Science - 31 January 2020

see fig. S14B for the somata). Around half of
the 326 proteins encoded by monosome-
preferring transcripts exhibited protein abun-
dances that were greater than the average (Fig.
6D and table S1), indicating that monosome-
preferring transcripts can also encode highly
abundant proteins. We next examined the
properties of the high-abundance proteins
encoded by monosome-preferring transcripts
(“mono-high”;n= 177). To investigate whether
the mono-high protein abundance is related
to mRNA abundance in the neuropil, we used
RNA-seq to estimate local transcript levels (fig.
S13B). Consistent with the correlation be-
tween the local transcriptome and proteome
(R^2 =0.26;Pvalue < 2.2 × 10−^16 ), the mono-
high genes had higher mRNA levels (Fig. 6E;
see fig. S14C for the somata). We then looked
at the relationship between mono-high pro-
tein abundance and local translation rates, a
measurement obtained from neuropil total
footprint libraries (without biochemical frac-
tionation) (fig. S13C). Perhaps predictably,
we observed that mono-high transcripts were
among the most highly translated mRNAs
within the neuropil, which agrees with the
overall positive correlation between the neu-
ropil proteome and local translatome (R^2 =
0.33;Pvalue < 2.2 × 10−^16 ) (Fig. 6F; see fig. S14D
for the somata). Thus, monosome-translated
transcripts can contribute to the neuropil
proteome composition by encoding a full
range of low- and high-abundance proteins,

depending on their expression level and

translation rate.

Discussion

In this work, we investigated the translational

landscape in neuronal processes and identi-

fied local translation on 80Smonosomes as an

essential source of synaptic proteins. To date,

knowledge about the conformation of the

translational machinery near synapses has

originated primarily from electron micro-

graphs. In these studies ribosomes are un-

ambiguously identified when organized as a

polysome cluster formed by more than three

ribosomes ( 45 ). The sparse distribution of

polysomes in dendrites and spines apparent

in electron micrographs has led some to sug-

gest that local proteinsynthesis represents a

minor source of synaptic protein under basal

conditions ( 46 ). Indeed, until the recent de-

tection of mRNAs and the machinery needed

for their translation ( 5 , 47 , 48 ), the inability

to identify polysomes in electron microscopy

(EM) images from mature axons led to asser-

tions that mature axons obtain protein exclu-

sively via intracellular transport from the soma.

Although a previous EM study suggested the

putative visualization of monosomes in den-

dritic spines ( 45 , 49 ), monosomes have not

been identified with certainty, because their

small size (10 to 25 nm) makes it difficult to

distinguish them from other dark-staining

cytoplasmic particles ( 45 ). A previous study

using a fluorescent reporter suggested that

monosome translation might be associated

with sporadic (isolated) translation events in

cultured neuron processes ( 50 ). In this study,

we detected substantial levels of ongoing pro-

tein synthesis in the synaptic neuropil in vivo,

andhereweprovidedirectevidenceforthe

preferential translation of many pre- and pos-

tsynaptic transcripts by monosomes. This find-

ing thus bridges the gap between the relative

paucity of visualized translational machinery in

neuronal processes and actual measurements

of local translation.

Dendritic spines and their associated presyn-

aptic boutons that comprise the excitatory syn-

apse are small subcellular compartments, often

<100 nm^3 for spines ( 51 ). The relatively large

dimensions of a polysome [~100 to 200 nm

( 7 )] limit the possibilities for high ribosome

occupancy in spines and axon terminals. In-

deed, each dendritic spine has been estimated

to contain, on average, one polyribosome ( 52 ).

The observed low density of polysomes at syn-

apses could be due to a limited pool of avail-

able ribosomes in neuronal processes compared

with cell bodies. In agreement with this con-

cept, we observed a decrease in the percentage

of ribosomal RNA (rRNA) relative to total

RNA as well as a de-enrichment of ribosomal

proteins in the neuropil compared with the

somata. Translation via smaller machines (i.e.,

monosomes) allows for more protein synthesis

sites within synaptic compartments. Polysomes

Bieveret al.,Science 367 , eaay4991 (2020) 31 January 2020 5of14

AB

Atp6v1b2Atp6v1a

Atp6v1c1

Gabra1*Gabrb2

Gabrb3Gabrg2

AP1g1AP2a2

AP2b1AP3b2

AP3m2AP3d1

Synrg

Aak1

Clathrin aClathrin b

Glutamate receptor & interactors

Endocytosis

Docking & fusion

Inhibitory receptors

Vamp

Synaptotagmins

Synaptophysins

V-ATPase

GluGlu––HH++ Synaptogyrins

Synapsins 1/2

CAPS

NSF

aSNAP

Actin

Munc13a*

Bsn

Syntaxin6/12 Munc18

Rab3c MyH10

MyH10

Cell Adhesion

PSD & scaffold

SAP102Dlg2*

Lrcc7*

Dlgap3,4*Dlg4*

Mpp2*

Adcy2IP3R1

Ryr2*Calb2*

Camk2aCamkv

Prkar1aCalm1

NrgnHpca

Hpcal4*

Calcium signaling & kinases

Ppp2r1a,2a,5ePpp1r1a,9a

Ppp3ca

Ppp3cb*

Ppp1r14a,1b,9b,12b,16b,3c,3g*

Phosphatases

Ion channels

Cacnb1Kcnab2Cacna2d1Cacna2d3

Cacna1e

Transporters

Slc1a2*Slc4a2*Slc30a4,a5,a9Slc12a2

Slc4a10

Plcb1Pik3c3

Pik3r4Pi4ka

Dgkb

Dgkz

Phosphatidylinositol signaling

GABA AGABA B1

Microtubule dynamics

Tub1a

Map1a/b*Map2*

Mapa/b*Kif1a/b

Kif3a

Dync1i1Kifap3

Dync1li1Dync1i2

Tub1bKlc1/2

Dynactin 2Nefl Cargo

Cargo

Kinesin

Dynein

microtubule

Cplx1/2*

Pclo*

AP

AP

AP

Regulators of local translation

Eif3a,cEif2a

Rptor

Rpl4,Rpl7,Rpl15,Rpl36alRplp0*

Rpl3,11,14,18,19,24,27,28,34*Rpsa*

Rps3*

Eif3fEif4f

Eif4ebp1*Eif5a*

Eef1b2,dEef2*

Gria1Gria2*

Grin2b*Gria3

Grm1*

Grm5Cnih2*

Cacng7*Rasgrf1

Cacng8*Sh3glb2

Shisa7Grasp

Rgs14

NMDARs mGluRs AMPARs

Nlgn1* Dynamin

Nrxn1*

L1CAMNrCAM

Teneurin 2/3/4Adam 22/23

DCC

EphR 5/6

Ephrin B3

Actin dynamics

Pak1,3

LimkPak6

MarcksRhoA

Cfn

Waver1

Arpc3Arpc2

Pfn

Actin

TPM1/3

monosome-enriched

polysome-enriched

actin filament bundle

cell cortex part

myofibril

cell cortex

cytoplasmic region

distal axon

site of polarized growth

dendritic tree

plasma membrane bounded cell projection part

glutamatergic synapse

synapse part

somatodendritic compartment

plasma membrane region

cytoplasmic vesicle

neuron projection

intracellular vesicle

vesicle

synapse

0246

-log 10 FDR

Fig. 4. Monosomes translate many key synaptic transcripts in dendrites and axons.(A) GO terms representing the top 10 significantly enriched protein function
groups for monosome-enriched (cyan) or polysome-enriched (orange) transcripts. (B) Scheme of pre- and postsynaptic compartments highlighting some of the
transcripts preferentially translated by monosomes (cyan) or polysomes (orange). Asterisks denote key synaptic components that were manually added, owing to
their exclusion by the excitatory neuron-specific filter. (See table S1 for information about the fold changes.)

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