fraction, a one-way analysis of variance (ANOVA)
was performed. A significantPvalue rejects
the null hypothesis that all frames exhibit
the expected P-site coverage.
Genome browser track visualization
Footprint coverage was visualized as custom
tracks on the UCSC Genome Browser ( 94 ). Foot-
print alignments were converted into BedGraph
files (https://genome.ucsc.edu/goldenPath/
help/bedgraph.html) using Bedtools v2.26.0.
Gene ontology analysis
GO enrichment of monosome- or polysome-
preferring genes was performed using the
R package clusterProfiler ( 95 ) with a Benjamini-
Hochberg multiple testing adjustment and a
FDR cutoff of 0.05, using all expressed neu-
ronal genes in the neuropil or somata as back-
ground, respectively. The simplify function with
acutoffof0.7wasusedtoremoveredundancy
from enriched GO terms.
Correlation between the M/P fold change
and transcript attributes
DNA sequences were extracted from the rat
(rn6) genome. Only genes with valid values for
all transcript attributes were used for analy-
sis. The length of 3′UTRs and 5′UTRs was set
to a minimum of 10 nts.
GC content: The GC content was assessed by
counting the number of G or C bases in the
sequence and then dividing by the number of
bases in the predicted 5′UTR, CDS, or 3′UTR.
Minimum free energy (MFE): The ViennaRNA
package version 2.0 with RNAfold was used
to calculate the MFE per 5′UTR or 3′UTR
sequence ( 96 ). A method described by Trotta
( 97 ) was adapted to normalize MFE units
tothesequencelength.Thesequencelength
was restricted to a maximum of 500 nucleo-
tides in proximity to the start and stop codons.
Codon adaptation index: CAI values in the
neuropil were obtained for neuronal genes only,
following the procedure described in ( 98 ).
Initiation rate: The initiation rate per gene
was calculated on the basis of the neuropil
total ribosome footprint and RNA coverage,
as previously described ( 99 ). In short, the
initiation rate depends on the translational
efficiency (defined as described above), CDS
length, average time for a ribosome to tra-
verse the CDS, and normalized ribosome oc-
cupancy in the initial 10 codons of the CDS.
The average elongation rate was assumed to
be 4 codons/s ( 53 ). Axvalue of 0.0084 was
determined from the best-fit line to the av-
erage ribosome density of a transcript (from
polysome profiling) versus its translational ef-
ficiency (from ribosome profiling and RNA-seq).
Mean typical decoding rate: A per-gene
MTDRwascalculatedonthebasisoftheneu-
ropil total ribosome footprint coverage, as
previously described in ( 26 ). In short, each
amino acid decoding time was defined as
a convolution of an average decoding time
(a Gaussian component with the parame-
tersmands) and a pausing decoding time
(an exponential component with the param-
eterl). A model-fitting procedure was used
to deconvolve the two distributions and
identify the three parameters per amino acid.
The geometric mean of all average decoding
times (m) was calculated to determine the
per-gene MTDR.Upstream open reading frame (uORF)
To identify transcripts containing uORFs, neu-
ropil total ribosome footprint libraries from
three replicates were used. Only genes with
annotated 5′UTRs were considered. A string
match algorithm was used to identify sequen-
ces within annotated 5′UTRs that are flanked
by canonical in-frame start and stop codons.
Only sequences with a minimum length of
three codons and at least 10 raw footprints in
all three replicates were considered as uORFs.Prediction of protein secondary structure
and protein domains
Appris transcript isoforms were translated
into amino acid sequences and used to pre-
dict secondary structures and protein domains.
Porter 5 was used to predict protein secondary
structures in three classes (ahelix,bstrand,
and coil) ( 100 ). Spans of coils were defined as
unstructured, whereas helices and strands
were defined as structured sequences. Tran-
sitions from structured to unstructured, and
vice versa, were counted and normalized to
the sequence length. Protein domains were
predicted using InterProScan5 based on the
Pfam database ( 101 ). Functional domains per
protein were merged into unique regions, and
their average length was compared between
monosome- and polysome-enriched genes.Codon pause score analysis
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before the stop codon) of neuropil monosome-
enriched genes, a pausescore was calculated
based on az-score–like quantity: pause score =
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