to 100% of cells displayed a strong nuclear
5-EU–labeled RNA signal that decreased
over time with a concomitant increase in the
cytoplasmic signal, indicative of RNA being
exported. After 6 hours, only ~5% of NUP358-
depleted cells exhibited nuclear retention of
labeled RNA, compared with <1% of control
cells (Fig. 5D). Because of this subtle effect, we
sought to confirm the result inAID::NUP358
DLD1 cells (Fig. 5D and figs. S66 and S67). Sim-ilar to theAID::NUP358HCT116 cell results,
only ~3% of NUP358-depleted DLD1 cells ex-
hibited nuclear RNA retention after a 6-hour
chase (Fig. 5D and fig. S68). By contrast, nu-
clear RNA retention was present in ~20% ofBleyet al., Science 376 , eabm9129 (2022) 10 June 2022 10 of 18
Fig. 5. NUP358 plays a
general role in translation
of exported mRNA.
(A) Subcellular localization
by immunofluorescent stain-
ing of endogenous nups
in synchronizedAID::NUP358
HCT116 cells at the indicated
time points after auxin-
induced NUP358 depletion.
Cytoplasmic puncta arise
from NUP88 overexpression
inherent to HCT116 cells.
(B) Subcellular localization
of N-terminally 3×HA-
tagged NUP358 variants in
AID::NUP358HCT116 cells
by immunofluorescence
microscopy. mAb414 staining
marks the nuclear envelope
rim location. The domain
structure of the transfected
NUP358 variants is shown
(top). (C) Schematic
illustrating the life cycle of
mRNPs from transcription
to maturation, export,
remodeling at the cyto-
plasmic face of the NPC,
and translation. Steps
associated with the NPC
are highlighted by the
red box. (D) Time-resolved
analysis of RNA nuclear
retention in synchronized
AID::NUP358HCT116 cells
upon auxin-induced NUP358
depletion visualizing 5-EU
metabolically pulse-labeled
RNA. Representative images
(left) and quantitation
(right) of the proportion of
cells (n > 200 per time
point) with nuclear RNA
retention are shown with
mean values (squares) and
the respective standard
error (shaded area) of
triplicate experiments. Quan-
titation from unsynchronized
AID::NUP358DLD1 and
NUP160::NG-AIDDLD1 cells
are also shown. (E) Time-
resolved Western blot analy-
sis of the expression of C-terminally 3×FLAG-tagged reporter proteins in synchronizedAID::NUP358HCT116 cells upon auxin-induced NUP358 depletion. Quantitated
reporter expression in auxin-treated cells was normalized to expression in control cells at the 10-hour time point. Experiments were performed in triplicate, with
mean and associated standard error shown. Scale bars, 10mm. Experimental timelines are shown above each experiment.
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