Activity-dependent spatially localized miRNA maturation in ...€¦ · REPORT NEUROSCIENCE Activity-dependent spatially localized miRNA maturation in neuronal dendrites Sivakumar

REPORT◥

NEUROSCIENCE

Activity-dependent spatially localizedmiRNA maturation in neuronal dendritesSivakumar Sambandan,1 Güney Akbalik,1* Lisa Kochen,1* Jennifer Rinne,2

Josefine Kahlstatt,2 Caspar Glock,1 Georgi Tushev,1 Beatriz Alvarez-Castelao,1

Alexander Heckel,2,3† Erin M. Schuman1,3†

MicroRNAs (miRNAs) regulate gene expression by binding to target messenger RNAs (mRNAs)and preventing their translation. In general, the number of potential mRNA targets in a cellis much greater than the miRNA copy number, complicating high-fidelity miRNA-targetinteractions.We developed an inducible fluorescent probe to explore whether the maturationof a miRNA could be regulated in space and time in neurons. A precursor miRNA (pre-miRNA)probe exhibited an activity-dependent increase in fluorescence, suggesting the stimulationof miRNA maturation. Single-synapse stimulation resulted in a local maturation of miRNA thatwas associated with a spatially restricted reduction in the protein synthesis of a target mRNA.Thus, the spatially and temporally regulatedmaturation of pre-miRNAs can be used to increasethe precision and robustness of miRNA-mediated translational repression.

MicroRNAs (miRNAs) regulate gene expres-sion by binding to the 3′ untranslatedregion (3′UTR) of target mRNAs and pre-venting their translation (1). A maturemiRNA is generated by the sequential

processing of two different precursors: the prima-rymiRNA (pri-miRNA) and the precursormiRNA(pre-miRNA). The pri-miRNA is processed in thenucleus (2) and then exported to the cytoplasm,where it is processed by Dicer (3) to generate themature miRNA. Bioinformatics allow the pre-diction of individual miRNA binding sites acrosstranscriptomes (4, 5), suggesting that eachmiRNAcan target multiple transcripts (6). The copy num-ber of most miRNAs is relatively low (7, 8) whencompared with the copy number and abundanceof mRNA targets (9), raising the question of howa miRNA can effectively regulate a particular tar-get mRNA. One means to enhance the efficiencyof regulationwould be to promote the spatial prox-imity of the miRNA and its target mRNA, thusincreasing the probability ofmiRNA-target inter-actions. This spatial colocalization could be accom-plished by regulating miRNA maturation—thatis, by first localizing the pre-miRNA and then con-trolling its maturation in both space and time.We tested this idea in neurons, where local

translation (10) and miRNAs (7, 11–15) have beenreported to regulate synaptic function in mor-phologically complex axons and dendrites. To de-termine the number of potential mRNA targets

that dendritic and axonal miRNAs might bind,we evaluated the expression and abundance ofmiRNAs in the hippocampal neuropil by usingNanoString (16) (Fig. 1A and table S1) and thenidentified the number of potential dendriticmRNA targets, using 3′UTR isoform sequencesfrom neuropil-localized mRNAs (Fig. 1B) (e.g., 17).For a given neuronalmiRNA, we detected a broadrange in the number of potential targets, rangingfrom 31 to 1077, with a median of 503. If a pre-miRNA is processed locally, then both the precur-sor and the machinery required for processingshould be present in the dendrites. Dicer (18) andpre-miRNAs have been observed in dendrites andnear synapses (19–21). We found that the precur-sor for miR-181a, a neuronal miRNA that is highlyexpressed in the hippocampus (Fig. 1A) (7, 13, 20),is present in both the cell bodies and dendrites ofcultured hippocampal neurons and hippocampalslices (Fig. 1, C to F, and fig. S1). When comparedwithmaturemiR-181a, there is a greater abundanceof pre–miR-181a in the cell bodies, but a rough-ly equal number of particles in the neuropil(fig. S2).To determinewhether there is active processing

of pre-miRNAs in dendrites, we made an induci-ble fluorescent sensor (22), using the structure ofpre–miR-181a as a backbone (Fig. 2A). A fluoro-phore was placed in the double-stranded back-bone of the miRNA, and a quencher was addedto the loop region. In the intact pre–miR-181aprobe, the fluorophorewas quenched.WhenDicerprocessed the pre-miRNA, the loop region wascleaved, liberating the quencher and enabling themeasurement of fluorescence. Endogenous Diceractivity was sufficient to stimulate probe cleavagein a cellular lysate prepared from control or con-ditional Dicer knockout mice (23) (Fig. 2B). InArgonaute pull-down experiments from hippo-campal lysates spikedwith the pre–miR-181a probe,

we observed a significant increase in fluorescenceassociated with Argonaute, relative to a controlpull-down (Fig. 2C). Thus, the probe works asdesigned to report the Dicer-dependent cleavageof pre–miRNA-181a.We introduced the fluorescent probe to indi-

vidual hippocampal neurons via a patch pipettetogether with a spectrally distinct fluorescent dye(Alexa 488) (Fig. 2E). Under basal conditions, theprocessing of the pre-miRNA probe, as indicatedby an increase in probe fluorescence over time,was evident primarily in the cell body over tens ofminutes (Fig. 2, E, G, andH). To examinewhetherthematuration of pre–miR-181a is sensitive to neu-ral activity, we elicited action potentials (Fig. 2D)and continued tomonitor the probe fluorescence(Fig. 2F). Depolarization led to a dramatic increasein pre-miRNA maturation, as indicated by an in-crease in fluorescence observed in both the somaand dendrites (Fig. 2, F toH). In neurons inwhichDicer expression levels were knocked down byusing a short hairpin RNA (figs. S3 and S4D), theactivity-induced fluorescence increase was sig-nificantly reduced (Fig. 2, G and H, and fig. S5).Calcium can stimulate Dicer activity (21), andincreases in some miRNAs have been observedafter activation of NMDA (N-methyl-D-aspartate)receptors (13, 24). To examinewhether an activity-induced increase in intracellular Ca2+ (mediatedby NMDA receptors) is important for pre-miRNAmaturation, we conducted experiments in thepresence of a NMDA receptor antagonist, APV(D,L-2-amino-5-phosphonovaleric acid; 50 mM)(Fig. 2, G and H). NMDA receptor antagonismcompletely prevented the stimulation-induced in-crease in the pre–miR-181a probe and reduced thebasal level of probe maturation observed in den-drites (Fig. 2, G and H).These data indicate that neural activity can

stimulate the Dicer-dependent maturation of pre-miRNAs. To examinewhether local synaptic activ-ity regulates the maturation of pre-miRNAs, weconducted glutamate uncaging experiments tocontrol the activation of individual synapses. Cul-tured hippocampal neurons were bathed in cagedglutamate, and an Alexa dye and the pre–miR-181aprobe were introduced into neurons as before(fig. S4, A and B). A dendritic region was chosenfor uncaging (Fig. 3A) on the basis of the ability oftest uncaging pulses to elicit a current (uncaging-evoked excitatory postsynaptic current, uEPSC)that resembled the waveform of a single sponta-neous EPSC (average uncaging spot size, 2 mm2;Fig. 3B).Whilemonitoring the probe fluorescencein the patched neuron, a train of uEPSCs wasevoked (~1 Hz, 20 pulses) by uncaging glutamatein a region close to the dendrite (fig. S4C). Coinci-dent with the activation of a single synapse, arapid and spatially localized fluorescence increasewas observed in the dendritic shaft immediatelyadjacent to the stimulated area (Fig. 3, C and D)or, in some cases, directly in the dendritic spine,suggesting the generation of a mature miRNA(Fig. 3, E and F). The probe fluorescence occur-red with an onset that was slightly variable withrespect to the start of the uncaging train, rangingfrom ~1 to 5 s, and began as a ~1-mm2 spot that

RESEARCH

Sambandan et al., Science 355, 634–637 (2017) 10 February 2017 1 of 4

1Max Planck Institute for Brain Research, Max-von-Laue Straße4, 60438 Frankfurt, Germany. 2Institute for Organic Chemistryand Chemical Biology, Goethe University, 60438 Frankfurt,Germany. 3Cluster of Excellence “Macromolecular Complexes inAction,” Goethe University, 60438 Frankfurt, Germany.*These authors contributed equally to this work.†Corresponding author. Email: [email protected] (A.H.);[email protected] (E.M.S.)

on

Febr

uary

14,

201

7ht

tp://

scie

nce.

scie

ncem

ag.o

rg/

Dow

nloa

ded

from

grew to ~3 mm2. After uncaging, themiRNA fluo-rescence persisted for several seconds before de-clining owing to diffusion and/or degradation.No fluorescence increasewas observed after photo-stimulation in Dicer KD neurons, when NMDA

receptors were blocked (Fig. 3D), or when cagedglutamate was omitted from the bath (Fig. 3Dand fig. S7, A and B).Does the local, activity-dependent maturation

of miR-181a at synapses have functional conse-

quences for a mRNA target of miR-181a?We useda recently described technique [Puro-PLA (25)]to pulse-label and visualize newly translated Ca2+-calmodulin protein kinase a (CamKIIa), a majorprotein at excitatory synapses (26) and a target ofmiR-181a (27). We conducted uncaging experi-ments in the presence of a low concentration of pu-romycin to label newly synthesized proteins andthen, using a specific antibody against CamKIIa,conducted Puro-PLA to detect nascent CamKIIathroughout the recorded neuron (fig. S6). Un-caging of glutamate increased probe fluorescencein the dendritic spine (Fig. 4, A and B), indicatingthe activity-dependent maturation of pre–miR-181a. As expected (25), there was abundant newlysynthesized CamKIIa evident in the cell body andthroughout the entire dendritic arbor (Fig. 4C). Inthe region ofmaturemiR-181a generated by localsynaptic activation and in the adjacent dendriticsegment, there was a marked reduction in nascentCamKIIa (Fig. 4, C to E), consistent with previousobservations that synaptic activity can suppresssynthesis of a CamKIIa-based reporter (28).We analyzed control dendrites (from the sameneurons used for uncaging) and observed no con-sistent reduction of signal at a “pseudo”-uncagingspot. In addition, we conducted experiments inneurons transfectedwith a green fluorescent protein(GFP) construct containing the 3′UTR of CamKIIa(with themiR-181a seed) and then conducted Puro-PLA to detect nascent GFP (Fig. 4F and fig. S6C).We observed a significant reduction in newly syn-thesized GFP in the uncaged region of the den-drite, whereas the within-neuron control dendritesexhibited no change. In Dicer KD neurons, therewas no reduction of nascent GFP in the uncagedregion of the dendrite (Fig. 4G and fig. S8). Toexamine whether the reduction in nascent GFPproduction requires the presence of amiR-181a seed,we expressed a GFP flanked by a short CamKIIa3′UTR isoform lacking themiR-181a seed. In theseneurons,weobservednoreduction innascentGFP inthe uncaged region of the dendrite (Fig. 4H and fig.S9); in control experiments, no reduction in nascentGFP was observed around a pseudo-uncagingspot (fig. S7, C and D).A role for miRNA-mediated regulation of syn-

aptic function is well documented (29). Here wedemonstrate directly that pre-miRNA processingoccurs in neuronal dendrites and is stimulated byneural activity at individual synapses, resulting ina local reduction in target protein synthesis. Oth-ers have demonstrated a rapid NMDA receptor–mediated regulation of various miRNAs (e.g.,30), includingmiR-181a (13, 24). In our study, thelocal maturation of pre–miR-181a, promoted bylow-frequency stimulation, led to a local down-regulation of a target, CamKIIa, consistent withdata showing a tonic repression of CamKIIasynthesis by spontaneous synaptic events (28). Onthe other hand, it is known that higher-frequencystimulation (e.g., 100 Hz) can lead to increasedCamKIIa synthesis (31), indicating the possibilityof frequency-dependent stimulation or repressionof Dicer activity. Taken together, our data indicatethat the spatially and temporally regulated con-trol of miRNA maturation is one mechanism by

Sambandan et al., Science 355, 634–637 (2017) 10 February 2017 2 of 4

Pre-miR-181aMAP2

C D

FE

individual miRNA species expression rank1 50 100 150 200 250 300 350 400 443m

iRN

A e

xpre

ssio

n [N

anoS

trin

g co

unts

]

0

10

100

1000

10000

detection threshold

miR-181a

num

ber

of ta

rget

mR

NA

tran

scrip

ts

miRNAs [relative count]0.0 0.2 0.4 0.6 0.8 1.0 median

0

200

400

600

800

1000

1200

503

A B

Pre-miR-181aMAP2DAPI

Scrmbl ctrlMAP2

Scrmbl ctrlMAP2DAPI

Fig. 1. Pre–miR-181a is present in neuronal dendrites. (A) Graph (means ± SEM) showing all miRNAsdetected in the neuropil of area CA1 of the rat hippocampus (purple dots; 138 of 443 present in the codeset) by using nCounter NanoString [n = 3; stars, positive spike-in; black dots, negative spike-in; graydots, not detected (305 of 443)]. (B) The predicted number of target mRNAs (y axis) for all miRNAsdetected in the hippocampal neuropil [(from (A)]. The median and quartiles are shown. (C) Rep-resentative in situ hybridization image (andmagnification of one dendrite, right panel) acquired using aprobe directed against the loop region of pre–miR-181a, showing pre–miR-181a (white) in the soma anddendrites of a cultured hippocampal neuron. Immunostaining with an antibody against MAP2 (microtubule-associated protein 2;magenta) identifies the dendrites. (D) Scrambled control for in situ hybridization. Scalebars in (C) and (D), 10 mm. (E)Representative in situ hybridization imageof pre–miR-181a in theCA1 regionof ahippocampal slice (and magnification of the boxed dendritic region, right panel). The in situ hybridizationsignal is abundant in the soma and neuropil layer. (F) Very low signal is observed in the scrambled control.Scale bars in (E) and (F), 50 mm. DAPI, 4′,6-diamidino-2-phenylindole.

RESEARCH | REPORT

on

Febr

uary

14,

201

7ht

tp://

scie

nce.

scie

ncem

ag.o

rg/

Dow

nloa

ded

from

Sambandan et al., Science 355, 634–637 (2017) 10 February 2017 3 of 4

20 mV1 s

-70 mV

2 min 10 min 15 min 25 min

2 min 10 min 15 min 30 min

E

200 pA

Alexa 488

F

miRNA probe

G H

Dic

er p

roce

ssin

g

A C

F

**

*

0

B

Control Dicer KO

***0.7

0.6

0.5

0.4

0.3

0.2

0.1

Q

Q

0 5 10 15 20 25 300.8

1.0

1.2

1.4

1.6

1.8

2.0

0 5 10 15 20 25 30

1

2

3

4

5

Time (min)Time (min)

BasalStimulated

Stimulated (+ APV)F

luor

esce

nce

(fol

d ch

ange

)

Stimulated (+ Dicer KD)

*

*

Somata DendritesC

ontr

olS

timul

ated

0

1

2

3

4

ecnecseroulf evit al eR

AGO IP Control

DF

Fig. 2. An inducible probe reports the activity-dependent matu-ration of a miRNA. (A) Schematic of the pre–miR-181a probeshowing the fluorophore (F) and quencher (Q) near the base of theloop. (B) In control lysates, the fluorescence generated by probeprocessing was significantly higher than that observed in Dicer KOlysates (see the methods section in the supplementary materials;***P<0.0001, n=3 animals). (C) Probe fluorescence after immuno-precipitation (IP) with either an Argonaute 2 (AGO2) or immuno-globulinG (IgG; control) antibody (one experimentwith four and tworeplicates for AGO2and IgG, respectively). (D) Representativewhole-cell recording showing intracellular current injection and actionpotentials. (E) Alexa dye–filled recorded neuron (left) and time-lapse images of pre–miR-181a probe fluorescence over time (right).An increase in probe fluorescence in the soma (white dotted line)was detectable over the course of 25 min, presumably reflectingbasal levels of Dicer activity. Scale bar, 20 mm. (F) Stimulated Alexadye–filled recorded neuron (left) and time-lapse images of probefluorescence over time (right). At 7min, the neuronwas depolarized,leading to a rapid increase in probe fluorescence in the soma (whitedotted line) and dendrites. Scale bar, 20 mm. (G) Group analysis(somata) showing the significant increase in probe fluorescenceafter stimulation [*P<0.05; one-way repeatedmeasures analysis ofvariance (rANOVA)], which was blocked by Dicer knockdown (*P <0.05; one-way rANOVA) or a block of NMDA receptors (APV; 50 mM)(*P<0.05; one-wayANOVA). (H)Group analysis (dendrites) showingthe significant increase in probe fluorescence after stimulation (*P <0.05; one-way ANOVA); either Dicer knockdown or APV reducedthe basalmaturation of the probe (*P <0.05; one-way ANOVA) andblocked the stimulation-induced increase (*P < 0.05; one-wayANOVA) (n= 10, 8, 8, and 7 for basal, stimulated, stimulated +DicerKD, and stimulated + APV groups, respectively). Arrows indicatethe time of stimulation. All values are means ± SEM.

Alexa 488

D

BA

F

50 pA

10 ms

uEPSCs sEPSCs

-10 0 10 20 30 40 50 600

1.0

2.0

3.0

4.0

Flu

ores

cenc

e ch

ange

Time (s)

Spine headDendritic shaft

***

E-5 s 5 s 10 s

15 s 20 s 25 s

-5 s -1 s 5 s1 s

20 s 30 s 60 s 8 min

C

Flu

ores

cenc

e ch

ange

Uncaged

Uncaged + APV

***

Uncaged + Dicer KD

Uncaging

Uncaging

-10 0 10 20 300.5

1.0

1.5

2.0

2.5

Laser only

Fig. 3. Local stimulation leads to the rapid and spatially re-strictedmaturation ofmiR-181a in dendrites and spines. (A) Alexadye–filled recorded neuron.The yellow box shows the dendritic seg-ment in (C) with the glutamate uncaging spot. Scale bar, 10 mm.(B) Representative spontaneous excitatory postsynaptic currents(sEPSCs) and uncaging-elicited currents (uEPSCs). (C) Time-lapseimages of pre–miR-181a probe fluorescence before and after localglutamate uncaging (yellow arrow). An increase in fluorescence wasevident within 1 s after uncaging. Scale bar, 2 mm. (D) Group analysisof dendrites showing the significant uncaging-induced increase inprobe fluorescence (***P< 0 .0001; two-way ANOVA), which wasblocked by Dicer knockdown or a block of NMDA receptors (APV;50 mM). In some cases, error bars are small and occluded by thesymbol. Photostimulation without caged glutamate did not producea fluorescence increase (n = 23, 6, 6, and 5 for uncaging, Dicer KD,uncaging + APV, and laser-only groups, respectively). (E) Time-lapseimages of probe fluorescence in a dendritic spine during localglutamate uncaging. Scale bar, 2 mm. (F) Group analysis of neuronaldendritic spines showing the significant increase in probe fluores-cence after local glutamate uncaging (***P<0.0004, n= 6; pairedt test). All values are means ± SEM.

RESEARCH | REPORT

on

Febr

uary

14,

201

7ht

tp://

scie

nce.

scie

ncem

ag.o

rg/

Dow

nloa

ded

from

which the potency and robustness of miRNA reg-ulation of gene expression can be achieved.

REFERENCES AND NOTES

1. D. P. Bartel, Cell 136, 215–233 (2009).2. Y. Lee et al., Nature 425, 415–419 (2003).

3. E. Bernstein, A. A. Caudy, S. M. Hammond, G. J. Hannon,Nature 409, 363–366 (2001).

4. S. Griffiths-Jones, H. K. Saini, S. van Dongen, A. J. Enright,Nucleic Acids Res. 36, D154–D158 (2007).

5. V. Agarwal, G. W. Bell, J. W. Nam, D. P. Bartel, eLife 4, e05005(2015).

6. L. P. Lim et al., Nature 433, 769–773 (2005).7. M. J. Kye et al., RNA 13, 1224–1234 (2007).8. U. Bissels et al., RNA 15, 2375–2384 (2009).9. B. Schwanhäusser et al., Nature 473, 337–342

(2011).10. C. E. Holt, E. M. Schuman, Neuron 80, 648–657

(2013).11. G. M. Schratt et al., Nature 439, 283–289 (2006).12. M. L. Baudet et al., Nat. Neurosci. 15, 29–38 (2012).13. M. van Spronsen et al., PLOS ONE 8, e74907 (2013).14. Y. Sasaki, C. Gross, L. Xing, Y. Goshima, G. J. Bassell, Dev.

Neurobiol. 74, 397–406 (2014).15. V. M. Ho et al., Mol. Cell. Neurosci. 61, 1–12 (2014).16. G. K. Geiss et al., Nat. Biotechnol. 26, 317–325

(2008).17. I. J. Cajigas et al., Neuron 74, 453–466 (2012).18. G. Lugli, J. Larson, M. E. Martone, Y. Jones, N. R. Smalheiser,

J. Neurochem. 94, 896–905 (2005).19. S. Bicker et al., Genes Dev. 27, 991–996 (2013).20. R. Saba et al., Mol. Cell. Biol. 32, 619–632 (2012).21. G. Lugli, V. I. Torvik, J. Larson, N. R. Smalheiser, J. Neurochem.

106, 650–661 (2008).22. B. P. Davies, C. Arenz, Bioorg. Med. Chem. 16, 49–55

(2008).23. J. Stubbusch et al., Dev. Biol. 400, 210–223 (2015).24. M. J. Kye et al., PLOS ONE 6, e24682 (2011).25. S. tom Dieck et al., Nat. Methods 12, 411–414

(2015).26. M. K. Bennett, N. E. Erondu, M. B. Kennedy, J. Biol. Chem. 258,

12735–12744 (1983).27. S. W. Chi, J. B. Zang, A. Mele, R. B. Darnell, Nature 460,

479–486 (2009).28. M. A. Sutton, N. R. Wall, G. N. Aakalu, E. M. Schuman, Science

304, 1979–1983 (2004).29. G. Schratt, Nat. Rev. Neurosci. 10, 842–849

(2009).30. K. Wibrand et al., Eur. J. Neurosci. 31, 636–645

(2010).

ACKNOWLEDGMENTS

We thank I. Bartnik, N. Fuerst, A. Staab, and C. Thum for thepreparation of cultured hippocampal neurons. We thank C. Hanusfor assistance with two-photon microscopy. E.M.S. is funded by theMax Planck Society, an Advanced Investigator award from theEuropean Research Council, and German Research Foundation(DFG) Collaborative Research Centre (CRC) 1080 (Molecular andCellular Mechanisms of Neural Homeostasis). E.M.S. and A.H. arefunded by DFG CRC 902 (Molecular Principles of RNA-basedRegulation) and by the DFG Cluster of Excellence “MacromolecularComplexes in Action,” Goethe University. J.R. and A.H. gratefullyacknowledge the stipend support of J.R. by Fonds der ChemischenIndustrie. S.S., G.A., L.K., J.R., J.K., C.G., and B.A.-C. designedand conducted experiments and analyzed results. G.T.conducted experiments and analyzed results. A.H. and E.M.S.designed experiments and supervised the project. E.M.S.wrote the manuscript. The authors declare no competingfinancial interests.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/355/6325/634/suppl/DC1Materials and MethodsFigs. S1 to S9Table S1References (31–36)Movies S1 and S2

16 April 2016; resubmitted 17 November 2016Accepted 5 January 201710.1126/science.aaf8995

Sambandan et al., Science 355, 634–637 (2017) 10 February 2017 4 of 4

A

DC

B

Nascent CamKIIα Nascent CamKIIαMAP2

-5 s 5 s 10 s 20 s 30 s

Ctrl Uncaged

E

GFP CDS CamKIIα long 3’UTR AAAA

mi181a

0.0

0.5

1.0

1.5

2.0

Nor

mal

ized

PLA

inte

nsity

Nor

mal

ized

PLA

inte

nsity

**

ns

Ctrl Uncaged Ctrl Uncaged Ctrl Uncaged0.0

0.5

1.0

1.5

2.0

2.5

AAAA

Dicer knockdown

0.0

0.5

1.0

1.5

0.0

0.5

1.0

1.5

Proximal segment

Uncaged segment

Proximal segment

Uncaged segment

Proximal segment

Uncaged segment

Proximal segment

Uncaged segment

F G H

ns

0.0

0.5

1.0

1.5

2.0

2.5Nascent GFP

Nascent GFP

GFP CDS CamKIIα long 3’UTR AAAAGFP CDS short 3’UTR

**

0.0

0.5

1.0

1.5

2.0

0.0

0.5

1.0

1.5

2.0

0.0

0.5

1.0

1.5

2.0 Nascent GFP

Nascent CamKIIα

Fig. 4. The local activity-dependent maturation of miR-181a is associated with a decrease in thetranslation of a target mRNA, CamKIIa. (A) Alexa dye–filled recorded neuron showing the glutamateuncagingspot (small yellowbox indicatedby thewhite arrow).Scale bar, 10 mm. (B) Time-lapse imageof probefluorescence in a dendritic spine during local glutamate uncaging (yellowbox, uncaging spot). In this experiment,theAlexa spine signal increased as a result of uncaging, owing to either changes in spinemorphologyordiffusionof the dye into the spine head. Scale bar, 5 mm. (C) Representative image showing newly synthesized CamKIIaparticles after labelingandglutamateuncaging (arrow, regionofuncaging). (D)Highermagnificationof theboxedarea in (C) (green, Puro-PLA signal for nascent CamKIIa; blue, dendrites immunostained with antibody againstMAP2; white box, uncaging spot). A paucity of newly synthesized CamKIIa particles is observed in the region ofuncaging, as well as the adjacent dendritic shaft. Scale bars in (C) and (D), 20 mm. (E) Summary graphsindicating the mean fluorescent intensity obtained in the proximal and uncaged dendritic segments (see themethods; n= 6 pairs of dendrites, **P=0.0054). In (E) to (H), uncaged andwithin-neuron control dendrites areshown in green and gray, respectively. (F) Schematic (top) and analysis (bottom) of newly synthesized GFPparticles in neurons transfected with the GFP-CamKIIa 3′UTR construct after labeling with puromycin andglutamateuncaging. (n=9pairsofdendrites, **P=0.0017). (G)Schematic (top) andanalysis (bottom)ofnewlysynthesized GFP particles in Dicer knockdown neurons transfected with the GFP-CamKIIa 3'UTR con-struct. [n = 6, P = 0.84, not significant (ns)]. (H) Schematic (top) and analysis (bottom) of newly syn-thesized GFP particles in neurons transfectedwith a construct lacking themiRNA seed region in the 3′UTR(n = 6, P = 0.57, ns). All P values are from unpaired t tests. Bars represent means (longer bars) and SEM.

RESEARCH | REPORT

on

Febr

uary

14,

201

7ht

tp://

scie

nce.

scie

ncem

ag.o

rg/

Dow

nloa

ded

from

(6325), 634-637. [doi: 10.1126/science.aaf8995]355Science (February 9, 2017) Alvarez-Castelao, Alexander Heckel and Erin M. SchumanRinne, Josefine Kahlstatt, Caspar Glock, Georgi Tushev, Beatriz Sivakumar Sambandan, Güney Akbalik, Lisa Kochen, Jenniferneuronal dendritesActivity-dependent spatially localized miRNA maturation in

 Editor's Summary

, this issue p. 634Sciencematuration can modulate target gene expression with local and temporal precision.miRNA was indeed associated with a local reduction in protein synthesis. Thus, localized miRNAactivity-dependent maturation of the probe in both the soma and dendrites. This local maturation of the introduced a fluorescent miRNA maturation reporter into hippocampal neurons and detectedused high-resolution in situ hybridization to detect precursor miRNA in rat neuronal dendrites. They

et al.How then can a miRNA effectively regulate translation of a particular target mRNA? Sambandan In cells and tissues, mRNA copy numbers far exceed the number of micro RNAs (miRNAs).

Intraneuronal control of protein expression

This copy is for your personal, non-commercial use only.

Article Tools

http://science.sciencemag.org/content/355/6325/634article tools: Visit the online version of this article to access the personalization and

Permissionshttp://www.sciencemag.org/about/permissions.dtlObtain information about reproducing this article:

is a registered trademark of AAAS. ScienceAdvancement of Science; all rights reserved. The title Avenue NW, Washington, DC 20005. Copyright 2016 by the American Association for thein December, by the American Association for the Advancement of Science, 1200 New York

(print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last weekScience

on

Febr

uary

14,

201

7ht

tp://

scie

nce.

scie

ncem

ag.o

rg/

Dow

nloa

ded

from