1

Supplemental Materials and Methods

Hydroxyl Radical Footprinting

The 28-nt zipcode flanked by 5′ and 3′ nucleotide extensions that were added for optimal resolution of RNA cleavage products by denaturing polyacrylamide gel electrophoresis was prepared by T7 transcription using synthetic DNA oligonucleotide templates (Invitrogen). The sequence of the sense template was 5′-

GGGAATGGATCCACATCTACGAACCGGACTGTTACCAACACCCACACCCCTTCACTGCAGACTTG

ACGAAGCTT-3′ and the sequence of the antisense template was 5′-

AAGCTTCGTCAAGTCTGCAGTGAAGGGGTGTGGGTGTTGGTAACAGTCCGGTTCGTAGATGTGGA

TCCATTCCC-3′. Italicized nucleotides indicate the zipcode[1-28] sequence. Templates were amplified by PCR using the T7 promoter sequence containing 41.30 primer (5′-

GATAATACGACTCACTATAGGGAATGGATCCACATCTACGA-3′) and the 24.30 primer (5′-

AAGCTTCGTCAAGTCTGCAGTGAA-3′). The transcribed RNA (5′-

GGGAATGGATCCACATCTACGAACCGGACTGTTACCAACACCCACACCCCTTCACTGCAGACTTG

ACGAAGCTT-3′) was gel-purified by denaturing polyacrylamide gel electrophoresis (8% [w/v] 29:1 acrylamide/Bis-acrylamide, 7M urea, 1X TBE), eluted, precipitated, and resuspended in CE buffer (10 mM sodium cacodylate pH 7.3, 0.1mM EDTA). RNA was dephosphorylated using Antarctic phosphatase (New England Biolabs) and then 5′-end labeled with 32P using T4 polynucleotide kinase

(New England Biolabs). Labeled RNA was gel purified, eluted, precipitated, and resuspended in CE buffer as described above.

Labeled 32P-RNA (0.01 uM) was equilibrated with recombinant MBP-ZBP1 KH34 (1µM, 0.1µM,

0.02µM, 0µM) in a buffer containing 10 mM sodium cacodylate (pH 7.3), 0.1 mM EDTA, 100 mM NaCl, and 0.01 mg mL-1 tRNA for approximately 3 h in order to ensure the binding reaction reached equilibrium. Cleavage reactions were initiated by mixing samples with freshly prepared Fe(II)-EDTA-

Ascorbate solution (5mM, 10mM, and 25mM in reaction, respectively) for 30 minutes at room 2 temperature. After phenol/chloroform extraction, RNA cleavage products were gel-separated using denaturing polyacrylamide gel electrophoresis (10% [w/v] 29:1 acrylamide/Bis-acrylamide, 7M urea, 1X

TBE) and imaged on storage phosphor screens using Storm 860 scanner (GE Healthcare). The hydroxyl radical reactivity of the nucleotides contacting ZBP1 KH34 were quantitated by SAFA individual peak fitting analysis(Das et al. 2005). The data for each protection were individually scaled to the integrated density of all bands in the respective lane(Shcherbakova et al. 2004).

EMSA

The sequences of the RNAs used in the ZBP1 KH34 EMSA experiments were:

Zipcode[1–28], 5′-Fl-ACCGGACUGUUACCAACACCCACACCCC-3′;

Zipcode[1–28]C3G, 5′-Fl-ACGGGACUGUUACCAACACCCACACCCC-3′;

Zipcode[1–28]C3A, 5′-Fl-ACAGGACUGUUACCAACACCCACACCCC-3′;

Zipcode[1–28]C3U, 5′-Fl-ACUGGACUGUUACCAACACCCACACCCC-3′;

Zipcode[1–28]G4A, 5′-Fl-ACCAGACUGUUACCAACACCCACACCCC-3′;

Zipcode[1–28]G4C, 5′-Fl-ACCCGACUGUUACCAACACCCACACCCC-3′;

Zipcode[1–28]G4U, 5′-Fl-ACCUGACUGUUACCAACACCCACACCCC-3′;

Zipcode[1–28]G5A, 5′-Fl-ACCGAACUGUUACCAACACCCACACCCC-3′;

Zipcode[1–28]G5C, 5′-Fl-ACCGCACUGUUACCAACACCCACACCCC-3′;

Zipcode[1–28]G5U, 5′-Fl-ACCGUACUGUUACCAACACCCACACCCC-3′;

Zipcode[1–28]A6G, 5′-Fl-ACCGGGCUGUUACCAACACCCACACCCC-3′;

Zipcode[1–28]A6C, 5′-Fl-ACCGGCCUGUUACCAACACCCACACCCC-3′;

Zipcode[1–28]A6U, 5′-Fl-ACCGGUCUGUUACCAACACCCACACCCC-3′;

Zipcode[1–28]C7G, 5′-Fl-ACCGGAGUGUUACCAACACCCACACCCC-3′;

Zipcode[1–28]C7A, 5′-Fl-ACCGGAAUGUUACCAACACCCACACCCC-3′;

Zipcode[1–28]C7U, 5′-Fl-ACCGGAUUGUUACCAACACCCACACCCC-3′;

Zipcode[1–28]A22C;A24C, 5′-Fl-ACCGGACUGUUACCAACACCCCCCCCCC-3′; 3

Zipcode[1–28]5RE-3RE Swap, 5′-Fl-ACACACCCGUUACCAACACCCCGGACUC-3′;

25RE spacing, 5′-Fl-AGCGGACUGUUACUGAAUUAUUAAAGCGUUUUACACCCUU-3′;

30RE spacing, 5′-Fl-AGCGGACUGUUACUGAUUCGAAUUAUUAAAGCGUUUUACACCCUU-3′.

Circular dichroism (CD) spectroscopy

All CD spectra were recorded on a Jasco J-815 spectrometer (Japan Spectroscopic Co., Tokyo,

Japan) equipped with a PTC-423 Peltier temperature-control system. Thermal unfolding was monitored in 1 cm path-length quartz cells (Hellma) by recording mean residue ellipticity at 222nm between 25°C and 85°C, at approximately 2°C intervals. Reversibility of unfolding was assessed by cooling to 25°C at the same rate. ZBP1 KH34 and ZBP1 KH34 F478A-F479A samples were prepared at 6uM concentration in 25 mM Tris pH 7.2, 500 mM NaCl. Data were fit to a modified version of the Gibbs-

Helmholtz equation for a two-state (folded-unfolded) transition model using SigmaPlot v11.0 (SyStat,

Chicago, IL, USA) as previously described(Greenfield 2006).

Wavelength scans from 240nm-190nm were performed in 0.1 cm path-length quartz cells

(Hellma) at 25°C. Protein samples were prepared at 35 uM concentration in 20 mM sodium phosphate buffer pH 7.2, 100 mM NaCl. To calculate approximate secondary structure, spectra were analyzed with the CDsstr algorithm(Johnson 1999; Sreerama and Woody 2000) using the DichroWeb server(Whitmore and Wallace 2004).

ZBP1 Immunoprecipitation

The gene symbols, accession numbers, and corresponding primer sequences used for PCR amplification of ZBP1 co-immunoprecipitated RNAs were: B2M, NM_009735, 5′-

ACAGTTCCACCCGCCTCACATT-3′, 5′-TAGAAAGACCAGTCCTTGCTGAAG-3′; RPL13A,

NM_009438, 5′-CTGCTCTCAAGGTTGTTCGGCT-3′, 5′-CCTTCCGTTTCTCCTCCAGAGT-3′; GAPDH,

NM_008084, 5′- GGTGAAGGTCGGTGTGAACG-3′, 5′-CTCGCTCCTGGAAGATGGTG-3′; ACTB,

NM_007393, 5′-GTCCCTCACCCTCCCAAAAG-3′, 5′- GCTGCCTCAACACCTCAACCC-3′; CSNK1D, 4

NM_027874, 5′-ATGCCATTTGGGTTTGTCAT-3′, 5′-CACCACACCTTTCTGGAGGT-3′; DTNA,

NM_207650, 5′-CGTTTGCACAAAGTTCGAGA-3′, 5′-AGCTCCACCGTGAATGTACC-3′; EFNB2,

NM_010111, 5′-CTAACCTCTCCTGCGCATTC-3′, 5′-GACGCACAGGACACTTCTCA-3′; EN1,

NM_010133, 5′-ACACAACCCTGCGATCCTAC-3′, 5′-GATATAGCGGTTTGCCTGGA-3′; GNAO1,

NM_010308, 5′-AGCAAGGCGATTGAGAAAAA-3′, 5′-ACGTCACACACCATCTTGGA-3′; PPP1R9B,

BC029672, 5′-GCAACAGCCCTCAACTTCTC-3′, 5′-CTCTGCAGGCACACCATCTA-3′; IRX5,

NM_018826, 5′-TCTATCCGGGCTACACGAAC-3′, 5′-GGGATACCGCACCAGAGTTA-3′; NTNG1,

NM_030699, 5′-AGCATTCTTCGCTGTCAGGT-3′, 5′-ACAGGGGTATGACTGGCTTG-3′; BARHL2,

NM_001005477, 5′-CAACCTCACTGACACCCAAGTC-3′, 5′-AAGGCGACGGAAACATCCTCTG-3′;

HES1, NM_008235, 5′-GGAAATGACTGTGAAGCACCTCC-3′, 5′-GAAGCGGGTCACCTCGTTCATG-

3′; KCNAB2, AK143166, 5′-CGGCATCGTCTCAGGGAAGTAT-3′, 5′-CTCCTCACTCAGGATCTTGTCC-

3′; NLGN2, BC056478, 5′-CGATGTCATGCTCAGCGCAGTA-3′, 5′-CCACACTACCTCTTCAAAGCGG-

3′; OPRL1, AF043277, 5′-GCTCAGCACAAGTGGAGGATGA-3′, 5′-

GGCTGTAGCAGACAGAGATGATC-3′; RAVER1, NM_027911, 5′-CTGGCTTCAGTGATGTGGATGC-

3′, 5′-CATCTCTGCTGTCTCGTACTCC-3′; SEMA6A, BC062979, 5′-AATGGCAGCCTTTTCCTGGAGG-

3′, 5′-GCAACATAGAGTGAGCCACTCG-3′; SYT3, NM_016663, 5′-TCCCAGCAAAGGACTCCAATGG-

3′, 5′-GGCACCGAGAACTGAAACGTCT-3′; SEPT8, BC049819, 5′-GTTTGCCTCTACTTCATCACGCC-

3′, 5′-GAGATGGTGTCAGCCTTGGCAA-3′.

Fluorescence in situ hybridization

The sequences for anti-sense probes (amino-modified T are underlined) used to detect the spinophilin mRNA were:

5′-ACATCCTCATTGCGTCGGTCATAGTCCTCGTTGGAGTAGGTGCTGAATAC-3′,

5′-AGCTCCACAGGAAACAGCTCCAACCTCTCCACTCGCTTCTCTAGCTCATA-3′,

5′-GTCTCCTCATCATCCTCTCCATACTGGGCGTATCTCTGCTCCATCATCTC-3′,

5′-CCTTTCTCCTGCTCCAGACTCTGCAACTTCCGTTTCAGCTGCTGGATTTC-3′, 5

5′-GGAACTCGATCTCCTTTTGCTGGTAGTCCTTGATGAGACGCTTGGCCTTG-3′,

5′-TTGGAGTTCCTCAGTGTCTGCAGGTTTCCTTCCAGTTCTGAGATCTTGTC-3′,

5′-TTAGGGCAAGGGTGGTAATGTGGAAGTGGCAAGAGATGGATTCTGAAAGG-3′,

5′-ACTGATCTCACAAGGAATCCACCTCCCAGCAAAAGGGCTTCCTAGTTCTA-3′,

5′-GTTTGTCCATTTCCTCCTTCCTGGCTAGCTCCGACTCTTCCAGTACCCGA-3′,

5′-AAGTGGGGAAACTGAGTCAATTTCACTTAGAAAGCAGGCTGACAGCAAGC-3′.

The sequences for anti-sense probes (amino-modified T are underlined) used to detect the GFP mRNA were:

5′-GGGTCTTGTAGTTGCCGTCGTCCTTGAAGAAGATGGTGCG-3′,

5′-GGCTGTTGTAGTTGTACTCCAGCTTGTGCCCCAGGATGTT-3′,

5′-TCTTTGCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTGT-3′.

6

Supplemental References

Das R, Laederach A, Pearlman SM, Herschlag D, Altman RB. 2005. SAFA: semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments. RNA (New York, NY 11: 344-354. Greenfield NJ. 2006. Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions. Nat Protoc 1: 2527-2535. Johnson WC. 1999. Analyzing protein circular dichroism spectra for accurate secondary structures. Proteins 35: 307-312. Schmittgen TD, Livak KJ. 2008. Analyzing real-time PCR data by the comparative C(T) method. Nature protocols 3: 1101-1108. Shcherbakova I, Gupta S, Chance MR, Brenowitz M. 2004. Monovalent ion-mediated folding of the Tetrahymena thermophila ribozyme. J Mol Biol 342: 1431-1442. Sreerama N, Woody RW. 2000. Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem 287: 252-260. Whitmore L, Wallace BA. 2004. DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic acids research 32: W668-673.

7

Supplemental Figure Legends

Figure S1. The ZBP1 KH34 didomain recognizes a bipartite RNA element within the zipcode RNA. (A)

Hydroxyl radical footprint of ZBP1 KH34 bound to the 28 nucleotide zipcode RNA. Lane designations

(from left): 28 nucleotide zipcode RNA in the absence of ZBP1 KH34, and in the presence of 1, 0.1, and

0.02 µM of ZBP1 KH34, respectively. (B) Quantification of cleavage intensity difference versus nucleotide position for the 0.1 µM ZBP1-KH34 bound RNA.

Figure S2. ZBP1 KH34 recognizes alternate sequences at the 3′ zipcode binding site. (A)

Representative EMSA result for ZBP1 KH34 binding to 3′ site mutant zipcode RNA; positions 22 and 24 of the zipcode are both mutated from A to C. The filled triangle represents a 1:1 serial dilution of recombinant protein. Free RNA (**) and RNA-protein complexes (*) are labeled. (B) Quantification of the fraction of RNA bound in EMSA was fit to the Hill equation to measure the Kd,app and Hill coefficient.

Figure S3. Assignment of IMP1 KH34 amide-resonances. 15N-HSQC spectra of IMP1 KH34 labeled with peak assignments. Inset shows peak assignments within highlighted region of the spectra.

Figure S4. Conserved residues that link KH3 and KH4 stabilize the antiparallel orientation required for

RNA binding. (A) Mutation of two KH34 linker residues F478A,F479A reduces binding to 28 nt zipcode

RNA. The filled triangle represents a 1:1 serial dilution of recombinant protein. Free RNA (**) is labeled.

(B) CD signal at 222nm of wild type ZBP1 KH34 and ZBP1 KH34 F478A,F479A linker mutant recorded during a thermal denaturation experiment. Data were fit to the Gibbs-Helmholtz equation as previously described(Greenfield 2006) to measure the midpoint of the unfolding/folding transition, TM. Transition midpoints are indicated by dashed lines. Mean fits are indicated by solid black lines with error bars representing the standard deviation of the fits. (C) Circular dichroism spectra of ZBP1 KH34 and ZBP1

KH34 F478A,F479A. The experimental and CDsstr reconstructed spectra for the average of three 8

samples of ZBP1 KH34 (filled circle and open circle, respectively) and ZBP1 KH34 F478A,F479A (filled square and open square, respectively) are shown.

Figure S5. Determination of maximum spacing between 5RE and 3RE for recognition by ZBP1 KH34.

Representative EMSA results for ZBP1 KH34 binding to RNAs with 25nt (A) and 30nt (B) spacing between 5RE and 3RE. The filled triangle represents a 1:1 serial dilution of recombinant protein. Free

RNA (**) and RNA-protein complexes (*) are labeled.

Figure S6. Expression of ZBP1 KH34 target mRNAs is not altered in ZBP1-KO brains. Fold change in target gene expression in ZBP1-KO as compared to ZBP1 WT brain tissue was calculated using the comparative CT method (Schmittgen and Livak 2008). Error bars represent the SEM of three replicates using independent RNA isolations from littermate embryos.

Figure S7. Quantity of poly-A mRNAs in dendrites of ZBP1-KO mice is unaltered. (A) FISH analysis of poly-A containing transcripts [detected using oligo(dT) probes] in cultured hippocampal neurons of wild type and ZBP1-KO mice. Scale bars represent 10µm (B) Quantification of average poly-A mRNA FISH fluorescence intensity in dendrites of developing hippocampal neurons from wild-type (blue bars) and

ZBP1-KO (red bars) mice. Average fluorescence was normalized to dendrite length in microns. Error bars represent the SEM of three replicates from littermate embryos, p>0.6.

Figure S8. Calibration of single molecule FISH signal (A) Intensity distribution of individual, non- hybridized FISH probes (red bars) and multiple probes hybridized to spinophilin mRNA in neurons (blue bars).

Table S1. ZBP1 KH34 recognition element containing mRNA ligands conserved between human and mouse genomes. 9

Table S2. Consensus sequence alignment for selected genes demonstrating evolutionary conservation of the ZBP1 KH34 binding motif between the human and mouse genomes: *Genes with more than one unique ZBP1 KH34 binding motif ‡Genes with both actin-like and swapped ZBP1 KH34 binding motifs occurring within the same sequence space Supplemental Figure 1

A. B. Supplemental Figure 2

AA. [MBP-ZBP1 KH34]

*

**

B. Supplemental Figure 3 Supplemental Figure 4

A. [MBP-ZBP1 KH34 478A-479A]

** B.

C. Supplemental Figure 5

A. 5′-AGCGGACUGUUACUGAAUUAUUAAAGCGUUUUACACCCUU-3′

[MBP-ZBP1 KH34] 500nM 0.06nM *

** 25 spacing: Kd,app 9.3 ± 1.8nM

B. 5′-AGCGGACUGUUACUGAUUCGAAUUAUUAAAGCGUUUUACACCCUU-3′ [MBP-ZBP1 KH34] 500nM 0.06nM *

**

30 spacing: Kd,app 29.5 ± 6.9nM Supplemental Figure 6 Supplemental Figure 7

A. ZBP1 +/+ ZBP1 - / -

B. p>0.6 Supplemental Figure 8

60 15 Single Probes Single mRNA Particles ) % (

40 10 y c n e u q e r

F 20 5

0 0 0 1000 2000 3000 4000 5000 S pot intens ity (AU) Gene Symbol Mouse Genome Browser ID Human Genome Browser ID Protein Accession ID Entrez Gene Name Location Type(s) Orientation ACTB NM_007393 uc003soq.3 NP_001092 actin, beta Cytoplasm other β-actin like ARF5 NM_007480 uc003vmb.1 NP_001653 ADP-ribosylation factor 5 Cytoplasm transporter β-actin like ARHGEF10L AK028648 uc009vpe.1 NP_001011722 Rho guanine nucleotide exchange factor (GEF) 10-like Cytoplasm enzyme β-actin like BARX1 AJ297677 uc004aud.2 NP_067545 BARX homeobox 1 Nucleus transcription regulator β-actin like BAZ2A AK122243 uc001slo.1 NP_038477 bromodomain adjacent to zinc finger domain, 2A Nucleus transcription regulator β-actin like BRD2 AK158970 uc003ocn.3 NP_005095 bromodomain containing 2 Nucleus kinase β-actin like BRD4 AK147545 uc002nar.2 NP_490597 bromodomain containing 4 Nucleus kinase β-actin like C20orf160 NM_145536 uc002wxf.2 NP_542192 chromosome 20 open reading frame 160 unknown other β-actin like CAMK1D AK147657 uc001ilo.2 NP_705718 calcium/calmodulin-dependent protein kinase ID Cytoplasm kinase β-actin like CELF6 BC052406 uc002auh.2 NP_443072 CUGBP, Elav-like family member 6 unknown other β-actin like COL4A1 BC072650 uc001vqw.3 NP_001836 collagen, type IV, alpha 1 Extracellular Space other β-actin like CSNK1D NM_027874 uc002keh.2 NP_620693 casein kinase 1, delta Cytoplasm kinase β-actin like CTDSPL NM_133710 uc003chg.2 NP_001008393 CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase-like Nucleus other β-actin like DTNA AK147279 uc002kxw.2 NP_116757 dystrobrevin, alpha Plasma Membrane other β-actin like DZIP1 AK085147 uc001vmi.2 NP_055749 DAZ interacting protein 1 Cytoplasm other β-actin like EBF4 AF387630 uc002wgt.3 NP_001103984 early B-cell factor 4 Nucleus other β-actin like EFNB2 NM_010111 uc001vqi.2 NP_004084 ephrin-B2 Plasma Membrane other β-actin like ELFN2 NM_183141 uc003asq.3 NP_443138 extracellular leucine-rich repeat and fibronectin type III domain containing 2 unknown other β-actin like EN1 NM_010133 uc002tlm.2 NP_001417 engrailed homeobox 1 Nucleus transcription regulator β-actin like ESR1 AK036627 uc011eew.1 NM_001122742 estrogen receptor 1 Nucleus ligand-dependent nuclear receptor β-actin like FAM125B BC059907 uc004bqh.1 NP_258257 family with sequence similarity 125, member B unknown other β-actin like FAM19A5 NM_134096 uc003bim.3 NP_001076436 family with sequence similarity 19 (chemokine (C-C motif)-like), member A5 Extracellular Space other β-actin like FAM53B AK165022 uc001lhu.1 NP_055476 family with sequence similarity 53, member B unknown other β-actin like GDAP2 NM_010269 uc001ehf.2 NP_060156 ganglioside induced differentiation associated protein 2 unknown other β-actin like GM2A NM_010299 uc003ltr.3 NP_000396 GM2 ganglioside activator Cytoplasm enzyme β-actin like GNAL AK158831 uc002kqc.2 NP_892023 guanine nucleotide binding protein (G protein), alpha activating activity polypeptide, olfactory type Cytoplasm enzyme β-actin like GNAO1 NM_010308 uc002eit.3 NP_620073 guanine nucleotide binding protein (G protein), alpha activating activity polypeptide O Plasma Membrane enzyme β-actin like GTDC1 AK028468 uc002tvo.2 NP_001006637 glycosyltransferase-like domain containing 1 unknown other β-actin like INPP5A AK157558 uc001llp.2 NP_005530 inositol polyphosphate-5-phosphatase, 40kDa Plasma Membrane phosphatase β-actin like IRX5 NM_018826 uc002ehv.2 NP_005844 iroquois homeobox 5 Nucleus transcription regulator β-actin like JPH2 NM_021566 uc002xlj.2 NP_787109 junctophilin 2 Cytoplasm enzyme β-actin like KCNC1 AK162964 uc001mnl.1 NP_001106212 potassium voltage-gated channel, Shaw-related subfamily, member 1 Plasma Membrane ion channel β-actin like KIAA0930 NM_001033273 uc003bfv.1 NP_001009880 KIAA0930 unknown other β-actin like KIAA1024 NM_153509 uc002bew.1 NP_056021 KIAA1024 unknown other β-actin like KY NM_024291 uc003eqr.1 NP_848649 kyphoscoliosis peptidase Cytoplasm other β-actin like MAFB NM_010658 uc002xji.2 NP_005452 v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (avian) Nucleus other β-actin like MPZ NM_008623 uc001gaf.3 NP_000521 myelin protein zero Plasma Membrane other β-actin like NFATC2 AK161174 uc002xwc.2 NP_036472 nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 2 Nucleus transcription regulator β-actin like NFIC NM_008688 uc002lxo.2 NP_995315 nuclear factor I/C (CCAAT-binding transcription factor) Nucleus transcription regulator β-actin like NFKB2 DQ020183 uc001kva.2 NP_001070961 nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 (p49/p100) Nucleus transcription regulator β-actin like NTNG1 NM_030699 uc001dvc.3 NP_055732 netrin G1 Extracellular Space other β-actin like NTRK2 NM_001025074 uc004any.1 NP_001018076 neurotrophic tyrosine kinase, receptor, type 2 Plasma Membrane kinase β-actin like PKNOX2 BC050865 uc001qbu.2 NP_071345 PBX/knotted 1 homeobox 2 Nucleus other β-actin like PPP1R9B BC029672 uc002iqh.3 NP_115984 protein phosphatase 1, regulatory (inhibitor) subunit 9B Cytoplasm other β-actin like PRRG2 NM_022999 uc002pon.3 NP_000942 proline rich Gla (G-carboxyglutamic acid) 2 Extracellular Space other β-actin like PTGFRN NM_011197 uc001egv.1 NP_065173 prostaglandin F2 receptor negative regulator Plasma Membrane other β-actin like RAB11FIP1 AK030769 uc003xko.1 NP_001002233 RAB11 family interacting protein 1 (class I) Cytoplasm other β-actin like RAB11FIP4 NM_175543 uc002hgn.1 NP_116321 RAB11 family interacting protein 4 (class II) Cytoplasm other β-actin like RAB5C BC023027 uc002hyz.2 NP_958842 RAB5C, member RAS oncogene family Cytoplasm enzyme β-actin like SARDH AK087163 uc004cen.2 NP_009032 sarcosine dehydrogenase Cytoplasm enzyme β-actin like SBK1 NM_145587 uc002dpd.2 NP_001019572 SH3-binding domain kinase 1 unknown kinase β-actin like SCARB1 NM_016741 uc001ugm.3 NP_005496 scavenger receptor class B, member 1 Plasma Membrane transporter β-actin like SGSH AK128926 uc002jxy.2 NP_000190 N-sulfoglucosamine sulfohydrolase Cytoplasm enzyme β-actin like SIM2 D64135 uc002yvp.2 NP_005060 single-minded homolog 2 (Drosophila) Nucleus transcription regulator β-actin like SLC6A12 NM_133661 uc001qhx.2 NP_001116320 solute carrier family 6 (neurotransmitter transporter, betaine/GABA), member 12 Plasma Membrane transporter β-actin like SLIT3 NM_011412 uc003mab.2 NP_003053 slit homolog 3 (Drosophila) Extracellular Space other β-actin like SPEG AK164360 uc002vln.1 NP_005867 SPEG complex locus Nucleus kinase β-actin like SYT3 NM_016663 uc002pst.2 NP_115674 synaptotagmin III Cytoplasm transporter β-actin like TAPBP NM_001025313 uc003odx.1 NP_003181 TAP binding protein (tapasin) Cytoplasm transporter β-actin like TMEM121 NM_153776 uc001yrp.1 NP_079544 transmembrane protein 121 unknown other β-actin like TMEM127 NM_175145 uc002svq.2 NP_060319 transmembrane protein 127 unknown other β-actin like UBE2L3 NM_009456 uc002zuz.1 NP_937800 ubiquitin-conjugating enzyme E2L 3 Cytoplasm enzyme β-actin like USP47 AK030909 uc001mjr.2 NP_060414 ubiquitin specific peptidase 47 unknown peptidase β-actin like WNK1 AY455867 uc001qio.3 NP_061852 WNK lysine deficient protein kinase 1 Cytoplasm kinase β-actin like ZC3H18 NM_001029993 uc002fky.2 NP_653205 zinc finger CCCH-type containing 18 unknown other β-actin like ZNF282 NM_146175 uc003wfm.2 NP_003566 zinc finger protein 282 Nucleus transcription regulator β-actin like ZNRF1 NM_133206 uc002fdl.1 NP_115644 zinc and ring finger 1 Cytoplasm other β-actin like BARHL1 AK048063 uc004cbp.1 NP_064448 BarH-like homeobox 1 Nucleus transcription regulator Swapped BARHL2 NM_001005477 uc001dns.2 NP_064447 BarH-like homeobox 2 Nucleus transcription regulator Swapped BAZ2A AK122243 uc001slo.1 NP_038477 bromodomain adjacent to zinc finger domain, 2A Nucleus transcription regulator Swapped BRSK2 NM_029426 uc001lti.2 NP_003948 BR serine/threonine kinase 2 unknown kinase Swapped C15orf57 BC023926 uc001zma.1 NP_001074260 chromosome 15 open reading frame 57 unknown other Swapped CADM1 NM_001025600 uc001pph.3 NP_055148 cell adhesion molecule 1 Plasma Membrane other Swapped CAMK2N2 AK013788 uc003fnj.1 NP_150284 calcium/calmodulin-dependent protein kinase II inhibitor 2 Nucleus other Swapped CBFA2T3 AK148139 uc002fmk.1 NP_787127 core-binding factor, runt domain, alpha subunit 2; translocated to, 3 Nucleus transcription regulator Swapped CBLN1 NM_019626 uc002efq.2 NP_004343 cerebellin 1 precursor Cytoplasm other Swapped CC2D1A BC016188 uc002mxo.2 NP_060191 coiled-coil and C2 domain containing 1A unknown other Swapped CD300LG AK138920 uc002iek.1 NP_660316 CD300 molecule-like family member g unknown other Swapped CREB3L1 BC016447 uc001ncf.2 NP_443086 cAMP responsive element binding protein 3-like 1 Nucleus transcription regulator Swapped DTNA AK147279 uc002kxw.2 NP_116757 dystrobrevin, alpha Plasma Membrane other Swapped DZIP1 AK085147 uc001vmk.2 NP_945319 DAZ interacting protein 1 Cytoplasm other Swapped ELFN2 NM_183141 uc003asq.3 NP_443138 extracellular leucine-rich repeat and fibronectin type III domain containing 2 unknown other Swapped ESRRB NM_011934 uc001xsq.1 NP_004443 estrogen-related receptor beta Nucleus ligand-dependent nuclear receptor Swapped FOSB NM_008036 uc002pbw.2 NP_001107643 FBJ murine osteosarcoma viral oncogene homolog B Nucleus transcription regulator Swapped HES1 NM_008235 uc003ftq.1 NP_005515 hairy and enhancer of split 1, (Drosophila) Nucleus transcription regulator Swapped INMT NM_009349 uc003tbs.1 NP_006765 indolethylamine N-methyltransferase Cytoplasm enzyme Swapped INPP5A AK157558 uc001llp.2 NP_005530 inositol polyphosphate-5-phosphatase, 40kDa Plasma Membrane phosphatase Swapped KCNAB2 AK143166 uc001alv.1 NP_003627 potassium voltage-gated channel, shaker-related subfamily, beta member 2 Plasma Membrane ion channel Swapped KCNC1 AK162964 uc001mnl.1 NP_001106212 potassium voltage-gated channel, Shaw-related subfamily, member 1 Plasma Membrane ion channel Swapped KIAA0467 BC059842 uc001cjk.1 NP_056099 KIAA0467 unknown other Swapped KY NM_024291 uc003eqr.1 NP_848649 kyphoscoliosis peptidase Cytoplasm other Swapped LGI3 NM_145219 uc003xav.2 NP_644807 leucine-rich repeat LGI family, member 3 Extracellular Space other Swapped MPZ NM_008623 uc001gaf.3 NP_000521 myelin protein zero Plasma Membrane other Swapped MXD1 BC034860 uc002sfy.2 NP_002348 MAX dimerization protein 1 Nucleus transcription regulator Swapped NFATC2 AK161174 uc002xwc.2 NP_036472 nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 2 Nucleus transcription regulator Swapped NLGN2 BC056478 uc002ggt.1 NP_065846 neuroligin 2 Plasma Membrane enzyme Swapped NPAS3 AK162871 uc001wrs.2 NP_775182 neuronal PAS domain protein 3 Nucleus other Swapped NPTX1 NM_008730 uc002jyp.1 NP_002513 neuronal pentraxin I Extracellular Space other Swapped NR4A1 NM_010444 uc001rzs.2 NP_002126 nuclear receptor subfamily 4, group A, member 1 Nucleus ligand-dependent nuclear receptor Swapped NR5A2 M81385 uc001gvb.2 NP_995582 nuclear receptor subfamily 5, group A, member 2 Nucleus ligand-dependent nuclear receptor Swapped NTRK2 NM_001025074 uc004any.1 NP_001018076 neurotrophic tyrosine kinase, receptor, type 2 Plasma Membrane kinase Swapped OAF NM_178644 uc001pxb.2 NP_848602 OAF homolog (Drosophila) unknown other Swapped OPRL1 AF043277 uc002yic.2 NP_872588 opiate receptor-like 1 Plasma Membrane G-protein coupled receptor Swapped PHF15 NM_199299 uc003kzn.2 NP_056103 PHD finger protein 15 Nucleus other Swapped PI4K2A NM_145501 uc001kog.1 NP_060895 phosphatidylinositol 4-kinase type 2 alpha Cytoplasm kinase Swapped PITPNA BC056171 uc002fst.3 NP_006215 phosphatidylinositol transfer protein, alpha Cytoplasm transporter Swapped RAVER1 NM_027911 uc002moa.2 NP_597709 ribonucleoprotein, PTB-binding 1 Nucleus other Swapped RBFOX3 AK160816 uc010dhs.2 NP_001076044 RNA binding protein, fox-1 homolog (C. elegans) 3 unknown other Swapped SCARB1 NM_016741 uc001ugm.3 NP_005496 scavenger receptor class B, member 1 Plasma Membrane transporter Swapped SEMA6A BC062979 uc003krv.3 NP_065847 sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6A Plasma Membrane other Swapped SEPT8 BC049819 uc003kxs.1 NP_055961 septin 8 Extracellular Space other Swapped SGSH AK128926 uc002jxy.2 NP_000190 N-sulfoglucosamine sulfohydrolase Cytoplasm enzyme Swapped SLC6A9 NM_008135 uc010okm.1 NP_001020016 solute carrier family 6 (neurotransmitter transporter, glycine), member 9 Plasma Membrane transporter Swapped SLCO5A1 AK028758 uc003xyk.2 NP_001139480 solute carrier organic anion transporter family, member 5A1 unknown transporter Swapped SOLH AK143711 uc002chi.2 NP_005623 small optic lobes homolog (Drosophila) unknown peptidase Swapped SOST NM_024449 uc002iec.1 NP_079513 sclerostin Extracellular Space other Swapped SPEG AK164360 uc002vln.1 NP_005867 SPEG complex locus Nucleus kinase Swapped SPI1 L03215 uc001nfb.1 NP_001074016 spleen focus forming virus (SFFV) proviral integration oncogene spi1 Nucleus transcription regulator Swapped SYT3 NM_016663 uc002pst.2 NP_115674 synaptotagmin III Cytoplasm transporter Swapped TIMP1 X04684 uc004dif.2 NP_003245 TIMP metallopeptidase inhibitor 1 Extracellular Space other Swapped TMEM231 NM_001033321 uc002fek.2 NP_001070884 transmembrane protein 231 unknown other Swapped TMEM63B AK159673 uc003owr.2 NP_060896 transmembrane protein 63B unknown other Swapped TRIM66 AK147559 uc010rbo.1 NP_055633 tripartite motif containing 66 Nucleus transcription regulator Swapped UNC5C AK141181 uc003htp.1 NP_003719 unc-5 homolog C (C. elegans) Plasma Membrane transmembrane receptor Swapped WTIP NM_207212 uc002nvm.2 NP_001073905 Wilms tumor 1 interacting protein Plasma Membrane other Swapped ZDHHC3 NM_026917 uc003cod.2 NP_057682 zinc finger, DHHC-type containing 3 Cytoplasm peptidase Swapped ZNF384 NM_175557 uc001qpz.2 NP_001035005 zinc finger protein 384 Nucleus transcription regulator Swapped ZZEF1 BC099976 uc002fxk.1 NP_055928 zinc finger, ZZ-type with EF-hand domain 1 unknown other Swapped

Gene Symbol Sequence Spacing

Engrailed homeobox 1 EN1 AGACTCGGACAGGTGCTATGGGGGAAAAATAAACATCTATT 22 Protein phosphatase 1 regulatory subunit 9B* PPP1R9B TTGTACGGACCATGCCCTCTGTCAGGTCGTCCCCATAAAAG 22 Synaptotagmin IIIǂ SYT3 CTGCCCGGACTGTGAACTCATCTCCTCGAAGCCATAATGTC 21 β-Actin ACTB TCTAGGCGGACTGTTACTGAGCTGCGTTTTACACCCTTTCT 19 Dystrobrevin αǂ DTNA CCCCAACGGACTACAGTTACCTGCACTCCTCCACGCCTTGC 19 Ephrin-B2 EFNB2 CCCACGCGGACCTCCAAGCCATGCACCCAGCCACTCAGGCC 19 Casein kinase 1 Δ CSNK1D GTGGTGGCGGACCTGCGCTCCCTGATGATCCCACCGCAGCT 18 Iroquois homeobox 5 IRX5 GTATCCCCGGACTTTCCTAATTTATTAAAACCATGGCCTTG 18 Protein phosphatase 1 regulatory subunit 9B* PPP1R9B TCCTTGGCCGGACAATAATAATCCCCTCCCATTATCCTCAC 15 Gα activating polypeptide O GNAO1 CAAAAGAGCTGCGGACAGACTGACCACCATCCCTGGCTCAT 10 Netrin G1 NTNG1 TCTCTCAGAACCGGACAGCACATCGCACATCGGAGTTGAGA 10 BarH-like homeobox 2 BARHL2 AGTGCCACAGCCCTCCAGGTTCCCACCTCACCCCGGACGGC 25 Opiate receptor-like 1 OPRL1 TATCTGCCACCCTATCCGTGCCCTTGATGTTCGGACATCCA 21 ǂ Dystrobrevin α DTNA TATAAACCACAGGTGCCATAAGGTCCCCCAACGGACTACAG 21, 12 Ribonucleoprotein, PTB-binding 1 RAVER1 AAGAAAAGCCACGCTTCCTACTCCAGAGGACGGACTGCCCA 18

Semaphorin 6A SEMA6A CTTCCTTCACATCCACGTCATACAGCAGGTCGGACTCACGA 18, 14 K+ voltage-gated channel shaker-related β2 KCNAB2 GTCTACTCCCCACCCCTCGTGCCCCACCCGGACAGTTCCCG 15

Septin 8* SEPT8 ACCCTTCTCCCATAACATGGTGTGAGGACGGACTGGGAGCC 15, 10 Hairy and enhancer of split 1 HES1 AGTCCCTAGCCCACCTCTCTCTTCTGACGGACACTAAAAAC 13 Septin 8* SEPT8 AGCACGCACAGACATACATGTCGAGAGCGGACTTTAGGCTT 12 Synaptotagmin IIIǂ SYT3 ACCCTCAGGACCCATCCCTTCCTGCCCGGACTGTGAACTCA 11 Neuroligin 2 NLGN2 GGCTGGGGGACCCCACGATACAGCCACGGACTCCAATGCCC 10