well-studied pause in Xbp1, in each genotype
(Fig. 4, A and B; fig. S15A; tables S2, A to H; and
table S3) (16, 17). However, no significant differences in pause number occurred between genotypes.
We then determined the number of pauses at
AGA codons (Fig. 4C and fig. S15B). In the B6J.
B6Nn–Tr20 and B6J.B6Nn–Tr20; nmf205–/– cerebellum,
few strong AGA pauses were observed (Fig. 4D and
tables S2, I to P). Demonstrating the effect of
impaired n-Tr20 processing, a threefold increase
in strong AGA pauses was observed in the B6J
Gene ontology analysis of transcripts with strong
pauses in the B6J-nmf205–/– cerebellum showed
enrichment for translation-related genes, among
others (table S2Q).
Reads at AGA codons in B6J and B6J-nmf205–/–
cerebella were 1.6 and 2.8 times background
expectations, respectively, whereas unusual AGA
pausing was not observed in cerebellar libraries
from B6J.B6Nn–Tr20 and B6J.B6Nn–Tr20; nmf205–/–
mice (Fig. 4F). To determine whether the increase
in ribosome pausing in the B6J and B6J-nmf205–/–
mice is specific to AGA codons, we compared codon frequency at (P ≥ 10) pause sites to the overall codon usage in transcripts. Although minor
deviations were observed for several other codons,
458 25 JULY 2014 • VOL 345 ISSUE 6195
Fig. 4. The n-Tr20 mutation induces ribosome stalling, which is resolved
by GTPBP2. (A) Cumulative distribution of pauses at all codons averaged
between replicates. (B) The mean number of pauses ≥10 standard deviations
above the background translation levels of their genes (P ≥ 10). (C) Cumulative
distribution of pause scores at AGA codons averaged between replicates.
(D) The mean number of pauses (P ≥ 10) at AGA codons. (E) Read profile for
Zfp238 coding sequence. Asterisk (*) indicates an AGA pause with P = 45.
(See fig. S16.) Arrows indicate AGA codons. (F) Average pausing magnitude
at AGA codons, calculated by dividing genome-wide observed reads at AGA
codons by expected reads. Expectations are based on read densities in genes
containing an AGA. Error bars indicate standard deviations across biological
replicates. *P < 0.05 (two-tailed Student's t test). (G) Difference in the codon
frequency observed in the A site at pauses (P ≥ 10) versus the genome-wide