
Both the S. pombe La and human La proteins deleted of
their NREs produce indistinguishable effects (14), suggesting
that the NRE has been conserved to mask or functionally
override an equally conserved nuclear export element that
affects pre-tRNA processing. These findings coupled with dis-
coveries of cytoplasmic splicing and retrograde transport led to
a suggestion that La may be involved in tRNA transport (29).
Our laboratory uses a suppressor tRNASerUGA that sup-
presses a nonsense codon in ade6-704 to study the role of La in
tRNA biogenesis in the fission yeast, S. pombe (7, 11, 12, 14,
17). Unsuppressed ade6-704 results in red colonies, and sup-
pression results in pink to white colonies. Substitutions in pre-
tRNASerUGA that cause dependency on Sla1p or hLa for
maturation have been described (11). In this system, La-
dependent processing is a primary determinant of mature
tRNASerUGA levels, and suppression is dependent on accu-
mulation of mature tRNASerUGA (7, 12, 14, 17). We used this
system to investigate determinants of the nuclear retention and
export of Sla1p and hLa and the effects of altered localization
on tRNA processing. We first attempted to distinguish be-
tween two possible models: in the first, the La NRE would
contribute directly to pre-tRNA processing and its deletion
would secondarily lead to export of La-associated pre-tRNAs
that bypassed end processing in the nucleus. In the second
model, the NRE does not contribute directly to processing but
functions only to prevent export of La and associated pre-
tRNAs. We present data supporting the second model, in
which normal pre-tRNA processing by NRE-defective La pro-
tein was rescued by mutation of conserved residues in hLa and
Sla1p that are important for the nuclear export of La?NRE.
The observed rescue could also be obtained through inhibition
of La?NRE export by leptomycin B (LMB) treatment. We
characterized highly conserved residues both in the NRE and
in RNA recognition motif 1 (RRM1) that are required for
nuclear retention and export, respectively, of Sla1p and hLa in
fission yeast. We found mutations of conserved RRM1 resi-
dues that block nuclear export of NRE-defective La proteins
and restore normal pre-tRNA processing and tRNA-mediated
suppression and modeled these onto available high-resolution
hLa structures. Our data indicate that nuclear export of La is
detrimental to tRNA maturation. The data are consistent with
a model in which conserved structural features of La protein
mediate La nuclear export and retention and the interrelation-
ship of these trafficking elements determines the ability of La
to engage RNAs residing in different cellular locations.
MATERIALS AND METHODS
Northern blotting. Northern blotting using LysCUU-int (40) and U1 probes
was as described previously (17).
Site-specific mutagenesis. Site-specific mutagenesis was performed with
QuikChange XL (Stratagene) using pREP4-La and pRep4-Sla1 or their ?NRE
or KK derivatives as templates (17). All constructs were verified by sequencing.
Deletions of the La motif (LM), RRM1, and RRM2 were designed based on the
domain boundaries indicated for the published structures (1, 18); details are
in Table 1.
IF. Immunofluorescence (IF) using primary antibody (“Go” anti-hLa) or anti-
Sla1p, each at a 1:1,000 dilution, followed by fluorescein isothiocyanate (FITC)-
conjugated goat anti-human or anti-rabbit immunoglobulin G (Jackson Immu-
noresearch) at 1:200, was as described previously (14).
tRNA-mediated suppression assay. The tRNA-mediated suppression was as
described previously (17), using 10 mg/liter adenine. LMB was obtained from
Molecular modeling. Molecular modeling was done using MacPyMOL
(DeLano Scientific LLC, Palo Alto, CA).
Conserved residues in the NREs of Sla1p and hLa that are
important for function. Although the architectures of hLa and
Sla1p differ, their NREs are each located adjacent to an RRM
(Fig. 1A and B). Attempts to decipher the determinants of
the hLa NRE should consider the consensus bipartite NLS
[KKx(10)KxKxK] (Fig. 1A) at amino acids 316 to 332 (34). The
upstream KK residues of the NRE are highly conserved in
the predicted NREs of several La proteins (Fig. 1A), whereas
the downstream basic residues are less conserved (14). In a
prior investigation that used deletion mutagenesis, no distinc-
tion was made between the upstream and downstream basic
residues (14). Here, we examined full-length hLa proteins har-
boring either a double substitution (hLa-K316A/K317A) or a
triple substitution (hLa-K328A/K330A/K332A) and compared
them to the NRE deletion mutant (hLa?NRE), and wild-type
hLa by IF (Fig. 1C to J). hLa and hLa?NRE were nuclear and
cytoplasmic, respectively, as expected (14) (Fig. 1C versus E),
whereas hLa-K316A/K317A was cytoplasmic (Fig. 1G) and
hLa-K328A/K330A/K332A was nuclear (Fig. 1I). These data
suggest that the upstream, more highly conserved lysines at
positions 316 to 317 of hLa are important for NRE function,
while the more divergent basic residues at 328 to 332 are not.
Substitution of the Sla1p homologous NRE upstream lysines
also led to cytoplasmic accumulation, since Sla1p-K234A/
K235A was indistinguishable from Sla1p?NRE (Fig. 1K to P).
As will be seen below, the KK substitutions inactivate hLa and
Sla1p for tRNA-mediated suppression and cause disordered
pre-tRNA splicing indistinguishable from that of the NRE-
deleted proteins. We conclude that one or both of the con-
served NRE lysines, hLa K316/K317 and Sla1p K234A/K235A,
are important determinants of nuclear retention in fission
yeast. Hereafter, we also refer to the NRE-defective hLa-
K316A/K317A and Sla1p-K234A/K235A as hLaKK and
3304BAYFIELD ET AL.MOL. CELL. BIOL.
LMB rescues the tRNA-mediated suppression activity of
hLa?NRE. Nuclear accumulation of NRE-deleted La proteins
was restored in S. pombe by LMB, an inhibitor of Crm1-
mediated nuclear export (14). We reasoned that if inactivity of
tRNA-mediated suppression by hLa?NRE was due solely to
nuclear export, LMB might restore its suppression activity.
Alternatively, if the NRE was more directly required for tRNA
maturation, hLa?NRE may remain inactive even if restored to
the nucleus. S. pombe cells harboring hLa, hLa?NRE, or
empty vector were grown in the presence of LMB. LMB had to
be used at a lower concentration than typically used for an IF
assay to minimize toxicity during growth (data not shown).
Even so, LMB led to significant recovery of suppression for
hLa?NRE but not the control cells (Fig. 2). This suggested
that hLa?NRE can support tRNA-mediated suppression if
retained in the nucleus and therefore that we might be able to
identify second-site mutations in NRE-defective La proteins
that restore nuclear accumulation and tRNA-mediated sup-
Deletion of RRM1 restores nuclear accumulation of NRE-
defective La proteins. In an attempt to restore nuclear accu-
mulation of hLa?NRE, we separately deleted the LM, RRM1
and RRM2 (Fig. 3), based on the domain boundaries indicated
for the published structures (1, 18). Because of the physical
proximity of NRE and RRM2 we anticipated that RRM2 har-
bored a nuclear export determinant and that its deletion would
block export and restore nuclear accumulation. However, de-
letion of RRM2 did not restore nuclear accumulation (Fig.
3C). Moreover, cytoplasmic accumulation of the resulting pro-
tein, hLa?NRE?RRM2, was fully reversed to a nuclear pat-
TABLE 1. hLa and Sla1p constructs
hLa (wild type)
hLa?NRE?LM (hLa?7-107 ?316-332)
hLaKK?LM (hLaK316A/K317A ?7-107)
hLa?NRE?RRM1 (hLa?316-33 ?101-194)
hLaKK?RRM1 (hLaK316A/K317A ?101-194)
Sla1p (wild type)
aLocalization determined by immunofluorescence. N, nuclear; C, cytoplasmic; blank space, not determined.
btRNA-mediated suppression. ???, full suppression; ?, no suppression; blank space, not determined.
cD, disordered spliced pre-tRNA; N, no disordered spliced pre-tRNA; blank space, not determined.
dFrom reference 12.
VOL. 27, 2007MASKED NUCLEAR EXPORT ACTIVITY OF La PROTEINS 3305
FIG. 1. Point mutations of conserved residues in the NRE mimic an NRE deletion. (A) Schematic of human and S. pombe La proteins. The
?4 strand of hLa RRM2 and adjacent ?3 helix comprising the NRE are represented, with corresponding sequence below (18). A potential
consensus bipartite NLS at positions 316 to 332, whose upstream KK residues are in bold, is indicated below (see text). (B) NMR structure model
3306 BAYFIELD ET AL.MOL. CELL. BIOL.
tern by LMB, indicating the presence of a nuclear export
determinant (data not shown). Likewise, deletion of the LM
did not restore nuclear accumulation (Fig. 3A). By contrast to
deletion of the LM and RRM2, deletion of RRM1 did restore
nuclear accumulation, in the contexts of both hLa?NRE and
hLaKK (Fig. 3B and D). We conclude that RRM1 mediates
nuclear export of NRE-defective La proteins. The conserva-
tion of RRM1 fits with this conclusion, since hLa and Sla1p
responded indistinguishably, by multiple functional criteria, to
deletion of their NREs in fission yeast (14).
Conserved RRM1 residues direct nuclear export of NRE-
defective hLa and Sla1p. While most proteins that undergo
Crm1-dependent export contain a nuclear export sequence
(NES) that is recognized by Crm1p, others do not (21, 38, 39).
We were unable to identify a functional NES of the con-
sensus sequence, L-X(2,3)-(LIVFM)-X(2,3)-L-X-(LI) (22), in
hLa?NRE and Sla1p?NRE, despite examinations of many
mutations (not shown).
Mapping of the nuclear export activity to RRM1 raised the
issue of whether this activity would overlap with or interfere
with the principal activity attributed to an RRM, RNA binding,
which maps to the ?-sheet surface of the RRM and adjacent
loops (28). We showed that the ?-sheet surface of RRM1 is
required for normal tRNA maturation and tRNA-mediated
suppression (11). Accordingly, La?NRE?RRM1, although
nuclear, was inactive for suppression (Table 1). We therefore
sought to isolate site-specific mutations in RRM1 that would
inactivate nuclear export of NRE-defective La proteins and
restore functional pre-tRNA processing and tRNA-mediated
suppression. This would indicate that the nuclear export activ-
ity and the tRNA maturation activity of La RRM1 might be
distinct and/or separable.
A series of alanine substitutions of conserved surface side
chains in RRM1 of hLa were introduced into hLaKK and
hLa?NRE (Table 1, proteins 9 to 23) as well as Sla1pKK and
Sla1p?NRE (Table 1, proteins 31 to 37) proteins. Some of
these substitutions restored nuclear accumulation, while others
did not (Fig. 4; Table 1). Especially noteworthy are the hLa
E132/D133 and homologous Sla1p E177A/E178A substitutions
in ?NRE and NRE-substituted (KK) proteins, as well as hLa
F150 and homologous Sla1p F196 substitutions, which largely
restored nuclear accumulation (Fig. 4; Table 1). We note that
Sla1p?NRE-E177A/E178A was nuclear in the majority of the
cells, while it was cytoplasmic in others (Fig. 4Q). A greater
majority of hLa?NRE-E132/D133 was nuclear (Fig. 4G). Al-
of hLa RRM2 (18). The RRM is blue, except for ?4 and the ?3 helix in black. The side chains of the conserved lysines at positions 316 and 317
are green, and the backbone chain of the lysines in the KXKXK sequence is shown in red. (C to P) Anti-La indirect IF using fluorescence-
conjugated secondary antibody (FITC, green) and DAPI (4?,6?-diamidino-2-phenylindole) (blue). (C) hLa, FITC. (D) hLa, DAPI. (E) hLa??RE,
FITC. (F) ??RE DAPI. (G) hLa-K316A/K317A, FITC. (H) hLa-K316A/K317A, DAPI. (I) hLa-K328A/K330A/K332A, FITC. (J) hLa-K328A/
K330A/K332A, DAPI. (K) Sla1p, FITC. (L) Sla1p, DAPI. (M) Sla1p??RE, FITC. (N) Sla1p??RE, DAPI. (O) Sla1p-K234A/K235A FITC. (P)
Sla1p-K234A/K235A DAPI. Panels K to P are at a slightly higher magnification than panels C to J.
FIG. 2. LMB restores tRNA-mediated suppression activity to
hLa?NRE. tRNA-mediated suppression of ade6-704 in the presence
or absence of LMB is shown. Cells were transformed with empty
vector, full-length hLa, or hLa?NRE. Lighter color indicates that
LMB increases suppression by hLa?NRE but not hLa or empty vector.
FIG. 3. RRM1 is required for nuclear export of hLa??RE. IF of S.
pombe cells transformed with various hLa NRE-defective constructs
that lack the LM (hLa??RE-?LM) (A), RRM1 (hLa??RE-?RRM1)
(B), RRM2 (hLa??RE-?RRM2) (C), or RRM1 in the context of
K316A/K317A (hLaK316A/K317A-?RRM1) (D) is shown.
VOL. 27, 2007 MASKED NUCLEAR EXPORT ACTIVITY OF La PROTEINS3307
though these observations might be explained by cell cycle-
dependent localization, we note that nuclear staining was in
general less complete with anti-Sla1p antibodies, even in wild-
type cells, than with anti-hLa (data not shown).
Mutations that restore nuclear accumulation of NRE-defec-
tive La proteins also reverse accumulation of disordered
spliced pre-tRNA. Northern analysis was performed to address
two issues: whether hLaKK causes disordered spliced pre-
FIG. 4. Conserved RRM1 residues important for nuclear export of hLa??RE and Sla1p?NRE. IF of S. pombe cells transformed with various
hLa NRE-defective constructs, as described for Fig. 1, is shown. (A) hLa, FITC. (B) hLa, DAPI. (C) hL,a??RE FITC. (D) hLa??RE, DAPI.
(E) hLa??RE-F150A, FITC. (F) hLa??RE-F150A, DAPI. (G) hLa??RE-E132A/D133A, FITC. (H) hLa??RE-E132A/D133A, DAPI.
(I) hLa??RE-I140A/M142A, FITC. (J) hLa??RE-I140A/M142A, DAPI. (K) Sla1p, FITC. (L) Sla1p, DAPI. (M) Sla1p?NRE, FITC.
(N) Sla1p??RE, DAPI. (O) Sla1p??RE-F196A, FITC. (P) Sla1p??RE-F196A, DAPI. (Q) Sla1p??RE-E177A/E178A, FITC. (R) Sla1p??RE-
E177A/E178A, DAPI. (S) Sla1p??RE-V186A/M188A, FITC. (T) Sla1p??RE-V186A/M188A, DAPI.
3308BAYFIELD ET AL.MOL. CELL. BIOL.
tRNA, similar to the case for hLa?NRE, and whether the
RRM1 mutations that restore nuclear accumulation of NRE-
defective proteins also reverse the accumulation of the disor-
dered spliced pre-tRNA.
Probes directed to distinct regions of tRNALysCUU precur-
sors have characterized multiple pre-tRNA intermediates in
Sla1? cells expressing ectopic La proteins (11, 14, 17, 40). hLa
produces a pattern in which nascent pre-tRNALysCUU is the
most prominent species (Fig. 5A, lane 2, band 1), as noted
previously (14). In sharp contrast to the pattern with hLa,
hLaKK led to the disordered spliced pre-tRNA (Fig. 5A, lane
3, band 3) that is characteristic of hLa?NRE (lane 8) (14).
RRM1 mutations that restored nuclear accumulation of
hLaKK-derived proteins led to much less, if any, of the disor-
dered spliced pre-tRNA (Fig. 5A, lanes 4 to 7). In addition,
some of the RRM1 mutants led to more of the nascent pre-
tRNA (band 1) than others (Fig. 5A, lanes 4 to 7), a repro-
ducible characteristic more similar to the pattern for hLa and
also observed for hLa?NRE RRM1 mutated proteins (Table 1
and data not shown). Immunoblot analysis indicated that the
FIG. 5. Second-site mutations of NRE-defective La proteins that restore nuclear accumulation, prevent accumulation of disordered spliced
pre-tRNA, and rescue tRNA-mediated suppression. (A) Northern blot of RNA from control cells (lanes 1 and 9) or cells expressing the La proteins
indicated above lanes 2 to 8. Bands corresponding to the pre-tRNALysCUU intermediates as previously characterized are numbered and
schematically represented on the right, band 3 reflects the disordered spliced pre-tRNA species (14), and the thick horizontal line represents the
3? trailer probe used for detection. (B) The same blot as in panel A probed for U1 snRNA. (C and D) Suppression data for selected mutants, as
indicated above the spots.
VOL. 27, 2007MASKED NUCLEAR EXPORT ACTIVITY OF La PROTEINS3309
wild-type and mutated proteins were expressed at comparable
levels (data not shown). Sla1p?NRE-homologous RRM1 mu-
tants that restored nuclear accumulation also lost the disor-
dered spliced pre-tRNA (e.g., proteins 35 and 36 in Table 1).
A subset of RRM1 mutations restore nuclear accumulation
and tRNA-mediated suppression to NRE-defective La pro-
teins. Some RRM1 mutations that restored nuclear accumu-
lation also restored tRNA-mediated suppression, while others
did not (Fig. 5C; Table 1). hLa?NRE-E132A/D133A restored
nuclear accumulation and suppression, as did the homolog,
Sla1p?NRE-E177A/E178A (Fig. 5C). It is noteworthy that
hLaKK-E132/D133, which led to more of band 1 than the
other hLaKK-RRM1 mutants (Fig. 5A), as also observed for
hLa?NRE-E132/D133 (not shown), also showed the highest
suppression activity among the hLa RRM1 mutants (Fig. 5C).
hLa?NRE-F150A and its homolog Sla1p?NRE-F196A also
restored some suppression (Fig. 5C). These RRM1 mutants
corroborate the LMB data which suggested that nuclear accu-
mulation of NRE-defective La proteins can enable them for
Multiple simultaneous substitutions are referred to in ab-
breviated form as A2, A5, A6, etc. (Table 1). We also examined
some of the RRM1 mutations that restored nuclear retention,
but not suppression, in the context of otherwise wild-type La
proteins. hLa carrying only the A5 or A6 mutations was nu-
clear but inactive for suppression (Fig. 5D, spots 4 and 5; Table
1, proteins 26 and 27), indicating these to be previously un-
known residues that are important for tRNA-mediated sup-
La proteins are associated with a variety of RNAs in differ-
ent cellular compartments. In this work, we have attempted to
detail the different trafficking elements important for La nu-
cleocytoplasmic transport by using a fission yeast system that
can assess function in tRNA maturation. We uncovered con-
served determinants of the nuclear export and nuclear reten-
tion activities of hLa and Sla1p proteins and demonstrated
opposing effects of these trafficking elements on La-dependent
tRNA maturation. Substitution of two highly conserved lysines
in the NRE produced phenocopies of NRE deletions, which
are characterized by nuclear exclusion of La, disordered pre-
tRNA splicing, and failure to support tRNA-mediated sup-
pression. These substitution data validate the NRE as a func-
tional element that maintains La in the nucleus, where it
promotes tRNA maturation. We then found that deletion of
RRM1 from hLa?NRE and hLaKK returned these La pro-
teins to the nucleus (Fig. 3). The data indicate that the NRE
has been conserved to functionally mask or override an equally
conserved nuclear export activity of the RRM1s of hLa and
However, since RRM1 is required for tRNA-mediated sup-
pression activity of La (11), its complete deletion could not tell us
if its nuclear export and tRNA-related activities were distinct or
overlapping. We therefore attempted to identify site-specific mu-
tations in RRM1 that could restore nuclear accumulation and
tRNA-mediated suppression activity to the NRE-defective La
proteins. Mutation of certain conserved RRM1 residues inacti-
vated nuclear export of both the Sla1p and hLa NRE-defective
proteins. These results led to two relevant conclusions. First,
hLa?NRE and Sla1p?NRE proteins lacking the NRE could be
made competent for tRNA-mediated suppression by RRM1 mu-
tations that prevented nuclear export (e.g., hLa?NRE-E132A/
D133A and Sla1p?NRE-E177A/E178A). This further suggested
that the NRE does not contribute directly to tRNA maturation
but rather serves to prevent untimely nuclear export of La and
dysfunctional processing of associated pre-tRNAs. Second, the
tRNA-related activity of RRM1 and its nuclear export activity
could be functionally separated.
Residues in RRM1 can direct nuclear export but are other-
wise masked or overridden by the NRE. It is noteworthy that
RRM1 of the Saccharomyces cerevisiae La protein, Lhp1p, was
suspected of having nuclear export activity (31), suggesting that
the conservation of nuclear export activity by RRM1 may ex-
tend to budding yeast.
Mutation of some RRM1 residues restored nuclear accumu-
lation of NRE-defective La proteins, prevented disordered
pre-tRNA splicing, and restored a more orderly pathway of
processing and tRNA-mediated suppression. RRM1 mutations
that restored nuclear accumulation and suppression activity to
the NRE-defective proteins (e.g., E132A/D133A) did not im-
pair nuclear accumulation or suppression when introduced
into wild-type La (Fig. 5D, lane 3, and Table 1). These data
suggest that the conserved nuclear export activity of RRM1 is
not required for the functional maturation of intron-containing
pre-tRNAs. Rather, functional unmasking or overriding of the
nuclear export activity of La by the NRE appears to be detri-
mental to tRNA maturation. Thus, the data in this report do
not support a positive role for La in tRNA nucleocytoplasmic
transport, as speculated previously (29). The fact that we could
identify mutations of conserved residues that block export
without impairing tRNA maturation (e.g., hLa E132/D133 and
Sla1p E177/E178) suggests that these residues were conserved
to function in an important process other than tRNA matura-
tion that involves nuclear export.
Although we have not identified a nuclear export factor for
hLa or Sla1p, we have addressed the possibility that pre-tRNA
binding may affect export. Examination of hLa26-408 and
other (point) mutants that are severely impaired for UUU-OH
binding and tRNA-mediated suppression revealed no localiza-
tion defects in the context of either wild-type La or La con-
taining other trafficking mutations (data not shown). While
other data argue for a RNA-binding-dependent effect on lo-
calization in human cells (10), our analysis with fission yeast
does not suggest that the capacity for pre-tRNA binding is a
significant determinant of the nuclear export activity of La.
Some RRM1 mutations restored nuclear accumulation but
not suppression (see, e.g., proteins 9 to 11, 19, and 20 in Table
1). These would appear to reflect residues that contribute to
the RRM1-mediated tRNA maturation activity of La, since
these same mutations inactivated wild-type La (Fig. 5D and
Table 1, proteins 26 and 27). Other site-specific La RRM1
mutations that impair the maturation of some pre-tRNAs have
been described (11).
Functional determinants of the NRE ?-helix of hLa. Our
analysis, which included IF, tRNA-mediated suppression, and
Northern blotting, showed that the more highly conserved ba-
sic residues at hLa positions 316 and 317, but not the less
3310 BAYFIELD ET AL.MOL. CELL. BIOL.
conserved basic residues at positions 328 to 332, are important
for NRE function.
The hLa NRE corresponds to a well-ordered ?-helix (Fig.
1B, ?3) whose underside contacts the ?-sheet surface of
RRM2 (18). It was therefore anticipated that RRM2 harbored
a nuclear export determinant that was masked by the overlying
NRE and that deletion of RRM2 would block export and
restore nuclear accumulation of the NRE-defective hLa pro-
teins. However, we could not identify a functional NES any-
where in RRM2, and hLa?NRE?RRM2 accumulated in the
cytoplasm in an LMB-sensitive manner (data not shown), in-
dicating a nuclear export determinant elsewhere in hLa. We
are left with the conclusion that the NRE masks or functionally
overrides the nuclear export potential of RRM1.
In addition to K316 and K317, two other highly conserved
residues of the NRE are E320, which is on the same face of ?3
as K316, and E324, which is on the same face as K317 (14, 18).
These side chains are not directed toward the RRM2 ?-sheet
surface and would be potentially available for other interac-
Conserved and diverged features of La trafficking and tRNA
processing. While La proteins from yeast to human reside in
the nucleoplasm, nucleolus, and cytoplasm (23, 25, 31), some
of the trafficking mechanisms appear to have been conserved
while others have diverged. The endogenous NLS of S. cerevi-
siae Lhp1p does not coincide with the C-terminal location of
hLa and Sla1p NLSs, and Lhp1p uses a different karyopherin
for nuclear import than that used by hLa and Sla1p (31).
Implications of these differences have been discussed previ-
Other species-specific differences in La trafficking have also
been noted. Nuclear export of Sla1p?NRE and hLa?NRE is
sensitive to the Crm1p export inhibitor LMB in fission yeast
(with total reversal of the nuclear exclusion pattern to com-
plete nuclear accumulation by 2 h) (14). By contrast to fission
yeast, LMB only partially restores nuclear accumulation of
Sla1p?NRE and hLa?NRE in primate cells (14) and is inef-
fective in preventing nuclear export of hLa during nucleo-
cytoplasmic shuttling in primate cells (5). It is unknown
whether these differences reflect differences in the kinetics or
in the transport machineries used for La transport in the mam-
malian and yeast cells. In any case, an outstanding issue is
whether the nuclear export activity of RRM1 elucidated here
contributes to nucleo-cytoplasmic shuttling in HeLa cells (5).
Because tRNA splicing occurs in the cytoplasm in yeasts but
in the nucleus in vertebrates (reviewed in reference 8), we
should expect that while NRE-deficient La mutants undergo
nuclear export in yeast and vertebrates (14), the effects on
disordered splicing may be different.
We note that although phospho-hLa is nucleoplasmic
whereas non-phospho-hLa is nucleolar and cytoplasmic, prior
results indicate that phosphorylation is not a determinant of
localization, since nonphosphorylatable La mutants or phos-
phomemetic La mutants do not mislocalize in human cells (3,
15, 16) or yeast cells (16). Moreover, examination of hLa
proteins expressed in yeast revealed that the NRE deletion did
not affect S366 phosphorylation status (14). In addition, exam-
ination of a variety of hLa trafficking mutants both in the S366
native form and containing nonphosphorylatable or phos-
phomemetic residues failed to support a causative relationship
between phosphorylation and localization (reference 15 and
data not shown). Although we have not examined the S366
phosphorylation status of the hLa NES mutants characterized
here, the cumulative data suggest that any difference in S366
phosphorylation would be a consequence of localization rather
than a functional determinant of localization.
Structural modeling of residues involved in nuclear export
onto the hLa RRM1 structure. Many RRM1 substitutions did
FIG. 6. Views of the RRM1 residues important for nuclear export that are masked or functionally overridden by the NRE in the isolated RRM1
(A) and in the LM-RRM1-UUU-OH RNA complex (B). (A) The mutated RRM1 residues that restored nuclear accumulation to the NRE-
defective hLa proteins are shown in green; the mutated residues that restored nuclear accumulation and suppression (E132, D133, and F150) are
shown as side chains. Mutated residues that did not restore nuclear accumulation are shown in blue, including Y114 and F155, the highly conserved
aromatic side chains on the ?-sheet surface. (B) The LM is shown in gold. The 3? UUU-OH (U7 U8 U9 ) is shown in red, with the rest of
the RNA chain in blue.
VOL. 27, 2007 MASKED NUCLEAR EXPORT ACTIVITY OF La PROTEINS3311
not restore nuclear accumulation (Table 1, proteins 13 to 18, Download full-text
23, 34, and 37). We examined the spatial arrangement of mu-
tated residues on the hLa structure (Fig. 6). In general, resi-
dues that restored nuclear accumulation appear on a side of
the RRM facing away from the canonical RNA-binding sur-
face, whereas those that did not face in the same general
direction as the RNA-binding surface (Fig. 6A). Mutated res-
idues that restored nuclear accumulation are also shown in the
LM-RRM1-UUU-OH RNA structure; all are quite distant
from the LM and the RNA (Fig. 6B), consistent with the idea
that they may be recognized by a transport carrier. These data
support the idea that the nuclear export and tRNA-related
activities of RRM1 are separable.
We thank M. Blum for medium preparation, Laboratory of Molec-
ular Growth Regulation members for discussion and/or comments, and
Vera Cherkasova and the reviewers for comments on the manuscript.
This research was supported by the Intramural Research Program of
the NICHD, NIH. R.J.M. is a commissioned officer in the U.S. Public
1. Alfano, C., D. Sanfelice, J. Babon, G. Kelly, A. Jacks, S. Curry, and M. R.
Conte. 2004. Structural analysis of cooperative RNA binding by the La motif
and central RRM domain of human La protein. Nat. Struct. Mol. Biol.
2. Bertrand, E., F. Houser-Scott, A. Kendall, R. H. Singer, and D. R. Engelke.
1998. Nucleolar localization of early tRNA processing. Genes Dev. 12:2463–
3. Broekhuis, C. H., G. Neubauer, A. van der Heijden, M. Mann, C. G. Proud,
W. J. van Venrooij, and G. J. Pruijn. 2000. Detailed analysis of the phos-
phorylation of human La (SS-B) autoantigen. (De)phosphorylation does not
affect subcellular distribution. Biochemistry 39:3023–3033.
4. Fairley, J. A., T. Kantidakis, N. S. Kenneth, R. V. Intine, R. J. Maraia, and
R. J. White. 2005. Human La is found at RNA polymerase III-transcribed
genes in vivo. Proc. Natl. Acad. Sci. USA 102:18350–18355.
5. Fok, V., K. Friend, and J. A. Steitz. 2006. Epstein-Barr virus noncoding
RNAs are confined to the nucleus, whereas their partner, the human La
protein, undergoes nucleocytoplasmic shuttling. J. Cell Biol. 173:319–325.
6. Haeusler, R. A., and D. R. Engelke. 2006. Spatial organization of transcrip-
tion by RNA polymerase III. Nucleic Acids Res. 34:4826–4836.
7. Hamada, M., A. L. Sakulich, S. B. Koduru, and R. Maraia. 2000. Transcrip-
tion termination by RNA polymerase III in fission yeast: a genetic and
biochemical model system. J. Biol. Chem. 275:29076–29081.
8. Hopper, A. K. 2006. Cellular dynamics of small RNAs. Crit. Rev. Biochem.
Mol. Biol. 41:3–19.
9. Hopper, A. K., and E. M. Phizicky. 2003. tRNA transfers to the limelight.
Genes Dev. 17:162–180.
10. Horke, S., K. Reumann, M. Schweizer, H. Will, and T. Heise. 2004. Nuclear
trafficking of La protein depends on a newly identified NoLS and the ability
to bind RNA. J. Biol. Chem. 279:26563–26570.
11. Huang, Y., M. A. Bayfield, R. V. Intine, and R. J. Maraia. 2006. Separate
RNA-binding surfaces on the multifunctional La protein mediate distin-
guishable activities in tRNA maturation. Nat. Struct. Mol. Biol. 13:611–618.
12. Huang, Y., R. V. Intine, A. Mozlin, S. Hasson, and R. J. Maraia. 2005.
Mutations in the RNA polymerase III subunit Rpc11p that decrease RNA 3?
cleavage activity increase 3?-terminal oligo(U) length and La-dependent
tRNA processing. Mol. Cell. Biol. 25:621–636.
13. Inada, M., and C. Guthrie. 2004. Identification of Lhp1p-associated RNAs
by microarray analysis in Saccharomyces cerevisiae reveals association with
coding and noncoding RNAs. Proc. Natl. Acad. Sci. USA 101:434–439.
14. Intine, R. V., M. Dundr, T. Misteli, and R. J. Maraia. 2002. Aberrant nuclear
trafficking of La protein leads to disordered processing of associated pre-
cursor tRNAs. Mol. Cell 9:1113–1123.
15. Intine, R. V., M. Dundr, A. Vassilev, E. Schwartz, Y. Zhao, M. L. Depamphilis,
and R. J. Maraia. 2004. Nonphosphorylated human La antigen interacts with
nucleolin at nucleolar sites involved in rRNA biogenesis. Mol. Cell. Biol. 24:
16. Intine, R. V., S. A. Tenenbaum, A. S. Sakulich, J. D. Keene, and R. J. Maraia.
2003. Differential phosphorylation and subcellular localization of La RNPs
associated with precursor tRNAs and translation-related mRNAs. Mol. Cell
17. Intine, R. V. A., A. L. Sakulich, S. B. Koduru, Y. Huang, E. Pierstorrf, J. L.
Goodier, L. Phan, and R. J. Maraia. 2000. Transfer RNA maturation is
controlled by phosphorylation of the human La antigen on serine 366. Mol.
18. Jacks, A., J. Babon, G. Kelly, I. Manolaridis, P. D. Cary, S. Curry, and M. R.
Conte. 2003. Structure of the C-terminal domain of human La protein
reveals a novel RNA recognition motif coupled to a helical nuclear retention
element. Structure (Cambridge) 11:833–843.
19. Kenan, D. J., and J. D. Keene. 2004. La gets its wings. Nat. Struct. Mol. Biol.
20. Krogan, N. J., W. T. Peng, G. Cagney, M. D. Robinson, R. Haw, G. Zhong,
X. Guo, X. Zhang, V. Canadien, D. P. Richards, B. K. Beattie, A. Lalev, W.
Zhang, A. P. Davierwala, S. Mnaimneh, A. Starostine, A. P. Tikuisis, J.
Grigull, N. Datta, J. E. Bray, T. R. Hughes, A. Emili, and J. F. Greenblatt.
2004. High-definition macromolecular composition of yeast RNA-processing
complexes. Mol. Cell 13:225–239.
21. Kutay, U., and S. Guttinger. 2005. Leucine-rich nuclear-export signals: born
to be weak. Trends Cell Biol. 15:121–124.
22. la Cour, T., R. Gupta, K. Rapacki, K. Skriver, F. M. Poulsen, and S. Brunak.
2003. NESbase version 1.0: a database of nuclear export signals. Nucleic
Acids Res. 31:393–396.
23. Long, K. S., T. Cedervall, C. Walch-Solimena, D. A. Noe, M. J. Huddleston,
R. S. Annan, and S. L. Wolin. 2001. Phosphorylation of the Saccharomyces
cerevisiae La protein does not appear to be required for its functions in
tRNA maturation and nascent RNA stabilization. RNA 7:1589–1602.
24. Lund, E., and J. E. Dahlberg. 1998. Proofreading and aminoacylation of
tRNAs before export from the nucleus. Science 282:2082–2085.
25. Maraia, R. J. 2001. La protein and the trafficking of nascent RNA poly-
merase III transcripts. J. Cell Biol. 153:F13–F17.
26. Maraia, R. J., and M. A. Bayfield. 2006. The La protein-RNA complex
surfaces. Mol. Cell 21:149–152.
27. Maraia, R. J., and R. V. Intine. 2001. Recognition of nascent RNA by the
human La antigen: conserved and diverged features of structure and func-
tion. Mol. Cell. Biol. 21:367–379.
28. Maris, C., C. Dominguez, and F. H. Allain. 2005. The RNA recognition
motif, a plastic RNA-binding platform to regulate post-transcriptional gene
expression. FEBS J. 272:2118–2131.
29. Phizicky, E. M. 2005. Have tRNA, will travel. Proc. Natl. Acad. Sci. USA
30. Reiner, R., Y. Ben-Asouli, I. Krilovetzky, and N. Jarrous. 2006. A role for the
catalytic ribonucleoprotein RNase P in RNA polymerase III transcription.
Genes Dev. 20:1621–1635.
31. Rosenblum, J. S., L. F. Pemberton, N. Bonifaci, and G. Blobel. 1998. Nuclear
import and the evolution of a multifunctional RNA-binding protein. J. Cell
32. Schwartz, E., R. V. Intine, and R. J. Maraia. 2004. CK2 is responsible for
phosphorylation of human La protein serine-366 and can modulate 5?TOP
mRNA metabolism. Mol. Cell. Biol. 24:9580–9591.
33. Shaheen, H. H., and A. K. Hopper. 2005. Retrograde movement of tRNAs
from the cytoplasm to the nucleus in Saccharomyces cerevisiae. Proc. Natl.
Acad. Sci. USA 102:11290–11295.
34. Simons, F. H., F. J. Broers, W. J. Van Venrooij, and G. J. Pruijn. 1996.
Characterization of cis-acting signals for nuclear import and retention of the
La (SS-B) autoantigen. Exp. Cell Res. 224:224–236.
35. Simons, F. H., G. J. Pruijn, and W. J. van Venrooij. 1994. Analysis of the
intracellular localization and assembly of Ro ribonucleoprotein particles by
microinjection into Xenopus laevis oocytes. J. Cell Biol. 125:981–988.
36. Takano, A., T. Endo, and T. Yoshihisa. 2005. tRNA actively shuttles between
the nucleus and cytosol in yeast. Science 309:140–142.
37. Teplova, M., Y.-R. Yuan, S. Ilin, L. Malinina, A. T. Phan, A. Teplov, and D. J.
Patel. 2006. Structural basis for recognition and sequestration of UUU-OH
3?-termini of nascent RNA pol III transcripts by La, a rheumatic disease
autoantigen. Mol. Cell 21:75–85.
38. Thomas, F., and U. Kutay. 2003. Biogenesis and nuclear export of ribosomal
subunits in higher eukaryotes depend on the CRM1 export pathway. J. Cell
39. Trotta, C. R., E. Lund, L. Kahan, A. W. Johnson, and J. E. Dahlberg. 2003.
Coordinated nuclear export of 60S ribosomal subunits and NMD3 in verte-
brates. EMBO J. 22:2841–2851.
40. Van Horn, D. J., C. J. Yoo, D. Xue, H. Shi, and S. L. Wolin. 1997. The La
protein in Schizosaccharomyces pombe: a conserved yet dispensable phos-
phoprotein that functions in tRNA maturation. RNA 3:1434–1443.
41. Wolin, S. L., and T. Cedervall. 2002. The La protein. Annu. Rev. Biochem.
42. Wolin, S. L., and A. G. Matera. 1999. The trials and travels of tRNA. Genes
43. Yoo, C. J., and S. L. Wolin. 1997. The yeast La protein is required for the 3?
endonucleolytic cleavage that matures tRNA precursors. Cell 89:393–402.
44. Yoshihisa, T., K. Yunoki-Esaki., N. Tanaka, and T. Endo. 2003. Possibility of
cytoplasmic pre-tRNA splicing: the yeast tRNA splicing endonuclease
mainly localizes on the mitochondria. Mol. Biol. Cell 14:3266–3279.
45. Zaitseva, L., R. Myers, and A. Fassati. 2006. tRNAs promote nuclear import
of HIV-1 intracellular reverse transcription complexes. PLoS Biol. 4:e332.
3312BAYFIELD ET AL.MOL. CELL. BIOL.