RNA Detection

information on the untranslated regions (UTRs) of isoforms, which

would be lost by RNase treatment, although the ribosome profiling

can reveal the number of ribosomes bound to a single mRNA

species which would accurately reflect the translational rate.

Previously, we developed axon-TRAP, a subcellular TRAP

method that allows specific isolation of ribosome-bound mRNAs

from the distal RGC axons inXenopustadpoles in vivo [4]. We

recently extended this technique to mouse, using RiboTag [6], a

knockin mouse line in which Cre-mediated recombination labels

the 60S subunit ribosomal protein L22 (Rpl22) with hemaggluti-

nin (HA) tags (Rpl22-HA). We crossed this mouse with a Pax6-

alpha-Cre [9], in which distal neural retinal progenitors transiently

express Cre, leading to permanent HA-labeling of ribosomes in

RGCs (Fig.1a, red area in the eye). This new technique, termed

axon-TRAP-RiboTag, involves affinity purification of HA-tagged

Retinal

axons

AB

HA-tagged

Superior ribosome

colliculus

Cre(-) Cre(+)

Nonspecifically

bound mRNA

+ RRL

Translating

ribosome

Stalled

ribosome

‘Run off’

Doesn’t run off

Dissection

Lysis and

affinity-

purification

RNA-

sequencing

Run-offNo Run-off

Affinity-

purification

RNA-

sequencing

DEG

analysis

Ribosome-bound

mRNAs

Translated

mRNAs

DEG

analysis

Split Cre (+) Iysate

Fig. 1Timeline of the experiments. (a) Axon-TRAP for RGCs. We crossed the RiboTag mouse with the Pax6-
alpha-Cre to express HA-tagged ribosomes in RGCs. The affinity purification of HA-tagged ribosome–mRNA
complexes from the superior colliculus (SCs), where RGC axons terminate, enables translational profiling of
distal RGC axons in vivo by deep sequencing. To exclude false-positive signals, we compared the RNA-seq
data from TRAPed mRNAs between the Cre-positive and -negative littermates. (b) In vitro ribosome run-off
assay. To distinguish translated mRNAs from translation-stalled mRNAs, we carried out in vitro ribosome run-
off assay, which allows translational elongation in vitro in the presence of rabbit reticulocyte lysate (RRL)

86 Toshiaki Shigeoka et al.