consistent with differences in Ltn1p-mediated
ubiquitination of the substrates (figs. S10 and S11).
Notably, the Ltn1p-accessible window defined
using four lysines (Fig. 2B) was fully consistent
with the refined window (fig. S9B). Thus, the
ability of Ltn1p to effectively access residues in a
limited window proximal to the exit tunnel is
primarily determined by the distance of the lysines from the C terminus, rather than local sequences or structure.
mRNAs lacking a stop codon (non-stop mRNAs)
are an important source of natural RQC substrates
with lysines sequestered in the exit tunnel (2). For
such messages, the ribosome translates the poly-adenine [poly(A)] tail, which appends AAA-encoded
lysines to the C terminus. The resulting lysines
are likely to be sequestered in the ribosome exit
tunnel, owing to the short length of yeast poly(A)
tails (median 27 nucleotides) (18, 19). Indeed, degradation of non-stop GFPLys-free with a defined
poly(A) tail of 30 nucleotides was dependent on
CAT-tails (Fig. 3E). Thus, CAT-tailing can expose
lysines that result from translation through the
poly(A) tract, allowing efficient ubiquitination and
degradation of non-stop decay substrates.
Having established lysine positioning as a critical determinant of CAT-tail–dependent degradation, we next computationally evaluated the
frequency with which endogenous substrates would
be expected to rely on CAT-tailing for degradation.
Although the sites of endogenous stalling remain
poorly defined, several processes such as mRNA
fragmentation, oxidative damage (20), or stress
from translation inhibitors can cause stalling at
any position along a message. We therefore considered the nascent chains produced if ribosomes
stall with uniform probability at each codon along
every coding sequence in the yeast genome (21),
and we calculated the fraction of potential stalling sites for which there are no lysines accessible
to Ltn1p but at least one lysine “hidden” in the
ribosome exit tunnel (fig. S12). Based on our experimental estimates of Ltn1p’s reach (~12 amino
acids) and previous measurements of the length
of the ribosome exit tunnel (~35 amino acids)
(22, 23), we estimate that CAT-tailing would substantially increase the fraction of RQC-degradable
substrates, from ~60% of possible nascent chains
to ~95% (Fig. 4A).
This analysis suggests that if CAT-tailing is
compromised, the accumulation of nondegradable
endogenous substrates will lead to growth defects.
Although yeast strains with deletions of RQC
components do not exhibit a growth defect in rich
media (5, 9), such strains have increased sensitivity to the translation inhibitor cycloheximide
(CHX) (24). Furthermore, deletion of mRNA-decay
factors (e.g., SKI2) stabilizes defective or truncated mRNAs, increasing the stalling burden
on the cell (25). Indeed, under conditions of widespread stochastic ribosome stalling induced by
CHX treatment and mRNA stabilization in a
ski2D background, rqc2mut cells exhibited a growth
defect intermediate between that of wt and RQC-deletion cells, consistent with the hypothesis that
a substantial fraction of the stalled nascent polypeptides depend on CAT-tailing for efficient degradation (Fig. 4B).
Collectively, our studies reveal an unanticipated
feature of Ltn1p, the key ubiquitin ligase responsible for RQC-mediated degradation of incomplete
nascent chains: Ltn1p can only efficiently access
lysines that lie within a narrow window of the
exit tunnel. This spatial specificity could protect
the cell from collateral damage [e.g., degradation
of ribosomal proteins or the translocation machinery, as well as unregulated quality control signaling
(26, 27)] caused by incidental ubiquitination by
Ltn1p. However, the limited reach of Ltn1p presents a challenge to the RQC machinery that must
deal with a diverse range of substrates: Without a
mechanism to relieve this restriction on lysine
positioning, many endogenous RQC substrates
would be resistant to Ltn1p-mediated degradation. Our studies reveal that, in addition to its
previously described role in promoting aggregation and inducing a heat shock response (9–11),
CAT-tailing acts as a fail-safe mechanism that enables the degradation of a far broader range of
substrates by exposing lysines sequestered in the
ribosome exit tunnel (Fig. 4C).
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We thank P. Shen for providing the Ltn1DRING strain and E. Costa
for advice on protein purification. We thank O. Brandman,
M. Jost, E. Costa, C. Gross, S. Torres, M. Chan, D. Santos,
N. Schirle Oakdale, P. Shen, and P. Walter for scientific input. This
work was supported by Howard Hughes Medical Institute (HHMI)
(J.S. W.), University of California San Francisco (UCSF) Discovery
Fellowship (K.K. and B.A.O.), NIH Center for RNA Systems
Biology (J.S. W.), HHMI Faculty Scholar award (A.F.), UCSF
Program for Breakthrough Biomedical Research (A.F. and D.E. W.),
the Searle Scholars Program (A.F.), and NIH grants
1DP2GM110772-01 (A.F.), DP5OD017895 (D.E. W.), and NIH
5R01AG041826-05 (J.S. W.). B.A.O. is a National Science
Foundation Graduate Research Fellow. J.A.H. is the Rebecca Ridley
Kry Fellow of the Damon Runyon Cancer Research Foundation
(DRG-2262-16). K.K. is a Howard Hughes Medical Institute
International Student Research fellow. A.F. is a Chan Zuckerberg
Biohub investigator. K.K., B.A.O., D.E. W., A.F., and J.S. W. conceived
the study. All authors evaluated the results and edited the
manuscript. K.K. and J.S. W. wrote the manuscript. K.K. and
K.L.H. performed all experiments. J.A.H. performed the
Materials and Methods
Figs. S1 to S12
Tables S1 to S3
Data File S1
15 January 2017; resubmitted 4 May 2017
Accepted 28 June 2017