Supporting Online Material For

www.sciencemag.org/cgi/content/full/317/5842/1220/DC1

Supporting Online Material for

A MicroRNA Feedback Circuit in Midbrain Dopamine Neurons

Jongpil Kim, Keiichi Inoue, Jennifer Ishii, William B. Vanti, Sergey V. Voronov, Elizabeth Murchison, Gregory Hannon, Asa Abeliovich*

*To whom correspondence should be addressed. E-mail: [email protected]

Published 31 August, Science 317, 1220 (2007) DOI: 10.1126/science.1140481

This PDF file includes:

Materials and Methods Figs. S1 to S6 Tables S1 to S3 References

Supplementary Online Materials

(1) Methods

Generation of Dicer conditional knockout ES cells

ES cells homozygous for a floxed allele of Dicer were derived from blastocysts generated by crossing mice homozygous for the Dicer floxed allele (1). Establishment of

ES cell lines from blastocysts was performed as described (2). Genotyping of Dicerflox/flox and DAT-Cre alleles was carried out by PCR.

ES cells carrying a conditional floxed allele of Dicer (1) were infected with CRE or

GFP lentivirus to generate Dicer knockout ES cells at the stage 4 as described (2).

Genomic DNA was extracted from stage 5 ES cells and CRE Infected cells were genotyped by using the following PCR primer pairs: for 23F (ATTGTTACCAGCGCT

TAGAATTCC); 458F (TCGGAATAGGAACTTCGTTTAA AC), and 460R

(GTACGTCTACAATTGTCTATG).

Quantitative real time RT-PCR

For quantitative real-time RT-PCR of miRNAs, total RNA from human brain and mouse brain were prepared with the mirVana miRNA Isolation Kit (Ambion). 0.5ug RNAs was used for the reverse transcription by using SuperScript II RTase (Invitrogen).

Quantitative real-time rtPCR was performed using the qRT-PCR detection kit with precursor miRNA-specific primers. PCRs were optimized to determine the linear amplification range by using a Stratagene MX3000P system with QuantiTect PCR mix

(Qiagen). The qPCR used a β-actin control and a standard curve. For the confirmation of the real time PCR result, Northern blot analysis for candidate miRNAs was performed with human and mouse brain RNA. Primers for precursors were designed based on the miRBase database (http://microrna.sanger.ac.uk/sequences/) using the OligoPerfect

Designer software (Invitrogen). Primers for miR133b is 5'- TGGTCAAACGGAACCAAGTC-3'(F), 5'-TTGCCAGCCCTGCTGTAG-3'(R, mus), 5'-

TCTCCAAGGACTGGGCATT-3'(R, human). Samples were controlled for β-actin using the following primers: 5'-GCTACAGCTTCACCACCACA-3'(F, mus), 5'-

TCTCCAGGGAGGAAGAGGAT-3'(R, mus), 5'-CTCTTCCAGCCTTCCTTCCT-3'(F, human), 5’-AGCACTGTGTGTTGGCGTACAG-3'(R, human).

Patient information for RNA samples:

Control Brain 1: Age: 89 years; Gender: male; Samples: mesencephalon, frontal cortex, cerebellum; Diagnosis: usual aging.

Control Brain 2: Age: 89 years; Gender: female; Samples: mesencephalon, frontal cortex, cerebellum; Diagnosis: usual aging.

Control Brain 3: Age: 63 years; Gender: male; Samples: frontal cortex; Diagnosis: cardiac arrest.

Control Brain 4: Age: 86 years; Gender: male; Samples: Substantia nigra; Diagnosis:

Congestive Heart failure.

Control Brain 5: Age: 68 years; Gender: male; Samples: Cerebellum; Diagnosis:

Coronary Atherosclerosis

Parkinson disease Brain 1: Age: 77 years; Gender: male; Samples: Cerebral cortex,

Mesencephalon, Cerebellum; Diagnosis: Diffuse Lewy body disease, limbic or transitional type (Parkinson disease –dementia).

Parkinson disease Brain 2: Age: 73 years; Gender: male; Samples: Cerebral cortex,

Mesencephalon, Cerebellum; diagnosis: Lewy body disease, limbic or transitional type

(Parkinson disease –dementia).

Parkinson disease Brain 3: Age: 60 years; Gender: male; Samples: prefrontal cortex, mesencephalon, Cerebellum; Diagnosis: Parkinson disease; cause of death: gunshot wound.

RNA transfection

For rescue experiments, small RNAs(<200bp) and large RNA(>200bp) were separately isolated with the mirVana RNA extraction kit (Ambion). After vCRE infection in mouse

ES cell derived dopamine neurons at stage 4, total RNAs were transfected at the end of stage 4 using an RNA transfection kit (TransMessenger Transfection Reagent, Qiagen) according to the manufacturer’s instructions.

TUNEL assay

Cryostat frozen sections (10 micrometer) embedded in OCT were prepared from control and mutant mouse brain. The TUNEL assay was performed using the ApopTag Plus In

Situ Apoptosis Fluorescein Detection Kit (Chemicon) according to the manufacturer’s instructions.

Mouse behavior

For open field test, 2 month-old male Dat-Cre: Dicer fl/+ and Dat-Cre: Dicer fl/fl mice were used (n = 4 for each group). Activity was monitored over 30 min by beam interruption (10 bins, 3 min per bin) in the novel environment of 17.0” X 17.0” (MED-

OFA-RS, Med Associates).

Northern blot analysis

Total RNA (20ug) was separated on 12% polyacrylamide gels containing 8M urea in vertical electrophoresis cells (Invitrogen). RNAs were transferred to Hybond-N+ nylon membranes (Amersham) in an electrophoretic transfer cell (BioRad) for 2 h at 200V.

Oligonucleotides complementary to miRNAs (as described in the Sanger microRNA registry) were end labeled and used as probes to detect their respective miRNAs.

Ribonuclease protection assay

RNase A/T1 protection assay (RPA) was performed using the mirVana miRNA Detection kit (Ambion) following the manufacturer's protocol. The RNA probes for LNA modified miR133a1 and miR133b were synthesized by Exiqon. The probes were generated by in vitro transcription using 20 pmoles of oligonucleotide as template. RPA reactions were performed using 5 µg total RNA and ~60,000 cpm of gel-purified probe as described by the manufacturer. After hybridization and digestion, probe was separated on a denaturing 15% polyacrylamide/urea gel. For imaging, gels were exposed and analyzed using a

Molecular Dynamics Storm Phosphorimager.

Luciferase assays pGL3 miR-133 sensor plasmid was constructed by insertion of two miR-133b binding sites in the XbaI site of the pGL3 promoter vector (Promega). The miR-133a and miR-

133b promoter plasmids were constructed by cloning of a 350bp miR-133a or miR-133b promoter sequences into the 5’ region of the pGL3 firefly luciferase assay vector. For luciferase assays, vectors were transfected using lipofectamine (Invitrogen) along with a

Renilla luciferase vector as internal control. 48 hours after transfection, cell extracts were analyzed for firefly luciferase activity and normalized with Renilla luciferase activity, as per the manufacturer’s instructions (Amersham).

Modified 2'-O-Methyl oligonucleotides siRNA duplexes and 2'-O-methyl oligonucleotides were purchased from a commercial vendor (IDT). 2'-O-methylated DNA oligonucleotides containing a 5' thiol group were reduced with TCEP (Sigma) at room temperature for 15 min. Penetratin (Qbiogene) was added and the mixture was incubated at 65 °C for 5 min followed by an inclubation at 37 °C for 60 min. Coupled oligonucleotides (90 mM) were heated at 65 °C for 15 min with polysalic acid and then added to cells.

Dopamine determination by reversed phase HPLC

Dopamine levels were determined in high KCL (56mM) medium and in control media essentially as described (2).

Flow cytometry

Flow cytometry was performed as previously described (3). Briefly, mice at postnatal day 10 were anesthetized, midbrains were removed, and dissociated for 45 minutes at

37C with Papain enzyme (Papain Dissociation System, Worthington Biochem) in EBSS according to the manufacture’s protocol. The dissociated mouse midbrain cells were then pelleted, resuspended in ice-cold 4% paraformaldehyde, and incubated for 15 min at 4°C. The cells were washed twice, resuspended at 106 cells/ml, and stained with appropriate antibody reagents in the presence of saponin (0.5%) for permeabilization. All flow cytometry was performed on a FACScan instrument (Becton-Dickinson). Data were analyzed with FlowJo software (TreeStar).

Lentiviral vector production

The lentiviral expression plasmids for miR-133b and miR-18 were constructed based on the Lentilox 3.7 plasmid (4). Sequences that generate hairpin repeats containing miRNAs were cloned into the HpaI/XhoI site of Lentilox 3.7. Lentivirus production was as described (2) and miRNA expression was confirmed by Northern blotting

(Supplementary Figure 5A).

Statistical analysis Results are given as mean ± S.E.M. Where appropriate, statistical analysis were performed with analysis of variance (ANOVA) test. Otherwise, comparisons between groups were conducted using Student’s t test. The null hypothesis was rejected at the

P<0.05 level.

(2) Supplementary Figures

Supplementary Figure 1

(A) PCR analysis of genomic DNA from murine Dicer conditional knockout embryonic stem cells (Dicer fl/fl) and wild-type ES cells (Dicer wt/wt). Dicer can be inducibly deleted (Del) at the terminal stage of differentiation in ES cell-derived dopamine neurons by transduction with a lentivirus that harbors the Cre recombinase (vCRE) but not control lentivirus (vGFP). Lentiviral infection leads to nearly 100% expression of Cre in ES cell culture. Lower panel is a schematic of the floxed Dicer gene with the oligonucleotide primers used in the PCR reactions. (B) Schematic of ES cell embryoid body differentiation protocol. Cultures are transduced with GFP or CRE lentiviral vectors. (C)

Cre lentivirus transduction had no affect on the differentiation of wild-type Dicer mouse

ES cells cells. Cells were transfected with CRE lentivirus during the differentiation of wild-type Dicer mouse ES cells. Data represent means ± S.E.M. n=3, (10 visual fields per set); Student’s t test, *p < 0.05. (D) As in Figure 1C. Dicer deletion phenotype could be rescued by transfection of total brain short RNAs. To confirm that the deficiency of miRNAs is responsible for the phenotype observed in the knock out cells, Dicer deficient cells were rescued by introducing small RNAs from midbrain. (Two independent experiment of 3 sets each with 10 visual fields per set), Data represent means ± S.E.M.

ANOVA-test, **p < 0.01. (Scale bars, 100µm).

(E) As in Figure 1D. DATCRE/+ :Dicerflox/flox display a progressive loss of midbrain DNs postnatally. Immunostaining of newborn (upper panel) and 3 week old (lower panel) mice for tyrosine hydroxylase (TH) demonstrates progressive loss of midbrain DNs in the substantia nigra (SN) and ventral tegmental area (VTA) and their axonal projections to the striatum relative to control littermates (DATCRE/+ : Dicerflox/+; N=3 for each genotype).

Note that in newborn mice, the striatal projections are not mature and express a low level of TH. Scale bars, 200µm.

Supplementary Figure 2

(A) See Figure 1C for details. TUNEL staining of midbrain from 6 weeks old DATCRE/+

:Dicerflox/flox mice Scale bar (100um). Quantitative analysis of TUNEL positive cells (n=3 for each genotype). Data represent means ± S.E.M. Student’s t test, *p < 0.05. (B) Locomotor activity of DATCRE/+ :Dicerflox/flox mice in the open field, as in Figure 1D. The ambulatory counts traveled during the test was significantly decreased in DATCRE/+

:Dicerflox/flox mice. (N=4 for each genotype), Data represent means ± S.E.M. Student’s t test, *p < 0.05, **p < 0.01.

(C) Quantification of TH+ neuron number in VTA and SN as in Fig1 (D), (N=3 for each genotype) Data represent means ± S.E.M. Student’s t test, *p < 0.05, **p < 0.01.

Supplementary Figure 3 (A) mRNA levels of DAT and TH in normal human brain and

Parkinson’s disease brain, as quantified by qPCR. n=3 independent experiments, data represent mean ± S.E.M. ANOVA test, **p < 0.01.

(B) (Left panel) Expression pattern of miRNA precursors in control and Parkinson’s disease brains. 224 miRNA precursors were analyzed by semi quantitative real time

PCR and 8 miRNAs appeared to be enriched (more than 2-fold) in midbrain dopamine neurons relative to cerebral cortex or cerebellum (Supplementary Table 1).

(Right panel) miRNA precursor expression patterns were confirmed by Northern blot analysis. miR-181 was widely expressed in all brain tissues and miR-153 and let7b were detectable in cerebral cortex. miR-132 and miR-34, however, were only expressed in cerebellum. miR-133b and miR-130 appeared to be specifically expressed in midbrain dopamine neurons. miR-18 was undetectable in adult mouse brain.

Supplementary Figure 4 miR-133b is enriched in midbrain and is deficient in PD samples.

(A) (Left panel) qPCR expression analysis for miR-133b precursor in cerebral cortex

(CX), midbrain (MB), and cerebellum (CB) of controls and Parkinson’s disease (PD) brain. n=3; data represent mean ± S.E.M. ANOVA test, *p < 0.05. (Right panel) Northern blot analyses for miR-133b expression in control or PD human brain.

(B) (Left panel) qPCR expression analysis for miR-133b precursor in cerebral cortex

(CX), midbrain (MB), or cerebellum (CB) of control and Aphakia mutant mice. n=3 independent experiments; data represent mean ± S.E.M. ANOVA test, *p < 0.05.

(Right panel) Northern blot analysis for miR-133b expression in control and Aphakia mutant mouse brain.

(C) (i) qPCR expression analysis of miR-133b precursor in cerebral cortex (CX), midbrain (MB), or cerebellum (CB) of control and 6-OHDA treated mice. (n=3 independent experiments; data represent mean ± S.E.M. ANOVA test, *p < 0.05. (ii)

Northern blot analysis for miR-133b expression in control and 6-OHDA treated mice.

(iii) RNA protection assay for miR133a1 and miR133b expression in control and 6-OHDA treated mice, as in (i).

(D) Regulation of the miR-133b promoter by Pitx3. Upper panel: upstream transcriptional regulatory sequences from miR-133a1 or miR-133b were inserted 5’ of a luciferase marker gene to generate the promoter assay vectors. Lower panel: Pitx3 induced expression of a luciferase marker gene driven by miR-133b but not miR-133a1 promoter sequences, whereas Nurr1 and control vector (GFP) failed to induce luciferase expression. n=3 independent experiments; data represent mean ± S.E.M. ANOVA test,

**p < 0.01.

Supplementary Figure 5

(A) (Left Panel) Functional analysis of miR-133b activity in COS cells. A luciferase reporter vector that harbors a precise predicted target sequence for miR-133b (miR133b sensor) was cotransfected with a miR-133b overexpression construct with or without the anti-miR-133b (or anti-miR-18) 2’-O-methyl modified oligonucleotide, and expression of the pGL3-miR-133b sensor was quantified. Data are from two independent experiments. (Right panel) A lentiviral vector was generated that harbors miR-133b precursor sequences, and expression of mature miR-133b was confirmed by Northern blot analysis with an oligonucleotide probe for miR-133b as in Supplementary Figure 4A.

(B) miR-133b overexpression reduced dopamine release in primary midbrain cultures (at day 7 in vitro; left panel), whereas miR-133b knockdown (as in [A]) induced dopamine release in primary midbrain cultures (right panel). MB: primary midbrain cultures. Data represent mean ± S.E.M. n=5; ANOVA test, *p < 0.05.

(TH) positive cells in mouse ES cell derived dopamine neurons, although this did not reach statistical significance. Data represent mean ± S.E.M. n=3 independent experiments; p=0.23, Student’s t test.

Supplementary Figure 6

(A) Left panel: Complementarity between miR-133b and the Pitx3 3’UTR was predicted by miRanda software for microRNA target prediction. Schematic of luciferase assay vectors, pGL3-Pitx3 and pGL3-Mut-Pitx3, that harbors 300bp Pitx3 3’-UTR sequences predicted to be subject to miR-133b regulation. pGL3-Mut-Pitx3 harbors mutations within the Seed region of compementarity, depicted by the red box.

Right panel: Overexpression of miR-133b (but not miR-18) precursor in HEK293 cells leads to decreased luciferase expression from pGL3-Pitx3 (but not control vector or pGL3-mut Pitx3). Data represent mean ± S.E.M. n=3 independent experiments;

Student’s t test, *p < 0.05.

133b in EB-differentiated ES cell cultures suppresses the phenotype (significantly reduced DAT gene expression) of miR-133b precursor-transduced cultures. Inhibition of

TH expression did not reach statistical significance, but shows a trend towards reduction

(as in Figure 3A). Viral transductions were performed at stage 4 of ES differentiation, and qPCR analysis performed at day 7 of stage 5. Data represent mean ± S.E.M. n=3;

ANOVA test, *p < 0.05.

(from postnatal day 10 animals) using TH and Pitx3 specific antibodies. (Upper panels)

Example of FACS profiles from DATCRE/+ :Dicerflox/flox and control mice. (Middle panels)

TH+ and Pitx3+ cells were significantly reduced in DATCRE/+ :Dicerflox/flox adult mice relative to control adult DATCRE/+ :Dicerflox/flox mice. Data represent mean ± S.E.M. n=3;

Student’s t test, *p < 0.05. (Lower panel) As is Figure 4C; an example of Pitx3 expression intensity in TH+ cells in control and DATCRE/+ :Dicerflox/flox mutant mice; increased Pitx3 expression intensity is seen in the remaining mutant TH-positive cells.

(D) Schematic of feedback circuitry. miR-133b and Pitx3 define a negative feedback loop in midbrain DN function and differentiation. It is likely that miR-133b has additional significant targets. (3) Supplementary Tables

Table 1. Control brain PD brain Brain pre-miRNAs CX MB CB CX MB CB pre-let7b 1 0.36 0.2 2.35 0.5 0.18 pre-miR9-1 1 0.91 0.41 2.27 1.41 0.44 pre-miR103 1 0.36 0.22 0.06 0.08 1.14 pre-miR105-1 1 0.19 0.16 1.69 1.29 0.16 pre-miR128a 1 0.26 0.79 10.3 0.56 1.26 miRNAs enriched pre-miR131 1 0.33 0.65 1.24 0.5 0.26 in cerebral cortex pre-miR132 1 0.03 0.45 0.99 2.14 0.23 pre-miR139 1 0.69 0.2 2.13 0.35 0.22 pre-miR153-1 1 0.14 0.54 0.25 0.16 1.48 pre-miR178 1 0.17 0.33 1.66 1.22 0.22 pre-miR204 1 0.66 0.12 0.99 1.63 0.27 pre-miR370 1 0.33 0.55 0.79 0.33 2.55 pre-let7a-1 1 2.99 1.25 2.38 3.29 1.46 pre-miR7-2 1 2.17 1.87 1.66 3.24 1.56 pre-miR99a 1 3.14 1.79 0.91 2.22 4.99 miRNAs enriched pre-miR130 1 6.15 1.35 0.7 5.86 7.21 in midbrain pre-miR133b 1 2.34 0.97 0.9 0.36 1.01 pre-miR136 1 13.1 8.11 2.62 17.9 14.7 pre-miR224 1 2.66 1.96 0.3 7.67 0.29 pre-miR143 1 5.03 1.41 1.6 4.89 86.2 pre-let7g 1 1.8 13.5 2.66 2.23 9.99 pre-let7i 1 2.3 4.79 1.75 2.93 11.6 pre-miR7-3 1 9.78 61.4 7.16 402 133 pre-miR15-1 1 4.44 22.5 1.17 3.32 9 pre-miR17 1 0.3 3.63 1.11 1.17 3.56 pre-miR24-1 1 0.57 3.61 0.55 0.3 4.06 pre-miR25 1 1.31 3.43 1.18 1.35 5.35 pre-miR26a 1 0.69 4.14 0.84 0.46 5.24 pre-miR26b 1 1.27 6.33 1.9 2.22 5.26 pre-miR33 1 1.67 56.5 2.16 1.57 207 miRNAs enriched pre-miR34a 1 1.37 14.7 4.03 3.61 1.39 in cerebellum pre-miR125a 1 1.72 3.89 1.02 1.22 4.08 pre-miR138-2 1 0.7 5.33 2.33 1.65 10.4 pre-miR142 1 1.49 7.94 0.83 0.97 15.7 pre-miR144 1 0.85 3.92 0.33 0.96 18.5 pre-miR200a 1 2.19 41.6 0.94 0.26 0.2 pre-miR210 1 1.35 7.57 1.01 1.32 8.88 pre-miR218-2 1 5.32 19.3 3.22 0.22 12.4 pre-miR219-1 1 0.24 3.95 1.36 0.12 5.13 pre-miR320 1 1.04 6.82 0.37 1.84 17.4 pre-miR326 1 0.99 2.83 0.81 2.33 0.8 pre-miR331 1 0.81 15.2 0.82 1.33 11.6 pre-miR368 1 1.19 4.66 0.65 1.71 14.9 pre-miR15b 1 2.1 0.09 1.06 0.11 0.29 pre-miR16-2 1 1.52 0 1.88 1.27 0.56 pre-miR23a 1 0.93 0.49 0.11 0.79 0.29 miRNAs enriched in pre-miR30a 1 1.35 0.34 1.24 1.68 0.12 cerebral cortex and pre-miR137 1 1.36 0 0.96 0.59 0 midbrain pre-miR138-1 11.44 0.12 1.38 0.64 0.51 pre-miR204 1 0.74 0.06 0.07 0.33 0.4 pre-miR208 1 3.18 0.58 0.84 0.74 0.55 pre-miR218-1 1 1.04 0.02 1.27 0.63 0.02 pre-let7c 1 0.72 2.43 0.84 0.32 3.89 pre-let7e 1 0.66 2.6 1.01 1.2 7.16 pre-miR27a 1 0.49 2.3 0.37 0.49 3.46 pre-miR29c 1 0.3 2.62 0.64 0.38 6.15 pre-miR124a1 1 0.27 1.17 1.6 0.3 0.47 miRNAs enriched in pre-miR124a2 1 0.65 1.37 1.29 0.33 2.66 cerebral cortex and pre-miR153 1 0.27 1.66 2.36 0.99 1.22 cerebellum pre-miR184 10.6 1 1.27 0.56 0.33 pre-miR191 1 0.43 2.5 1.04 0.02 2.79 pre-miR212 1 0.37 0.97 0.6 0.48 1.32 pre-miR221 1 0.15 2.03 2.43 0.02 0.24 pre-miR16-1 1 10.1 16 2.87 2.22 8.28 pre-miR19a 1 5.7 2.03 1.19 1.13 4.59 pre-miR93 1 4.47 5.13 1.67 1.52 3.89 pre-miR101-1 1 8.06 9.13 0.14 0.18 3.76 pre-miR107 1 22.8 62.2 1.21 1.58 101 pre-miR127 1 7.57 7.11 0.95 2.62 8.11 miRNAs enriched pre-miR128b 1 1.97 3.41 0.97 1.44 3.32 in midbrain and pre-miR152 1 6.59 3.92 8.69 5.06 6.77 cerebellum pre-miR199 12.43 3.07 0.97 1.06 9.99 pre-miR200b 1 6.23 5.26 2.65 3.26 5.22 pre-miR330 1 2.04 1.77 1.27 0.54 2.5 pre-miR335 1 10.6 16.7 1.19 0.94 12.4 pre-miR345 1 9.78 7.06 4.11 0.36 13.6 pre-let7a-1 1 1.7 1.27 0.8 0.91 0.22 pre-let7a-2 1 1.46 1.03 0.38 0.66 0.62 pre-let7a-3 1 1.73 1.85 1.1 1.92 1.56 pre-let7d 1 1.27 0.9 1.13 2.32 1.03 pre-let7f-1 1 1.11 0.96 0.79 1.65 2.16 pre-mR1-1 1 1.59 1.04 0.07 2.41 0.07 pre-mR1-2 1 1.78 1.73 1.01 0.75 1.67 pre-miR7-1 1 0.85 1.38 0.26 0.95 0.83 pre-miR10a 1 1.56 2.08 1.13 0.9 1.74 pre-miR10b 1 2.07 1.35 0.61 1.31 1.01 pre-miR19a 1 1.52 1.65 0.42 0.57 1.14 pre-miR19b1 1 1.99 1.73 0.02 3.36 1.13 pre-miR21 1 1.67 0.9 0.79 0.57 2.2 pre-miR22 1 2.51 0.5 1.02 0.65 0.66 pre-miR23b 1 1.13 1.24 0.96 0.87 1.45 pre-miR27b 1 0.95 1.68 2.33 1.32 1.91 pre-miR28 1 2.2 2.83 0.68 1.16 0.86 pre-miR29a 1 1.02 2.64 0.76 0.74 2.43 pre-miR29b1 1 1.69 0.69 4.03 1.37 1 pre-miR30b 1 0.9 1.27 1.57 0.33 1.33 pre-miR30d 1 0.99 1.11 1.27 1.24 1.62 pre-miR30e 1 0.65 1.27 1.27 1.65 1.22 pre-miR32 1 2.07 1.93 0.71 0.78 5.66 pre-miR95a 1 1.41 1.92 2.58 1.39 1.66 pre-miR101-2 1 1.64 1.21 1.66 0.66 0.94 pre-miR106a 1 1.48 1.75 1.78 3.34 2.13 pre-miR106b 1 0.99 1.24 1.26 0.27 1.36 pre-miR108 1 0.84 2.73 0.2 1.93 8.4 pre-miR126 1 1.93 1.31 1.16 2.3 0.3 pre-miR129-2 1 0.87 1.11 0.67 2.01 1.27 pre-miR133a1 1 1.3 1.56 2.73 0.64 2.28 pre-miR133a2 1 1.79 1.84 0.92 1.64 1.44 pre-miR134 1 0.75 1.18 1.07 0.77 1.4 pre-miR135 1 0.98 1.69 1.39 0.81 3.29 No difference in cerebral pre-miR140 1 1.78 1.92 1.35 1.84 1.79 cortex,midbran and pre-miR147 1 1.3 2.08 1.13 0.32 1.39 cerebellum pre-miR148a 11.66 1.51 0.48 0.71 0.47 pre-miR149 1 0.89 1.62 1.68 0.75 2.57 pre-miR151 1 1.58 0.59 1.75 0.94 1.2 pre-miR154 1 0.8 1.75 1.38 1.06 2.23 pre-miR155 1 0.95 1.97 0.9 0.66 3.05 pre-miR181a1 1 2.07 2.81 1.91 2.43 3.16 pre-miR181b1 1 1.99 0.97 2.56 1.27 0.3 pre-miR181c 1 0.78 0.63 1.37 1.27 0.64 pre-miR185 1 1.75 1.75 1.36 2.53 0.97 pre-miR186 1 2.01 1.96 0.68 0.7 4.76 pre-miR187 1 1.04 1.02 0.04 1.33 2.79 pre-miR190 1 1.2 0.7 1.12 1.27 0.56 pre-miR193 1 1.67 2.33 1.56 2.85 3.41 pre-miR194 1 1.95 1.43 0.82 0.98 3.81 pre-miR195 1 0.9 1.28 0.57 0.7 1.73 pre-miR197 1 1.14 1.48 0.63 0.62 1.68 pre-miR211 1 1.09 0.93 0.6 0.84 0.81 pre-miR213 1 0.81 2.25 1.12 1.42 1.05 pre-miR215 1 2.46 1.78 0.84 0.73 2.33 pre-miR220 1 0.56 1.36 2.56 1.26 0.33 pre-miR242 1 1.03 2.02 1.65 0.62 1.97 pre-miR291 1 1.27 1 2.02 1.09 1.52 pre-miR301 1 1.71 1.8 1.06 0.45 3.36 pre-miR302b 1 1.27 1.56 0.96 1.56 0.36 pre-miR302 1 1.33 1.77 2.07 0.52 2.19 pre-miR302c 1 1.12 0.67 0.66 1.03 1 pre-miR323 1 1.33 1.65 2.07 0.52 1.08 pre-miR324 1 1.51 1.97 1.34 0.54 3.94 pre-miR325 1 1.16 0.85 0.97 2.19 1.02 pre-miR329-1 1 0.78 1.32 0.99 1.66 1.65 pre-miR337 1 0.85 1.57 1.11 0.8 1.21 pre-miR338 1 2.16 0.91 1.84 0.81 1.06 pre-miR340 1 2.1 1.69 1.05 2.11 3.1 pre-miR361 1 1.12 1.66 0.32 1 2.16 pre-miR369 1 1.21 1.35 2.2 1.02 1 pre-miR372 1 1.27 1.75 1.75 3.34 1.36 pre-miR373 1 1.66 1.68 4.59 6.92 2.35 pre-miR374 1 1.96 2.3 0.67 0.96 4.72 pre-miR376a1 1 1.65 1.27 0.66 1.24 1 pre-miR377 1 0.78 1.19 0.55 0.46 1.23 pre-miR378 1 0.92 5.43 1.29 1.57 5.94 pre-miR379 1 1.77 1.88 2.99 7.89 9.25 pre-miR380 1 1.76 1.79 0.97 1 2.15 pre-miR381 1 1.33 1.67 1.09 1.19 2.69 pre-miR383 1 0.75 1.19 1.01 0.95 1.48 pre-miR384 1 1.78 1.42 0.65 1.61 1.39 pre-miR423 1 0.72 1.09 0.33 1.13 1.41 pre-miR425 1 1.93 1.28 1.11 2.71 4.35 pre-miR9-2 00000 0 pre-miR9-3 00000 0 pre-miR18 00000 0 pre-miR19-2 00000 0 pre-miR20 00000 0 pre-miR24-2 00000 0 pre-miR26a2 00000 0 pre-miR29b2 00000 0 pre-miR30c1 00000 0 pre-miR31 00000 0 pre-miR34b 00000 0 pre-miR34c 00000 0 pre-miR92-1 00000 0 pre-miR92-2 00000 0 pre-miR92b 00000 0 pre-miR96 00000 0 pre-miR99b 00000 0 pre-miR122 00000 0 pre-miR124a3 00000 0 pre-miR125b1 00000 0 pre-miR125b2 00000 0 pre-miR129-1 00000 0 pre-miR141 00000 0 pre-miR145 00000 0 pre-miR146a 00000 0 pre-miR146b 00000 0 pre-miR148b 00000 0 Not detectable in brain pre-miR150 00000 0 pre-miR182 00000 0 pre-miR183 00000 0 pre-miR188 00000 0 pre-miR192 00000 0 pre-miR196a1 00000 0 pre-miR200c 00000 0 pre-miR202 00000 0 pre-miR203 00000 0 pre-miR205 00000 0 pre-miR206 00000 0 pre-miR222 00000 0 pre-miR223 00000 0 pre-miR214 00000 0 pre-miR216 00000 0 pre-miR217 00000 0 pre-miR219-2 00000 0 pre-miR229 00000 0 pre-miR296 00000 0 pre-miR299 00000 0 pre-miR328 00000 0 pre-miR342 00000 0 pre-miR371 00000 0 pre-miR376a2 00000 0 pre-miR382 00000 0 pre-miR409 00000 0 pre-miR410 00000 0 pre-miR411 00000 0 pre-miR422 00000 0 pre-miR424 00000 0 pre-miR449 00000 0 pre-miR450-1 00000 0 pre-miR450-2 00000 0 pre-miR451 00000 0 pre-miR484 00000 0 pre-miR485 00000 0 pre-miR496 00000 0

Supplementary Table 1 miRNA expression determined by quantitative real-time (qPCR) for a panel of 224 precursor miRNAs in RNA samples from PD patients and normal controls. Expression level in midbrain (MB) and cerebellum (CB) is presented normalized to the cerebral cortex (CX) level determined as described in the supplementary methods.

Oligonucleotide sequences used for the amplifications are presented.

Table 2. pri-let7a1-forward primer 5'-TGGTGCTCAACTGTGATTCC-3' pri-let7a1-reverse primer 5'-TATCTCCCAGTGGTGGGTGT-3' pri-let7a2-forward primer 5'-TGCTCCCAGGTTGAGGTAGT-3' pri-let7a2-reverse primer 5'-GGAATCATGATCGTTCTCACC-3' pri-let7a3-forward primer 5'-CCCTTTGGGGTGAGGTAGTA-3' pri-let7a3-reverse primer 5'-GTTCCAGACGCTCTGTCCAC-3' pre-let7b-forward primer 5'-GGGTGAGGTAGTAGGTTGTGTG-3' pre-let7b-reverse primer 5'-CAGGGAAGGCAGTAGGTTGT-3' pre-let7c1-forward primer 5'-GTGTGCATCCGGGTTGAG-3' pre-let7c1-reverse primer 5'-TGTGCTCCAAGGAAAGCTAGA-3' pre-let7d-forward primer 5'-AGGAAGAGGTAGTAGGTTGCATAG-3' pre-let7d-reverse primer 5'-TAAGAAAGGCAGGTCGT-3' pre-let7e-forward primer 5'-GGGCTGAGGTAGGAGGTTGT-3' pre-let7e-reverse primer 5'-GGAAAGCTAGGAGGCCGAAT-3' pre-let7i-forward primer 5'-TGGCTGAGGTAGTAGTTTGTGC-3' pre-let7i-reverse primer 5'-AGCTTGCGCAGTTATCTCCA-3' pre-let7f-forward primer 5'-GATTGTATAGTTGTGGGGTAGTGA-3' pre-let7f-reverse primer 5'-TCAGGGAAGGCAATAGATTGTA-3' pre-let7g-forward primer 5'-GGCTGAGGTAGTAGTTGTACAGTT-3' pre-let7g-reverse primer 5'-AGGCAGTGGCCTGTACAGTTA-3' pre-miR1-1-forward primer 5'-CACCGCTTGGGACACATAC-3' pre-mR1-1-reverse primer 5'-GCCTGAAATACATACTTCTTTACATT-3' pri-miR1-2-forward primer 5'-TGTACCCATATGAACATACAATGC-3' pri-mR1-2-reverse primer 5'-TTCACGTAGAAAGAAGCAAGAGC-3' pre-miR7-1-forward primer 5'-TGGATGTTGGCCTAGTTCTG-3' pre-miR7-1-reverse primer 5'-TGGCAGACTGTGATTTGTTG-3' pre-miR7-2-forward primer 5'-GGCCCCATCTGGAAGACTA-3' pre-miR7-2-reverse primer 5'-ATGGCTGGCACCATTAGGTA-3' pre-miR7-3-forward primer 5'-GGCTGTGGTCTAGTGCTGTG-3' pre-miR7-3-reverse primer 5'-AAGGGAGTCTGCGCTATGAG-3' pre-miR9-1-forward primer 5'-GGGGTTGGTTGTTATCTTTGG-3' pre-miR9-1-reverse primer 5'-GGGGTTATTTTTACTTTCGGTTA-3' pre-miR9-2-forward primer 5'-GAAGCGAGTTGTTATCTTTGGTT-3' pre-miR9-2-reverse primer 5'-TGAAGGAGTTTTTACTTTCGGTTA-3' pre-miR9-3-forward primer 5'-GAGGCCCGTTTCTCTCTTTG-3' pre-miR9-3-reverse primer 5'-TGAGAATCATTTCTACTTTCGGTTA-3' pre-miR10a-forward primer 5'-ATACCCTGTAGATCCGAATTTGT-3' pre-miR10a-reverse primer 5'-AGAGCGGAGTGTTTATGTCAA-3' pre-miR10b-forward primer 5'-CCCTGTAGAGAATTTGTG-3' pre-miR10b-reverse primer 5'-TGAAGTTTTTGCATCGACCA-3' pre-miR15a-forward primer 5'-CACCCTTGGAGTAAAGTAGCAG-3' pre-miR15a-reverse primer 5'-TCCTTGTATTTTGAGGCAGC-3' pre-miR15b-forward primer 5'-AGTACTGTAGCAGCACATCATGG-3' pre-miR15b-reverse primer 5'-TTTCTTTAAATTTCTAGAGCAGCA-3' pre-miR16-forward primer 5'-GTCAGCAGTGCCTTAGCAGC-3' pre-miR16-reverse primer 5'-GTCAACCTTACTTCAGCAGCAC-3' pre-miR16-2-forward primer 5'-TCCACTCTAGCAGCACGTAAA-3' pre-miR16-2-reverse primer 5'-GTCACACTAAAGCAGCACAGTAA-3' pre-miR17-forward primer 5'-ATGTCAAAGTGCTTACAGTGCAG-3' pre-miR17-reverse primer 5'-GTCACCATAATGCTGCTACAAGTGC-3' pre-miR18-forward primer 5'-GATGCTTTTGAGCTGCTTCTT-3' pre-miR18-reverse primer 5'-TGCCAGAAGGGGCATTTA-3' pre-miR19A-forward primer 5'-CCTCTGTTAGTTTTGCATAGTTGC-3' pre-miR19A-reverse primer 5’-CAGGCCACCATCAGTTTTG-3’ pre-miR19-2-forward primer 5'-TTACAATTAGTTTTGCAGGTTTGC-3' pre-miR19-2-reverse primer 5'-TTATCACAATCAGTTTTGCATGG-3' pre-miR19b-forward primer 5'-TGGTTAGTTTTGCAGGTTTGC-3' pre-miR19b-reverse primer 5'-CACTACCACAGTCAGTTTTGCAT-3' pre-miR20-forward primer 5'-GTAGCACTAAAGTGCTTATAGTGCAG-3' pre-miR20-reverse primer 5'-GCAGTACTTTAAGTGCTCATAATGCA-3' pre-miR21-reverse primer 5'-TGTCAGACAGCCCATCGA-3' pre-miR21-forward primer 5'-TGTCGGGTAGCTTATCAGACTG-3' pre-miR22-forward primer 5'-GCTGAGCCGCAGTAGTTCTT-3' pre-miR22-reverse primer 5'-GCAGAGGGCAACAGTTCTTC-3' pre-miR23a-forward primer 5'-CACCTGGGGTTCCTGGG-3' pre-miR23a-reverse primer 5'-GTTGGAAATCCCTGGCAAT-3' pre-miR23b-forward primer 5'-CAGGTGCTCTGGCTGCTT-3' pre-miR23b-reverse primer 5'-GTGGTAATCCCTGGCAATGT-3' pre-miR24-forward primer 5'-CACCTCCGGTGCCTACTG-3' pre-miR24-reverse primer 5'-CTCCTGTTCCTGCTGAACTGA-3' pre-miR24-2-forward primer 5'-CTCCCGTGCCTACTGAGC-3' pre-miR24-2-reverse primer 5'-CCCTGTTCCTGCTGAACTGA-3' pre-miR25-forward primer 5'-AGTGTTGAGAGGCGGAGACT-3' pre-miR25-reverse primer 5'-GCACTGTCAGACCGAGACAA-3' pre-miR26a1-forward primer 5'-CACCTGGCCTCGTTCAAG-3' pre-miR26a1-reverse primer 5'-CGTCCCCGTGCTGCAAGTAAC-3' pre-miR26a2-forward primer 5'-AGCTGCCTCCAGAAACAAGT-3' pre-miR26a2-reverse primer 5'-CGTCCCCGTGCTGCAAGTAAC-3' pre-miR26b-forward primer 5'-CGGGACCCAGTTCAAGTAAT-3' pre-miR26b-reverse primer 5'-GTCCCCGAGCCAAGTAATG-3' pre-miR27a-forward primer 5'-GAGGAGCAGGGCTTAGCTG-3' pre-miR27a-reverse primer 5'-CAGGGGGCGGAACTTAGC-3' pre-miR27b-forward primer 5'-TGCAGAGCTTAGCTGATTGG-3' pre-miR27b-reverse primer 5'-CCTTCTCTTCAGGTGCAGAAC-3' pre-miR28-forward primer 5'-CCCTCAAGGAGCTCACAGTC-3' pre-miR28-reverse primer 5'-CCCTCCAGGAGCTCACAAT-3' pre-miR29a-forward primer 5'-ATGACTGATTTCTTTTGGTGTTCA-3' pre-miR29a-reverse primer 5'-ATAACCGATTTCAGATGGTGCT-3' pre-miR29b1-forward primer 5'-GGAAGCTGGTTTCACATGGT-3' pre-miR29b1-reverse primer 5'-CCTAAAACACTGATTTCAAATGG-3' pre-miR29b2-forward primer 5'-CTTCTGGAAGCTGGTTTCAC-3' pre-miR29b2-reverse primer 5'-CTCCTAAAACACTGATTTCAAATGG-3' pre-miR29c-forward primer 5'-CACAGGCTGACCGATTTCTC-3' pre-miR29c-reverse primer 5'-CCCCTACATCATAACCGATTTC-3' pri-miR30a-forward primer 5'-GCGACTGTAAACATCCTCGAC-3' pri-miR30a-reverse primer 5'-GCAGCTGCAAACATCCGA-3' pre-miR30b-forward primer 5'-ACCAAGTTTCAGTTCATGTAAACA-3' pre-miR30b-reverse primer 5'-CAGCTGAAGTAAACATCCACCTC-3' pre-miR30c1-forward primer 5'-ACCATGCTGTAGTGTGTGTAAAC-3' pre-miR30c1-reverse primer 5'-TCCATGGCAGAAGGAGTAAAC-3' pri-miR30d-forward primer 5'-CCCGACTGGAAGCTGTAAGA-3' pri-miR30d-reverse primer 5'-TCCACTCCGGGACAGAATTA-3' pre-miR30e-forward primer 5'-GGCAGTCTTTGCTACTGTAAACAT-3' pre-miR30e-reverse primer 5'-GCCTGCCGCTGTAAACAT-3' pre-miR31-forward primer 5'-GGAGAGGAGGCAAGATGCT-3' pre-miR31-reverse primer 5'-GGAAAGATGGCAATATGTTGG-3' pre-miR32-forward primer 5'-GGAGATATTGCACATTACTAAGTTGC-3' pre-miR32-reverse primer 5'-GAAATTATCACACACATCAAATTGCAT-3' pre-miR33-forward primer 5'-TGGTGCATTGTAGTTGCATT-3' pre-miR33-reverse primer 5'-TGTGATGCACTGTGGAAACAT-3' pre-miR34a-forward primer 5'-CACCAGCTGTGAGTAATTCTTTG-3' pre-miR34a-reverse primer 5'-ACAATGTGCAGCACTTCTAGG-3' pre-miR34b-forward primer 5'-GCTCGGTTTGTAGGCAGTGT-3' pre-miR34b-reverse primer 5'-CTTGTTTTGATGGCAGTGGA-3' pre-miR34c-forward primer 5'-AGTCTAGTTACTAGGCAGTGTA-3' pre-miR34c-reverse primer 5'-TCTTTTTACCTGGCCGTGTG-3' pre-miR92-1-forward primer 5'-CTTTCTACACAGGTTGGGATCG-3' pre-miR92-1-reverse primer 5'-CCAAACTCAACAGGCCGG-3' pre-miR92-2-forward primer 5'-CATCCCTGGGTGGGGATT-3' pre-miR92-2-reverse primer 5'-CCGGGACAAGTGCAATATTT-3' pri-miR92b-forward primer 5'-GCGGTGCAGTGTTGTTTTT-3' pri-miR92b-reverse primer 5'-GGGATGGAGAGCCAGGAG-3' pre-miR93-forward primer 5'-GGGGCTCCAAAGTGCTGT-3' pre-miR93-reverse primer 5'-GAAGTGCTAGCTCAGCAGTAGG-3' pre-miR95a-forward primer 5'-CACAGTGGGCACTCAATAATG-3' pre-miR95a-reverse primer 5'-CACAGAGTGGGTGCTCAATAAA-3' pre-miR96-forward primer 5'-CCGATTTTGGCACTAGCAC-3' pre-miR96-reverse primer 5'-CATATTGGCATGCACATGA-3' pre-miR99a-forward primer 5'-TGGCATAAACCCGTAGATCC-3' pre-miR99a-reverse primer 5'-CACACTGACACAGACCCATAGA-3' pre-miR99b-forward primer 5'-CTGGACTCCTGGGTTCCTT-3' pre-miR99b-reverse primer 5'-GACACGGACCCACAGACAC-3' pre-miR101-1-forward primer 5'-CCCTGGCTCAGTTATCACAGT-3' pre-miR101-1-reverse primer 5'-TGCCATCCTTCAGTTATCACA-3' pre-miR101-2-forward primer 5'-TCCTTTTTCGGTTCTCATGG-3' pre-miR101-2-reverse primer 5'-CCACCATTCTTCAGTTATCACA-3' pre-miR103-forward primer 5'-TACTGCCTTCGGCTTCTTTA-3' pre-miR103-reverse primer 5'-CAATGCCTTCATAGCCCTGT-3' pre-miR105-1-forward primer 5'-TGCATCGTGGTCAAATGCT-3' pre-miR105-1-reverse primer 5'-CACCGTAGCACATGCTCAAA-3' pre-miR106a-forward primer 5'-CCTTGGCCATGTAAAAGTGC-3' pre-miR106a-reverse primer 5'-CCATGGTAATGTAAGAAGTGCTTAC-3' pre-miR106b-forward primer 5'-CACCTGCTGGGACTAAAGTGC-3' pre-miR106b-reverse primer 5'-CCTGCTGGAGCAGCAAGTA-3' pre-miR107-forward primer 5'-TCTCTGCTTTCAGCTTCTTTACA-3' pre-miR107-reverse primer 5'-TCTGTGCTTTGATAGCCCTGT-3' pre-miR108-forward primer 5'-CACTGCAAGAACAATAAGGATTTT-3' pre-miR108-reverse primer 5'-CAGCAAGAAAAATGAGGGACTT-3' pre-miR122-forward primer 5'-AGCAGAGCTGTGGAGTGTGA-3' pre-miR122-reverse primer 5'-GCCTAGCAGTAGCTGTTTAGTGTGA-3' pre-miR124a1-forward primer 5'-CACCATCAAGATCAGAGACTCTG-3' pre-miR124a1-reverse primer 5'-TTCAAGTGCAGCCGTAGG-3' pre-miR124a2-forward primer 5'-AGAGGCTCTGCTCTCCGTGT-3' pre-miR124a2-reverse primer 5'-TTCAAGTGCAGCCGTAGG-3' pre-miR124a3-forward primer 5'-TGAGGGCCCCTCTGCGTGTTC-3' pre-miR124a3-reverse primer 5'-GCCTCTCTTGGCATTCACC-3' pre-miR125a-forward primer 5'-CTCTAGGTCCCTGAGACCCTTT-3' pre-miR125a-reverse primer 5'-GGCTCCCAAGAACCTCACCT-3' pre-miR125b1-forward primer 5'-TGCGCTCCTCTCAGTCCCTGAGA-3' pre-miR125b1-reverse primer 5'-GACTCGCAGCTCCCAAGA-3' pre-miR125b2-forward primer 5'-ACCAGACTTTTCCTAGTCCCT-3' pre-miR125b2-reverse primer 5'-AGGTCCCAAGAGCCTGACTT-3' pre-miR126-forward primer 5'-CTGGCGACGGGACATTATTA-3' pre-miR126-reverse primer 5'-CGTGGACGGCGCATTATTA-3' pre-miR127-forward primer 5'-TGTGATCACTGTCTCCAGCC-3' pre-miR127-reverse primer 5'-GATGATGAGACTTCCGACCAG-3' pre-miR128a-forward primer 5'-ATTGGCCTTGTTCCTGAGC-3' pre-miR128a-reverse primer 5'-CCAGGAAGCAGCTGAAAAAG-3' pre-miR128b-forward primer 5'-GGCCGATACACTGTACGAGA-3' pre-miR128b-reverse primer 5'-GCTCTTTGGATAACGGCATT-3' pre-miR129-1-forward primer 5'-CACCTGGATCTTTTTGCGGT-3' pre-miR129-1-reverse primer 5'-TAGATACTTTTTGGGGTAAGGGC-3' pre-miR129-2-forward primer 5'-TGCCCTTCGCGAATCTTTTT-3' pre-miR129-2-reverse primer 5'-GCAAATGCTTTTTGGGGTAA-3' pre-miR130-forward primer 5'-GGCCAGAGCTCTTTTCACAT-3' pre-miR130-reverse primer 5'-GGCCAATGCCCTTTTAACAT-3' pre-miR131A1-forward primer 5'-CTAAAGCTGGTAAAATGGAACC-3' pre-miR131A1-reverse primer 5'-AGCTGGTTGAAGGGGACCA-3' pre-miR132-forward primer 5'-ACCGTGGCTTTCGATTGTTA-3' pre-miR132-reverse primer 5'-GGCGACCATGGCTGTAGACT-3' pre-miR133a-forward primer 5'-AAATGCTTTGCTAGAGCTGGT-3' pre-miR133a-reverse primer 5'-GCCATGAATGCACAGCTACA-3' pre-miR133a2-forward primer 5'-GGAGCCAAATGCTTTGCTAGA-3' pre-miR133a2-reverse primer 5'-GCCATCAATGCACAGCTACA-3' pre-miR133b-forward primer 5'-TGGTCAAACGGAACCAAGT-3' pre-miR133b-reverse primer 5'-TCTCCAAGGACTGGGCATT-3' pre-miR134-forward primer 5'-CAGGGTGTGTGACTGGTTGA-3' pre-miR134-reverse primer 5'-GAGGGTTGGTGACTAGGTGG-3' pre-miR135-forward primer 5'-GGCCTCGCTGTTCTCTATG-3' pre-miR135-reverse primer 5'-ACGGCTCCAATCCCAATATGA-3' pre-miR136-forward primer 5'-CCTCGGAGGACTCCATTTGT-3' pre-miR136-reverse primer 5'-GAACCCTCTGAAGACTCATTTG-3' pre-miR137-forward primer 5'-ACTCTCTTCGGTGACGGGTA-3' pre-miR137-reverse primer 5'-CGCTGGTACTCTCCTCGACT-3' pre-miR138-forward primer 5'-GGGCAGCTGGTGTTGTGA-3' pre-miR138-reverse primer 5'-GTGTGGCCCTGGTGTTGT-3' pre-miR138-1-forward primer 5'-CACCTCTAGCATGGTGTTGTG-3' pre-miR138-1-reverse primer 5'-CTTGCAGTGCAGTGTGGC-3' pre-miR138-2-forward primer 5'-CTGCAGCTGGTGTTGTGAAT-3' pre-miR138-2-reverse primer 5'-CAACCCTGGTGTCGTGAAAT-3' pre-miR139-forward primer 5'-GTGTATTCTACAGTGCACGTGTC-3' pre-miR139-reverse primer 5'-GTTACTCCAACAGGGCCG-3' pre-miR140-forward primer 5'-CTCTCTGTGTCCTGCCAGTG-3' pre-miR140-reverse primer 5'-GTGCCCCGGTATCCTGTC-3' pre-miR141-forward primer 5'-CCTGGGTCCATCTTCCAGTA-3' pre-miR141-reverse primer 5'-ACCCGGGAGCCATCTTTAC-3' pre-miR142-forward primer 5'-CACCCTAAAGTAGAAAGCAC-3' pre-miR142-reverse primer 5'-CATAAAGTAGGAAACACTACACCC-3' pre-miR143-forward primer 5'-CTGTCTCCCAGCCTGAGGT-3' pre-miR143-reverse primer 5'-TGCAGAACAACTTCTCTCTTCC-3' pre-miR144-forward primer 5'-GCCCTGGCTGGGATATCAT-3' pre-miR144-reverse primer 5'-TGCCCGGACTAGTACATCATC-3' pre-miR145-forward primer 5'-CTTGTCCTCACGGTCCAGT-3' pre-miR145-reverse primer 5'-TGACCTCAAGAACAGTCTTTCCA-3' pre-miR146a-forward primer 5'-CGATGTGTATCCTCAGCTTTGA-3' pre-miR146a-reverse primer 5'-TCCCAGCTGAAGAACTGAATTT-3' pre-miR146b-forward primer 5'-CCTGGCACTGAGAACTGAAT-3' pre-miR146b-reverse primer 5'-GCACCAGAACTGAGTCCACA-3' pre-miR147-forward primer 5'-AGACAACATTTCTGCACACACA-3 pre-miR147-reverse primer 5'-AGCAGAAGCATTTCCACACA-3' pre-miR148a-forward primer 5'-GAGGCAAAGTTCTGAGACACTC-3' pre-miR148a-reverse primer 5'-GAGACAAAGTTCTGTAGTGCACTGA-3' pre-miR148b-forward primer 5'-CAAGCACGATTAGCATTTGAG-3' pre-miR148b-reverse primer 5'-TCGAGACAAAGTTCTGTGATGC-3' pre-miR149-forward primer 5'-CTCTGGCTCCGTGTCTTCA-3' pre-miR149-reverse primer 5'-GTCCCTCCCTCCCTCCTC-3' pre-miR150-forward primer 5'-CCTGTCTCCCAACCCTTGTA-3' pre-miR150-reverse primer 5'-GTCCCCAGGTCCCTGTCC-3' pre-miR151-forward primer 5'-CCTCGAGGAGCTCACAGTCT-3' pre-miR151-reverse primer 5'-ATGACCATCCCTGTCCTCAA-3' pre-miR152-forward primer 5'-CGGCCCAGGTTCTGTGATAC-3' pre-miR152-reverse primer 5'-CTTCCGGGCCCAAGTTCT-3' pre-miR153-forward primer 5'-CACCGGTGTCATTTTTGTGA-3' pre-miR153-reverse primer 5'-CAATGATCACTTTTGTGACTATGC-3' pre-miR154-forward primer 5'-CACCGAAGATAGGTTATCCGTG-3' pre-miR154-reverse primer 5'-AAAAATAGGTCAACCGTGTATGATTC-3' pre-miR155-forward primer 5'-CTGTTAATGCTAATCGTGATAGGG-3' pre-miR155-reverse primer 5'-CTGTTAATGCTAATATGTAGGAGTCAG-3' pre-miR181-forward primer 5'-CACCATGGAACATTCAACG-3' pre-miR181-reverse primer 5'-CCAAGGTACAGTCAACGGTC-3' pre-miR181b-1-forward primer 5'-AAAAGGTCACAATCAACATTCA-3' pre-miR181b-1-reverse primer 5'-GGCCACAGTTGCATTCATT-3' pre-miR181c-forward primer 5'-GGAAAATTTGCCAAGGGTTT-3' pre-miR181c-reverse primer 5'-GCCTCAGGGTCCACTCAAC-3' pre-miR183-forward primer 5'-CGCAGAGTGTGACTCCTGTT-3' pre-miR183-reverse primer 5'-TGCTCTGTTTATGGCCCTTC-3' pre-miR182-forward primer 5'-CCGTTTTTGGCAATGGTAGA-3' pre-miR182-reverse primer 5'-TGAGTCCTCGCCCCATAGT-3' pre-miR184-forward primer 5'-CAGTCACGTCCCCTTATCACT-3' pre-miR184-reverse primer 5'-CCTTATCAGTTCTCCGTCCAA-3' pre-miR185-f-forward primer 5'-GCGAGGGATTGGAGAGAAAG-3' pre-miR185-reverse primer 5'-GAGGGAAGGACCAGAGGAAA-3' pre-miR186-forward primer 5'-TGCTTGTAACTTTCCAAAGAATC-3' pre-miR186-reverse primer 5'-AGCTCAAACTTCCCAAAAAATC-3' pre-miR187-forward primer 5'-GGGCTCACCATGACACAGT-3' pre-miR187-reverse primer 5'-CTCCGGCTGCAACACAAG-3' pre-miR188-forward primer 5'-TCCCTCTCTCACATCCCTTG-3' pre-miR188-reverse primer 5'-CATCCTGCAAACCCTGCAT-3' pre-miR190-forward primer 5'-GCAGGCCTCTGTGTGATATG-3' pre-miR190-reverse primer 5'-GGCAAGACACTGTAGGAATATGT-3' pre-miR191-forward primer 5'-GGCAACGGAATCCCAAAAG-3' pre-miR191-reverse primer 5'-AGGAGAGCAGGGGACGAAAT-3' pre-miR192-forward primer 5'-TGCACAGGGCTCTGACCTAT-3' pre-miR192-reverse primer 5'-GCATTGAGGCGAACATACCT-3' pre-miR193-forward primer 5'-AGCTGAGGGCTGGGTCTT-3' pre-miR193-reverse primer 5'-GCCGAGAACTGGGACTTTGT-3' pre-miR194-forward primer 5'-ATGGTGTTATCAAGTGTAACAGCA-3' pre-miR194-reverse primer 5'-TTGGTAACCATCAAAAGTAACAGC-3' pre-miR195-forward primer 5'-GCTTCCCTGGCTCTAGCA-3' pre-miR195-reverse primer 5'-CTGGAGCAGCACAGCCAATA-3' pre-miR196a1-forward primer 5'-AATTAGGTAGTTTCATGTTGTTGG-3' pre-miR196a1-reverse primer 5'-GAATCGGGTGGTTTAATGTTG-3' pre-miR197-forward primer 5'-GCTGTGCCGGGTAGAGAG-3' pre-miR197-reverse primer 5'-CCATGCTGGGTGGAGAAG-3' pre-miR199a-forward primer 5'-GCCAACCCAGTGTTCAGACT-3' pre-miR199a-reverse primer 5'-GCCTAACCAATGTGCAGACTACT-3' pre-miR200a-forward primer 5'-TGTGAGCATCTTACCGGACA-3' pre-miR200a-reverse primer 5'-GGGTCACCTTTGAACATCGT-3' pre-miR200b-forward primer 5'-CCGTGGCCATCTTACTGG-3' pre-miR200b-reverse primer 5'-TCCGCCGTCATCATTACC-3' pre-miR200c-forward primer 5'-CCCTCGTCTTACCCAGCAG-3' pre-miR200c-reverse primer 5'-CCATCATTACCCGGCAGTAT-3' pre-miR202-forward primer 5'-GTATAGGGCATGGGAAAACG-3' pre-miR202-reverse primer 5'-GTGGAGTCCCAAGTCAGGAG-3' pre-miR203-forward primer 5'-GCTGGGTCCAGTGGTTCTTA-3' pre-miR203-reverse primer 5'-GCCGGGTCTAGTGGTCCTA-3' pre-miR204-forward primer 5'-TGACTCGTGGACTTCCCTTT-3' pre-miR204-reverse primer 5'-CAATTGAACGTCCCTTTGC-3' pre-miR205-forward primer 5'-TCCTCAGACAATCCATGTGC-3' pre-miR205-reverse primer 5'-AGCTCCATGCCTCCTGAACT-3' pre-miR206-forward primer 5'-CACCAGGCCACATGCTTC-3' pre-miR206-reverse primer 5'-CCAAAACCAACAACTTCCTTACA-3' pre-miR208-forward primer 5'-CTTTTGGCCCGGGTTATAC-3' pre-miR208-reverse primer 5'-AACAAGCTTTTTGCTCGTCTT-3-3' pre-miR210-forward primer 5'-CGTGCCCCAGACCCACTGT-3' pre-miR210-reverse primer 5'-CTGCCCAGGCACAGATCA-3' pre-miR211-forward primer 5'-TCACCTGGCCATGTGACTT-3' pre-miR211-reverse primer 5'-CTCCGTGCTGTGGGAAGT-3' pre-miR212-forward primer 5'-GGCACCTTGGCTCTAGACTG-3' pre-miR212-reverse primer 5'-GCCGTGACTGGAGACTGTTA-3' pre-miR213-forward primer 5'-TGAGGTTGCTTCAGTGAACATT-3' pre-miR213-reverse primer 5'-TGATGGTTAGCCATAGGGTACA-3' pre-miR214-forward primer 5'-GGCCTGGCTGGACAGAGT-3' pre-miR214-reverse primer 5'-AGGCTGGGTTGTCATGTGA-3' pre-miR215-forward primer 5'-TCATTCAGAAATGGTATACAGGAA-3' pre-miR215-reverse primer 5'-CAGAATATTGGCCTAAAGAAATGA-3' pre-miR216-forward primer 5'-GGCTTAATCTCAGCTGGCAAC-3' pre-miR216-reverse primer 5'-TCGTGAGGGCTAGGAAATTG-3' pre-miR217-forward primer 5'-TTTGATGTCGCAGATACTGCAT-3' pre-miR217-reverse primer 5'-CTTGTTTAGATGCTGAAGGCAAT-3' pre-miR218-forward primer 5'-GCGAGATTTTCTGTTGTGCTT-3' pre-miR218-reverse primer 5'-TAGAAAGCTGCGTGACGTTC-3' pre-miR218-2-forward primer 5'-GGGGCTTTCCTTTGTGCT-3' pre-miR218-2-reverse primer 5'-CTTTCCGCGGTGCTTGAC-3' pre-miR219-1-forward primer 5'-CGGCTCCTGATTGTCCAAA-3' pre-miR219-1-reverse primer 5'-CGGGACGTCCAGACTCAACT-3' pre-miR219-2-forward primer 5'-ACTCAGGGGCTTCGCCACTGA-3' pre-miR219-2-reverse primer 5'-GGAGCTCAGCCACAGATGTC-3' pre-miR220-forward primer 5'-GCATTGTAGGGCTCCACACC-3' pre-miR220-reverse primer 5'-TCCGTGAGGAGTTCCCAGAC-3' pre-miR221-forward primer 5'-CACCATCCAGGTCTGGGG-3' pre-miR221-reverse primer 5'-TTCCAGGTAGCCTGAAACCC-3 pre-miR222-forward primer 5'-AAGGTGTAGGTACCCTACATGG-3' pre-miR222-reverse primer 5'-CCATCAGAGACCCAGTAGC-3' pre-miR223-forward primer 5'-AGTGCCACGCTCCGTGTAT-3' pre-miR223-reverse primer 5'-CGCACTTGGGGTATTTGACA-3' pre-miR224-forward primer 5'-GGGCTTTCAAGTCACTAGTGGT-3' pre-miR224-reverse primer 5'-GGGCTTTGTAGTCACTAGGGC-3' pre-miR291-forward primer 5'-CACCTATGTAGCGGCCATCA-3' pre-miR291-reverse primer 5'-CCATCAGTGGCACACAA-3' pre-miR296-forward primer 5'-AGGACCCTTCCAGAGGGC-3' pre-miR296-reverse primer 5'-AGAGCCCTTCAGGAGAGCC-3' pre-miR299-forward primer 5'-CACCAAGAAATGGTTTACCGTC-3' pre-miR299-reverse primer 5'-AAGCGGTTTACCGTCCCA-3' pre-miR301-forward primer 5'-ACTGCTAACGAATGCTCTGACT-3' pre-miR301-reverse primer 5'-CCTGCTTTCAGATGCTTTGAC-3' pre-miR302a-forward primer 5'-CCACCACTTAAACGTGGATGT-3' pre-miR302a-reverse primer 5'-CCATCACCAAAACATGGAAG-3' pre-miR302b-forward primer 5'-CTTCAACTTTAACATGGAAGTGC-3' pre-miR302b-reverse primer 5'-ACTCCTACTAAAACATGGAAGCA-3' pre-miR302c-forward primer 5'-CCTTTGCTTTAACATGGGG-3' pre-miR302c-reverse primer 5'-CCTCCACTGAAACATGGAAG-3' pre-miR320-forward primer 5'-CTCCCCTCCGCCTTCTCT-'3 pre-miR320-reverse primer 5'-CTCATCCTTTTTCGCCCTCT-3' pre-miR323-forward primer 5'-TTGGTACTTGGAGAGAGGTGG-3' pre-miR323-reverse primer 5'-GATTAGATACTGCAAAGAGGTCGA-3' pre-miR324-forward primer 5'-CTGACTATGCCTCCCCGC-3' pre-miR324-reverse primer 5'-GACTACAACCCCCAGCAGC-3' pre-miR325-forward primer 5'-TGCTTGGTTCCTTAGTAGGTGTC-3' pre-miR325-reverse primer 5'-GCACAGTGCTTGATTGATAGGA-3' pre-miR326-forward primer 5'-CATCTGTCTGTTGGGCTGGA-3' pre-miR326-reverse primer 5'-CCCAGAGGCGATCTGAGC-3' pre-miR331-forward primer 5'-GAGTTTGGTTTTGTTTGGGTTT-3' pre-miR331-reverse primer 5'-GAGCTTAGGTTGGTTCTAGGATAGG-3' pre-miR337-forward primer 5'-GGTGGGAACGGCTTCATAC-3' pre-miR337-reverse primer 5'-TTGAAGGGGATGAAGAAAGG-3' pre-miR328-forward primer 5'-AGGAGGGGCTCAGGGAGA-3' pre-miR328-reverse primer 5'-GGACGGAAGGGCAGAGAG-3' pre-miR330-forward primer 5'-CCTCTCTGGGCCTGTGTCT-3' pre-miR330-reverse primer 5'-AGAGCGCTGCCTCTCTGC-3' pre-miR335-forward primer 5'-GGGGTCAAGAGCAATAACGA-3' pre-miR335-reverse primer 5'-GCAAATGAGAGGAGGTCAGG-3' pre-miR338-forward primer 5'-TCTCCAACAATATCCTGGTGC-3' pre-miR338-reverse primer 5'-TCTTCAACAAAATCACTGATGC-3' pre-miR340-forward primer 5'-CCTGGTGTGATTATAAAGCAATGA-3' pre-miR340-reverse primer 5'-CCAGGTATGGCTATAAAGTAACTGA-3' pre-miR342-forward primer 5'-AACTGGGCTCAAGGTGAGG-3' pre-miR342-reverse primer 5'-GGTGCGATTTCTGTGTGAGA-3' pre-miR345-forward primer 5'-AACCCTAGGTCTGCTGACTCC-3' pre-miR345-reverse primer 5'-AAACCCAGGCCTCCAGAC-3' pre-miR361-forward primer 5'-GGAGCTTATCAGAATCTCCAGG-3' pre-miR361-reverse primer 5'-GAAGCAAATCAGAATCACACCTG-3' pre-miR368-forward primer 5'-GGTGGATATTCCTTCTATGTTTATG-3' pre-miR368-reverse primer 5'-AAAACGTGGAATTTCCTCTATG-3' pre-miR369-forward primer 5'-AAGGGAGATCGACCGTGTTA-3' pre-miR369-reverse primer 5'-GAAAAGATCAACCATGTATTATTCG-3' pre-miR370-forward primer 5'-AGACAGAGAAGCCAGGTCACG-3' pre-miR370-reverse primer 5'-AGACAGACCAGGTTCCACCC-3' pre-miR371-forward primer 5'-GTGGCACTCAAACTGTGGG-3' pre-miR371-reverse primer 5'-GTAACACTCAAAAGATGGCGG-3' pre-miR372-forward primer 5'-GTGGGCCTCAAATGTGGA-3' pre-miR372-reverse primer 5'-GTGACGCTCAAATGTCGC-3' pre-miR373-forward primer 5'-GGGATACTCAAAATGGGGG-3' pre-miR373-reverse primer 5'-GGGACACCCCAAAATCGA-3' pre-miR374-forward primer 5'-TCGGCCATTATAATACAACCTG-3' pre-miR374-reverse primer 5'-CACAGACAATTACAATACAATCTGA-3' pre-miR376a-forward primer 5'-AAGGTAGATTCTCCTTCTATGAGTACA-3' pre-miR376a-reverse primer 5'-CGTGCATTTTCCTCTATGATTAATC-3' pre-miR377-forward primer 5'-TTGAGCAGAGGTTGCCCT-3' pre-miR377-reverse primer 5'-CAAACAAAAGTTGCCTTTGTGT-3' pre-miR378-forward primer 5'-AGGGCTCCTGACTCCAGG-3' pre-miR378-reverse primer 5'-AGGCCTTCTGACTCCAAGTC-3' pre-miR379-forward primer 5'-AGAGATGGTAGACTATGGAACGTAG-3' pre-miR379-reverse primer 5'-AGAGTTAGTGGACCATGTTACATAGG-3' pre-miR380-forward primer 5'-AAGATGGTTGACCATAGAACATG-3' pre-miR380-reverse primer 5'-AAGATGTGGACCATATTACATACGAC-3' pre-miR381-forward primer 5'-TACTTAAAGCGAGGTTGCCCT-3' pre-miR381-reverse primer 5'-TACTCACAGAGAGCTTGCCCTT-3' pre-miR382-forward primer 5'-TACTTGAAGAGAAGTTGTTCGTGG-3' pre-miR382-reverse primer 5'-TACTGAAAAAAGTGTTGTCCGTG-3' pre-miR383-forward primer 5'-CTCCTCAGATCAGAAGGTGATTG-3' pre-miR383-reverse primer 5'-CTCTTTCTGACCAGGCAGTG-3' pre-miR384-forward primer 5'-TGTTAAATCAGGAATTTTAAACAATTC-3' pre-miR384-reverse primer 5'-TGTTACAGGCATTATGAACAATTTCT-3' pre-miR409-forward primer 5'-TGGTACTCGGGGAGAGGT-3' pre-miR409-reverse primer 5'-TGATACCGAAAAGGGGTTCA-3' pre-miR410-forward primer 5'-GGTACCTGAGAAGAGGTTG-3' pre-miR410-reverse primer 5'-CTGAAAACAGGCCATCTGTG-3' pre-miR411-forward primer 5'-TGGTACTTGGAGAGATAGTAGA-3' pre-miR411-reverse primer 5'-CGGGGATGGATTTGATACTG-3' pre-miR422-forward primer 5'-AGAGAAGCACTGGACTTAGGG-3' pre-miR422-reverse primer 5'-GAGGACAAAGCTTGGCTCAG-3' pre-miR423-forward primer 5'-AAAGGAAGTTAGGCTGAGGG-3' pre-miR423-reverse primer 5'-CGCGGGTTAGGAAGCAAG-3' pre-miR424-forward primer 5'-AGGGGATACAGCAGCAATTC-3' pre-miR424-reverse primer 5'-ACCTTCTACCTTCCCCACGA-3' pre-miR425-forward primer 5'-AAGCGCTTTGGAATGACA-3' pre-miR425-reverse primer 5'-AGAGCACTGGGCGGACAC-3' pre-miR449-forward primer 5'-CTGTGTGTGATGAGCTGGCA-3' pre-miR449-reverse primer 5'-GACAGCAGTTGCATGTTAGCC-3' pre-miR450-1-forward primer 5'- AAACGATACTAAACTGTTTTT-3' pre-miR450-1-reverse primer 5'-TACATGCAAAATGTTCCCAAT-3' pre-miR450-2-forward primer 5'-CCAAAGAAAGATGCTAAACTAT-3' pre-miR450-2-reverse primer 5'-TGATACAAAACTATGAATGCAAAATG-3' pre-miR451-forward primer 5'-CTTGGGAATGGCAAGGAA-3' pre-miR451-reverse primer 5'-TCTGGGTATAGCAAGAGAACCA-3' pri-miR484-forward primer 5'-CCTCCCGATAAACCCCTAAA-3' pri-miR484-reverse primer 5'-GGTTCCTTTCGACTCCACAA-3' pri-miR485-forward primer 5'-AGGCAATGGATTTCTCACCA-3' pri-miR485-reverse primer 5'-CCGAGGCAGAATTTGACACT-3'

Supplementary Table 2

Oligonucleotide sequences used in PCR analyses for miRNA precursors as per Table 1.

Table 3 TuJ1+ positive cells Dicer/C Dicer/GFP RE Field1 289 213 Field2 266 146 Field3 259 172

TH+ positive cells Dicer/C Dicer/GFP RE Field1 19 1 Field2 16 2 Field3 11 3

GABA+ positive cell GFP CRE Field1 285 125 Field2 315 113 Field3 250 148

Supplementary Table 3

As in Figure 1A: absolute number of cells for TH, TujI, and GABA in Dicer conditional knockout ES cells by CRE infection at stage 5. Cultures transduced with a lentiviral Cre vector (vCre) were significantly decreased in TH+ neurons to a greater extent than the decrease in TujI+ and GABA+ neurons. (4) Supplementary References

1. E. P. Murchison, J. F. Partridge, O. H. Tam, S. Cheloufi, G. J. Hannon, Proc Natl

Acad Sci U S A 102, 12135 (Aug 23, 2005).

2. C. Martinat et al., Proc Natl Acad Sci U S A 103, 2874 (Feb 21, 2006).

3. M. K. Lobo, S. L. Karsten, M. Gray, D. H. Geschwind, X. W. Yang, Nat Neurosci

9, 443 (Mar, 2006).

4. D. A. Rubinson et al., Nat Genet 33, 401 (Mar, 2003).