Science - 31 January 2020

that a large proportion of transcripts prefer
thesametypeofribosomeoccupancyinboth
compartments (Fig. 5A, monosome-enriched
in quadrant I or polysome-enriched in quad-
rant III). An overlap of the genes classified as
monosome- or polysome-preferring in the
somata (fig. S7A) and neuropil (Fig. 3A) re-
vealed that many but not all genes exhibited
a similar preference between compartments
(fig. S11, A and B). Using DESeq2 ( 18 ), we
identified transcripts that exhibited signifi-
cant differences in the M/P ratio between
somata and neuropil (Fig. 5A and table S1).
Only a handful of transcripts (e.g.,Arc;Fig.
5B) exhibited a significantly lower M/P ratio
in the neuropil than in the somata (Fig. 5A,
purple dots, and Fig. 5C). The majority of
transcripts (e.g.,Serpini1; Fig. 5D) with dif-
ferential ribosome occupancy between com-
partments displayed significantly elevated
M/P fold changes in the neuropil (Fig. 5A, cyan
dots, and Fig. 5E). Overall, we observed a shift

toward a higher monosome preference in the

neuropil (Fig. 5F and fig. S11C), which could

result, at least in part, from a lower ribosome

abundance in the neuropil than in the somata

(fig.S12,AandB).

Notably, we also identified some transcripts

with opposing M/P ratios between somata

and neuropil (i.e., monosome-preferring in

one compartment and polysome-preferring

in the other), some of which were key reg-

ulators of synaptic plasticity (Fig. 5A, quad-

rants II and IV; fig. S12, C and D; and table S1)

( 32 – 41 ). Thus, neuropil-localized transcripts

are, in general, more likely to be translated on

monosomes than somatic transcripts.

Monosome translation contributes

to the neuropil proteome

Individual synapses are small independent in-

formation processing units, each endowed

with their own complement of proteins, rang-

ing in copy numbers from tens to a thousand

or so ( 42 , 43 ). We observed that previously

published protein copy numbers in the rat

presynapse (Fig. 6A) ( 43 ) and postsynapse

(Fig. 6B) ( 42 ) were poorly correlated with

the neuropil M/P preference. To understand

the contribution of monosome and polysome

translation to the overall proteome compo-

sition, we conducted mass spectrometry of

neuropil proteins (see Materials and methods)

and estimated their absolute protein abun-

dances using iBAQ (intensity-based absolute

quantification) ( 44 ) (fig. S13A). As might be

expected, we observed higher median iBAQ

values for proteins encoded by polysome-

preferring transcripts when compared with

proteins encoded by monosome-preferring

transcripts (Fig. 6C; see fig. S14A for the

somata). When we examined the relationship

between the abundance of neuropil proteins

and their respective M/P ratios, however,

we observed a surprisingly weak correlation

(R^2 = 0.021;Pvalue = 2.944 × 10−^11 ; Fig. 6D;

Bieveret al.,Science 367 , eaay4991 (2020) 31 January 2020 4of14

0

0.05

0.1

0.15

0.2

0.25

0.3

fraction

-5-4-3-2-1012345

pause-score per codon

2 kb

Kif1a

B

1 kb

58

0

58

0

Slc17a7

C D

Camk2a

0.5 kb

677

0

677

0

F

463 genes

372 genes

A

-3

-2

-1

0

1

2

3

monosome vs polysome

(log

FC) 2

10 15

mean expression (log 2 )

0 5

neuropil

-25 start 25 50 75 -50 -25center 25 50 -75 -50 -25 stop 25

nucleotide position

0

1

2

3

4

5

6

7

8

normalized p-site coverage

anova p = 2.26e-04

(^012) frame
-0.5
0
0.5
(obs - exp) / exp
E
neuropil monosome-enriched transcripts
0
370
37
more pausing
in polysome
no difference in
pausing
more pausing
in monosome
Fig. 3. Monosomes predominate on a subset of transcripts in dendrites
and axons.(A) MA plot showing transcripts with significantly enriched
monosome (cyan) or polysome (orange) footprint coverage in the central
portion of the ORF (region spanning 15 codons from the start site to 5 codons
before the stop site). DESeq2 was used for analysis, with a threshold of 0.05
on the adjustedPvalue (see Materials and methods). Gray dots denote
nonenriched transcripts. (BtoD) Genome browser views representing the
average monosome (top) or polysome (bottom) footprint coverage for three
transcripts:Kif1a(B),Camk2a(C), andSlc17a7(D). Theyaxis indicates the
number of normalized reads. (E) Metagene analysis showing the monosome
P-site coverage of transcripts that exhibit significant monosome enrichment in
the neuropil. The average normalized coverage is plotted per nucleotide position
around the 5′end (start), central portion (center), and 3′end (stop) of the ORF.
The standard deviation is shaded (n= 3 replicates). The inset shows the
observed (obs)–to–expected (exp) ratio of the footprint distribution in
different reading frames.P= 2.26 × 10−^4 , ANOVA. (F) A pause score was
computed for each codon located in the elongating ORF portion of the 463
monosome-enriched transcripts: pause score (z-score–like) = (normalized
footprint coverage in monosome library–normalized footprint coverage
in polysome library)/(normalized footprint coverage in polysome library)1/2
(n= 3 replicates). The graph shows the fraction of codons per pause score.
Dashed lines highlight pause score values of ±1.96 (P= 0.05), values between
these lines represent codons exhibiting similar coverage in monosome and
polysome libraries.
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