Supplemental Table 1: Examples of confirmed nuclear and nucleolar localization signals (NLS/NoLSs)
Protein Name / Organism / NLS/NoLS sequences / Reference PMIDNLS Sequences
Hepatitis B virus core antigen / 21SKCLGWLWG 29 / 2585603
Hepatitis D virus antigen / 66EGAPPAKRAR 75 / 17897693
HIV-1 Rev protein / 35RQARRNRRRRWR 46 / 9891055
HIV-1 Tat / 48GRKKRRQRRRAP59 / 9891055
Human T-cell leukemia virus type 1 (HTLV)-1 Rex protein / 1MPKTRRRPRRSQRKRPPT 18 / 9891056
Polyoma large T-antigen / 189VSRKRPRP 196 / 3000623
SV40 large T-antigen / 126PKKKRKV 132 / 6096007
Hrp1 / Yeast / 506RSGGNHRRNGRGGRGGYNRRNNGYHPY532 / 18343812
LYS14 / Yeast / 190LNPKNKKRRTSNAQRVKEFRKHSTSLDNDHNNARKRQHSSCKAEKKKKVRQNLSEDTTDPK250 / 10975256
Matα2 / Yeast / 1MNKIPIKDLLNPQ 13/ 148VRILESWFAKNI 159 / 6323016
MIH1 / Yeast / 31KKK33 / 18562688
Pho4 / Yeast / 144KVTKNKS150…157KRRGKPGP164 / 9732266
PKC1 / Yeast / 810KKRA813 / 15643058
RPT2 / Yeast / 11KKKKK15 and 33RKKRK37 / 15210724
SWI5 / Yeast / 636KKYENVVIKRSPRKRGRPRK655 / 7615496
Ty1 Integrase / Yeast / 595SKKRSLED602…625PPRSKKRI632 / 9448008
Nucleoplasmin / Xenopus / 154KRPAATKKAGQAKKKKLD171 / 3417784
p53 / Mouse / 311PPQPKKKPLDGE 322 / 2247074
glucocorticoid receptor / Rat / 497YRKCLQAGMNLEARKTKKKIKGIQQATA524 / 3123217
c-myc / Human / 320PAAKRVKLD 328 and 364RQRRNELKRSF 374 / 3054508
hnRNP A1 / Human / 267NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY 305 / 7730395
NF-κB p50 / Human / 367QRKRQK 372 / 1547506
NF-κB p65 / Human / 281EEKRKR 286 / 7679069
PLSCR1 / Human / 257GKISKHWTGI266 / 15611084
RCC1 / Human / 1MSPKRIAKRRSPPADAIPKSKKVKVSHR 28 / 10811825
SREBP2 / Human / 344RSSINDKIIELKDLVMGTDAKMHKSGVLRKAIDYIKYLQQVNHKLRQENMVLKLANQKNKL403 / 11283257
SRY / Human / 59KRPMNAFIVWSRDQRRK75 and 130RPRRK135 / 9346931
Ribosomal protein NLSs
RPS17A / Yeast / 2GRVRTK7 / 3939318
RPS22A / Yeast / 21GKRQVLIRP29 / 10386617
RPS25A / Yeast / 11AKAAAALAGGKKSKKKWSKKSMKDRA36 and 87GIIKPISKH95 / 10386617
RPL3 / Yeast / 1MSHRKYEAPRHGHLGFLPRKRA21 / 3931077
RPL25 / Yeast / 11KKAVVKG17 and 18TNGKKALKVRT28 / 1920406
RPL29 / Yeast / 6KTRKHRG13 and 23KHRKHPG29 / 2104804
RPL5 / Human / 21RRRREGKTDYYARKRLV37 and 255KKPKKEVKKKR265 / 10766838
RPL7 / Human / 18LKKKRRNFAE27, 28LKIKRLRKKFAQ39, 40KMLRKARRKLIY51 and 156KRGYGKINKKRI167 / 16797011
20828572
RPL22 / Human / 13KKKK16 and 88KKYLKK93 / 11056215
RPL23a / Human / 32VHSHKKKKIRTSPTFTTPKTLRLRRQPKYPRKSAPRRNKLDHY 74 / 9687515
RPS6 / Human / 167KKPR170 and 188KRRR191 and 230KRRR233 / 8590812
NoLS Sequences
Betanodavirus GGNNV-alpha / 23RRRANNRRR31 / 12642096
Coronavirus IBV N protein / 71WRRQARFK78 / 16734668
Herpese simplex virus type 1 gamma (1) 34.5 / 1MARRRRHRGPRRPRPP16 / 12186925
Herpese virus MDV MEQ protein / 62RRRKRNRDARRRRRKQ78 / 9060682
Herpese virus ORF57 / 91KRPR94 and 119RRPSRPFRKP128 (a bipartite NoLS) / 17005724
HIV-1 REV / 38RRNRRRRWRERQRQI52 / 2404140
HIV-1 Tat / 48RKKRRQRRRAHQ61 / 2108259
Porcine reproductive and respiratory syndrome virus (PRRSV) N protein / 41PGKKNKKKNPEKPHFPLATEDDVRHHFTPSER72 / 10500278
ApLLP / Slug / 1MAKSIRSKHRRQMRMMKRE19 / 12759182
MDM2 / Mouse / 466KKLKKRNK473 / 10707090
Angiogenin / Human / 53IMRRRGL59 / 11374889
FGF2 / Human / 249RSRKYTSWYVALKR262 / 15247275
HIC p40 / Human / 44GRCRRLANFGPRKRRRRRR62 / 11139147
LIMK2 / Human / 491KKRTLRKNDRKKR503 / 16820362
LYRIC / Human / 441KSKKKKKKKKKQGE454 and 569KQIKKKKKARRET582 / 19383828
MLF-1 / Human / 1MAPRGRRRPRPHRSEGARRSK21 / 17595757
NIK / Human / 143RKKRKKK149 / 15252129
Nuclear VCP-like protein / Human / 112KRKGKLKNKGSKRKK126 / 15469983
p120 / Human / 40SKRLSSRARKRAAKRRLG57 / 8089149
SAP30L / Human / 120RRYKRHYK127 / 16820529
Survivin / Human / 74MQRKPTIRRKNLRKLRRK91 / 15735764
Supplemental Table 2. Primers used for cloning (restriction sites are in bold and underlined)
ORF / Primer / Primer sequence / Restriction siteRPS8A / Forward / GAATTCATGGGTATTTCTCGTGAC / EcoRI
Reverse / GGATCCAGCAGCACCCTTGCCC / BamHI
RPL15A / Forward / GAATTCATGGGTGCGTACAAGTATG / EcoRI
Reverse / GGATCCACGGTAACGACGAAGGCTG / BamHI
RPL23aA / Forward / GCGGAATTCATGTCTCCGGCTAAAG / EcoRI
Reverse / GCGGGATCCGATGATGCCGATCTTGTTAG / BamHI
EGFP / Forward / GCGAAGCTTATGGTGAGCAAGGGCG / HindIII
Reverse / GCGACTAGTTTACTTGTACAGCTCGTCC / SpeI
AtFIBRILARIN2 / Forward / GCGGAATTCATGAGACCCCCAGTTACAG / EcoRI
Reverse / GCGGGATCCTGAGGCTGGGGTCTTTTG / BamHI
RPL23aA-N∆29 / Forward / GAATTCATGGCCTTCAAGAAGAAGGAC / EcoRI
Reverse / GCGGGATCCGATGATGCCGATCTTGTTAG / BamHI
RPL23aA-mid 91 / Forward / GAATTCATGGCCTTCAAGAAGAAGGAC / EcoRI
Reverse / GCGGGATCCCTTGGTCTGGATGTC / BamHI
RPL23aA-C∆34 / Forward / GCGGAATTCATGTCTCCGGCTAAAG / EcoRI
Reverse / GCGGGATCCCTTGGTCTGGATGTC / BamHI
RPL23aA-C∆64 / Forward / GCGGAATTCATGTCTCCGGCTAAAG / EcoRI
Reverse / GCGGGATCCGTCTTCAATCTTCTTC / BamHI
RPL23aA-C34 / Forward / GAATTCATGAAAGTGAACACACTCATC / EcoRI
Reverse / GCGGGATCCGATGATGCCGATCTTGTTAG / BamHI
RPL23aA-5’RR (native promoter) / Forward / GGGCCCTTTTCCGGCGGCGGAGAG AGACTTTG / ApaI
Reverse / GCGGAATTCGGCTTGAAATGATTCTTC / EcoRI
∆GST / Forward / GCGGAATTCATGTCCCCTATACTAG / EcoRI
Reverse / GCGGGATCCTACATGATCACCATTTAAATATG / BamHI
Supplemental Table 3. Primers used for site-directed mutagenesis (nucleotides corresponding to amino acid substitutions are underlined)
ORF / Primer ID / Mutation details / SequencepNLS1’-F / 10KKAD13 to 10AAAD13 / GAT ACT ACC GCG GCG GCT GAT CCT AAG
pNLS1’-R / CTT AGG ATC AGC CGC CGC GGT AGT ATC
pNLS1’’-F / 15KAKALK20 to 15AAAALA20 / GCT GAT CCT GCG GCC GCG GCC TTG GCG GCG GCA AAG
pNLS1’’-R / CTT TGC CGC CGC CAA GGC CGC GGC CGC AGG ATC AGC
pNLS2-F / 33KKDK36 to 33LADA36 / GGT CAA GCC TTC AAG CTG GCG GAC GCA AAG ATT AGG ACC AAG G
pNLS2-R / C CTT GGT CCT AAT CTT TGC GTC CGC CAG CTT GAA GGC TTG ACC
pNLS3-F / 36KKIR39 to 36AAIR39 / C AAG AAG AAG GAC GCA GCG ATT GCG ACC AAG GTC ACC TTC C
pNLS3-R / G GAA GGT GAC CTT GGT CGC AAT CGC TGC GTC CTT CTT CTT G
pNLS2, 3-F / 33KKDKKIR36 to 33LADAAIR36
(RPL23aA ORF with 33KKDK36 to 33LADA36 mutation was used as template) / C AAG CTG GCG GAC GCA GCG ATT GCG ACC AAG GTC ACC
pNLS2, 3-R / GGT GAC CTT GGT CGC AAT CGC TGC GTC CGC CAG CTT G
pNLS4-F / 86KK87 to 86AA87 / GAA TCT GCG ATG GCG GCG ATT GAA GAC AAC
pNLS4-R / GTT GTC TTC AAT CGC CGC CAT CGC AGA TTC
pNLS5-F / 105KKIK108 to 105AAIK108 / GCT GAC AAG GCG GCG ATT GCG GAT GCT GTT AAG
pNLS5-R / CTT AAC AGC ATC CGC AAT CGC CGC CTT GTC AGC
pNLS6-F / 112KK113 to 112AA113 / G GAT GCT GTT GCG GCG ATG TAT GAC ATC
pNLS6-R / GAT GTC ATA CAT CGC CGC AAC AGC ATC C
pNLS7-F / 120KK121 to 120AA121 / GAC ATC CAG ACC GCG GCA GTG AAC ACA CTC
pNLS7-R / GAG TGT GTT CAC TGC CGC GGT CTG GAT GTC
pNLS8-F / 132KK133 to 132AA133 / CCT GAT GGA ACC GCG GCG GCT TAC GTG AGG
pNLS8-R / CCT CAC GTA AGC CGC CGC GGT TCC ATC AGG
pRBS-F / 132KKAYVRL138 to 132AAAAAAA138
(RPL23aA ORF with 133KK134 to 133AA134 mutation was used as template) / ACC GCG GCG GCT GCC GCG GCG GCT ACA CCA GAC
pRBS-R
(Putative rRNA binding site) / GTC TGG TGT AGC CGC CGC GGC AGC CGC CGC GGT
RPL15A / pNLS1-F / 11RKK13 to 11AAA13 / CT GAG CTA TGG GCG GCG GCA CAG TCC GAT GTG ATG
pNLS1-R / CAT CAC ATC GGA CTG TGC CGC CGC CCA TAG CTC AG
pNLS2-F / 47KARR50 to 47AAAA50 / CT ACT CGT CCT GAT GCG GCT GCT GCT TTG GGT TAC AAG G
pNLS2-R / C CTT GTA ACC CAA AGC AGC AGC CGC ATC AGG ACG AGT AG
pNLS3-F / 65RVRR68 to 65AVAA68 / GTG TAC CGT GTA GCT GTG GCA GCT GGT GGA CGC AAG
pNLS3-R / CTT GCG TCC ACC AGC TGC CAC AGC TAC ACG GTA CAC
pNLS3, 4-F / 65RVRRGGRKR73 to 65AVAAGGAAA73
(RPL15A ORF with 65RVRR68 to 65AVAA68 mutation was used as template) / GTG GCA GCT GGT GGA GCC GCG GCG CCA GTG CCT AAG
pNLS3, 4-R / CTT AGG CAC TGG CGC CGC GGC TCC ACC AGC TGC CAC
pNLS5-F / 96RSKR98 to 96ASAA98 / CAA CTC AAG TTC CAG GCT AGC GCG GCT TCT GTT GCT GAG GAG
pNLS5-R / CTC CTC AGC AAC AGA AGC CGC GCT AGC CTG GAA CTT GAG TTG
pNLS6-F / 105RAGRK109 to 105AAGAA109 / GCT GAG GAG GCT GCT GGC GCG GCA TTG GGT GGT C
pNLS6-R / G ACC ACC CAA TGC CGC GCC AGC AGC CTC CTC AGC
pNLS7-F / 169KKNR172 to 169AANA172 / CC TCA GAG GGA GCG GCG AAC GCA GGT CTC CGC
pNLS7-R / GCG GAG ACC TGC GTT CGC CGC TCC CTC TGA GG
pNLS8-F / 188RR189 to 188AA189 / G AAC CGT CCA TCT GCC GCG GCT ACA TGG
pNLS8-R / CCA TGT AGC CGC GGC AGA TGG ACG GTT C
pNLS9-F / 193KK194 to 193AA194 / GCT ACA TGG GCG GCA AAC AAC TCT CTC
pNLS9-R / GAG AGA GTT GTT TGC CGC CCA TGT AGC
pNLS10-F / 201RRYR204 to 201AAYA204 (nucleotides in bold are part of the plasmid pBSKS+) / TCT CTC AGC CTT GCT GCT TAC GCT GGA TCC TTA TCG
pNLS10-R / CGA TAA GGA TCC AGC GTA AGC AGC AAG GCT GAG AGA
RPS8A / pNLS1-F / 10KRR12 to 10AAA12 / GAC TCT ATC CAC GCG GCG GCT GCC ACT GGA GGC AAG
pNLS1-R / CTT GCC TCC AGT GGC AGC CGC CGC GTG GAT AGA GTC
pNLS2-F / 22RKKRK26 to 22AAAAA26 / G CAG AAG CAA TGG GCG GCG GCG GCA GCG TAT GAG ATG GGA AGG C
pNLS2-R / G CCT TCC CAT CTC ATA CGC TGC CGC CGC CGC CCA TTG CT T CTG C
pNLS3-F / 45RRIR48 to 45AAIA48 / GC AAC AAG ACG GTC GCA GCA ATA GCA GTT CGT GGT GG
pNLS3-R / CC ACC ACG AAC TGC TAT TGC TGC GAC CGT CTT GTT GC
pNLS4-F / 75RKTR78 to 75AATA78 / GAA GCA ACT ACC GCC GCG ACC GCA GTC CTT GAT GTG G
pNLS4-R / C CAC ATC AAG GAC TGC GGT CGC GGC GGT AGT TGC TTC
pNLS5-F / 124RKKK127 to 124AAAA127 / GGT GTT GAG CTT GGG GCC GCG GCG GCG AGT GCT TCT TCC
pNLS5-R / GGA AGA AGC ACT CGC CGC CGC GGC CCC AAG CTC AAC ACC
pNLS6-F / 133KK134 to 133AA134 / GCT TCT TCC ACC GCG GCG GAC GGA GAG
pNLS6-R / CTC TCC GTC CGC CGC GGT GGA AGA AGC
pNLS7-F / 151KK152 to 151AA152 / CCT GAG GAG GTC GCG GCG AGC AAC CAC
pNLS7-R / GTG GTT GCT CGC CGC GAC CTC CTC AGG
pNLS8-F / 158RK159 to 158AA159 / CAC CTC CTG GCA GCG ATT GCA AGC CGT C
pNLS8-R / G ACG GCT TGC AAT CGC TGC CAG GAG GTG
pNLS9-F / 211KK212 to 211AA212 / GAG TTC TAC ATG GCG GCG ATC CAG AAG AAG
pNLS9-R / CTT CTT CTG GAT CGC CGC CAT GTA GAA CTC
pNLS9, 10-F / 211KKIQKKKGK219 to 211AAIQAAAGA219 (RPS8A ORF with 211KK212 to 211AA212 mutation was used as template) / GCG GCG ATC CAG GCG GCG GCG GGC GCG GGT GCT GC
pNLS9, 10-R / GC AGC ACC CGC GCC CGC CGC CGC CTG GAT CGC CGC
1
Supplemental Table 4. Percentage (± SE) of transformed cells showing the three different patterns of nucleolar localization in various pNoLS mutants of RPL23aA (n=30, r=3).
Details of mutations / % Reduction in total basic charge / Wild type localization / Disrupted nucleolar localizationpI / Diffused / Ring / Nucleolar exclusion / Total / P
Wild type RPL23aA / 0 / 10.20 / 97 ± 1.9 / 0 / 3 ± 1.9 / 0 / 3
m-pNoLS1*** / 10 / 10.12 / 99 ± 1.0 / 0 / 1 ± 1.0 / 0 / 1 / 0.19
m-pNoLS2 / 7.5 / 10.14 / 87 ± 3.8 / 1 ± 1.0 / 11 ± 2.9 / 1 ± 1.0 / 13* / 0.04
m-pNoLS3 / 7.5 / 10.11 / 92 ± 4.0 / 1 ± 1.0 / 7 ± 3.8 / 0 / 8 / 0.18
m-pNoLS4 / 5 / 10.16 / 99 ± 1.0 / 0 / 0 / 1 ± 1.0 / 1 / 0.19
m-pNoLS5 / 7.5 / 10.14 / 97 ± 3.3 / 1 ± 1.0 / 0 / 2 ± 2.0 / 3 / 0.50
m-pNoLS6 / 5 / 10.16 / 98±1.0 / 1±1.0 / 1±1.0 / 0 / 2 / 0.32
m-pNoLS7 / 5 / 10.16 / 98 ± 1.0 / 0 / 2 ± 1.0 / 0 / 2 / 0.32
m-pNoLS8 / 5 / 10.16 / 90 ± 1.9 / 0 / 10 ± 1.9 / 0 / 10* / 0.04
m-pNoLS5 + 6 / 12.5 / 10.10 / 88 ± 4.0 / 0 / 6 ± 2.0 / 7 ± 1.9 / 12 / 0.06
m-pNoLS2 + 5 + 6 / 20 / 10.02 / 96 ± 2.9 / 0 / 2 ± 2.0 / 2 ± 1.0 / 4 / 0.38
m-pNoLS2 + 5 + 6 + 3 / 25 / 9.94 / 100 / 0 / 0 / 0 / 0 / 0.08
m-pNoLS2 + 5 + 6 + 3 + 4 / 30 / 9.87 / 54 ± 2.0 / 20 ± 3.8 / 19 ± 2.9 / 7 ± 1.9 / 46** / 6.83E-05
m-pNoLS2 + 5 + 6 + 3 + 4 + 7 / 35 / 9.79 / 29 ± 4.8 / 58 ± 4.8 / 11 ± 1.0 / 2 ± 1.0 / 71** / 1.00E-04
m-pNoLS2 + 5 + 6 + 3 + 4 + 7 + 1’ / 40 / 9.70 / 0 / 96 ± 1.0 / 1 ± 1.0 / 3 ± 1.9 / 100** / 4.7E-07
m-pNoLS2 + 5 + 6 + 3 + 4 + 7 + 1’ + 1’’
(m-pNoLS1’ + m-pNoLS1’’ = m-pNoLS1) / 45 / 9.57 / 0 / 12 ± 2.0 / 0 / 88 ± 2.0 / 100** / 4.7E-07
m-pNoLS2 + 5 + 6 + 3 + 4 + 7 + 1 + 8
[m-pNoLS (all)] / 50 / 9.40 / 0 / 0 / 0 / 100 / 100** / 4.7E-07
* and ** indicate the % cells showing disrupted nucleolar localization is significantly different from wild type at p < 0.05 and 0.01, respectively.
*** pNLS1 (10KKADPKAKALK20) was mutated in two parts. The first, pNLS1' 10KKAD13 was mutated to 10AAAD13. Using this mutant ORF as a template, pNLS1” 15KAKALK20 was mutated to 15AAAALA20.
1
Supplemental Table 5. Percentage (± SE) of transformed cells showing the three different patterns of nucleolar localization when only pNoLS 8, 7, 4, and 1 of RPL23aA were serially mutated or when pNoLSs 2, 3, 5, and 6 were serially mutated, in addition to pNoLS 8, 7, 4, and 1 (n=30, r=3).
Details of mutations / % Reduction in total basic charge / Wild type Localization / Disrupted nucleolar localizationpI / Diffused / Ring / Nucleolar exclusion / Total / P
Wild type RPL23aA / 0 / 10.20 / 97 ± 1.9 / 0 / 3 ± 1.9 / 0 / 3
m-pNoLS8 / 5 / 10.16 / 90 ± 1.9 / 0 / 10 ± 1.9 / 0 / 10* / 0.04
m-pNoLS8 + 7 / 10 / 10.12 / 94 ± 2.9 / 3 ± 3.0 / 2 ± 2.0 / 0 / 6 / 0.28
m-pNoLS8 + 7 + 4 / 15 / 10.07 / 99 ± 1.0 / 1 ± 1.0 / 0 / 0 / 1 / 0.19
m-pNoLS8 + 7 + 4 + 1’ / 20 / 10.02 / 93 ± 0 / 0 / 7 ± 0 / 0 / 7 / 0.08
m-pNoLS8 + 7 + 4 + 1’ + 1’’ / 25 / 9.97 / 94 ± 2.0 / 4 ± 2.9 / 1 ± 1.0 / 0 / 6 / 0.24
m-pNoLS8 + 7 + 4 + 1’ + 1’’ + 2 / 32.5 / 9.86 / 0 / 71 ± 2.9 / 11 ± 2.9 / 18 ± 2.9 / 100** / 4.7E-07
m-pNoLS8 + 7 + 4 + 1’ + 1’’ + 2 + 3 / 37.5 / 9.75 / 0 / 92 ± 4.0 / 0 / 8 ± 4.0 / 100** / 4.7E-07
m-pNoLS8 + 7 + 4 + 1’ + 1’’ + 2 + 3 + 5 / 45 / 9.57 / 0 / 0 / 0 / 100 / 100** / 4.7E-07
m-pNoLS8 + 7 + 4 + 1’ + 1’’ + 2 + 3 + 5 + 6 [m-pNoLS (all)] / 50 / 9.40 / 0 / 0 / 0 / 100 / 100** / 4.7E-07
132KKAYVRL138 to 132AAAAAAA138 / 7.5 / 10.14 / 6± 2.2 / 89 ± 2.9 / 5 ± 1.1 / 0 / 94** / 3.23E-06
* and ** indicate the % cells showing disrupted nucleolar localization is significantly different from wild type at p < 0.05 and 0.01, respectively.
Supplemental Table 6. Percentage (± SE) of transformed cells showing the three different patterns of nucleolar localization when different segments of RPL23aA were deleted (n=30, r=3). N∆29 = deletion of N-terminal 29 amino acids. Mid 91 = fragment spanning amino acid residues 30 to 120. C∆34 = deletion of C-terminal 34 amino acids. C∆64 = deletion of C-terminal 64 amino acids.
Details of mutation / pNoLSs deleted / Wild type localization / Disrupted nucleolar localizationpI / Diffused / Ring / Nucleolar exclusion / Total / P
Wild type RPL23aA / 0 / 10.20 / 97 ± 1.9 / 0 / 3 ± 1.9 / 0 / 3
N∆29 / 1 / 10.14 / 100 / 0 / 0 / 0 / 0 / 0.08
Mid 91 / 1, 7, 8 / 10.24 / 0 / 9 ± 2.0 / 87 ± 3.3 / 4 ± 2.9 / 100** / 4.7E-07
C∆34 / 7, 8 / 10.30 / 4 ± 2.0 / 24 ± 11.8 / 70 ± 12.0 / 1 ± 1.0 / 96** / 3.08E-06
C∆64 / 5, 6, 7, 8 / 10.40 / 2 ± 2.0 / 62 ± 14.9 / 36 ± 15.7 / 0 / 98** / 2.8E-06
** indicates the % cells showing disrupted nucleolar localization is significantly different from wild type at p < 0.01.
1
Supplemental Table 7. Fluorescence intensities (arbitrary units 0-255) of RPL23aA-GST-EGFP and RPL23aA-m-pNoLS (all)-GST-EGFP. n = 10, r = 3.
Fluorescent fusion protein / Fluorescence intensity of EGFP fusionslaser strength = 10%, laser output = 100%, detector gain = 900, pinhole = 3µm / Fluorescence intensity of Co-expressed 35S-mRFP
laser strength = 10%, detector gain = 700, pinhole = 3µm / Relative fluorescence intensity
(fluorescence intensity of EGFP fusions/fluorescence intensity of 35S-mRFP)
Nucleus / Cytoplasm / Nucleus / Cytoplasm / Nucleus / Cytoplasm
RPL23aA-GST-EGFP / 47±5.4 / 33±3.0 / 103±0.7 / 57±2.7 / 0.46 / 0.58
RPL23aA-m-pNoLS (all)-GST-EGFP / 190±22.9 / 159±12.2 / 97±11.0 / 60±4.5 / 1.96 / 2.67
Supplemental Table 8. Fluorescence intensities of GST-EGFP, ΔGST-GST-EGFP and RPL23aA-m-pNoLS (all)-GST-EGFP. n = 10, r = 3.
Fluorescent fusion protein / Nuclear fluorescence intensity of EGFP fusionslaser strength = 10%, laser output = 100%, detector gain = 900, pinhole = 3µm / Nuclear fluorescence intensity of Co-expressed 35S-mRFP
laser strength = 10%, detector gain = 700, pinhole = 3µm / Relative fluorescence intensity
(fluorescence intensity of EGFP fusions/fluorescence intensity of 35S-mRFP)
GST-EGFP / 209±8.1 / 107±14.7 / 1.95
ΔGST-GST-EGFP / 36±3.0 / 156±12.3 / 0.23
RPL23aA-m-pNoLS (all)- GST-EGFP / 190±22.9 / 97±11.0 / 1.96
1
Supplemental Table 9. Fluorescence intensities of RPL15A-GST-EGFP and RPL15A- m-pNLSs-GST-EGFP. n = 10, r = 3.
Fluorescent fusion protein / Cytoplasmic fluorescence intensitylaser strength = 20%, laser output = 100%, detector gain = 1000, pinhole = 3µm / Nuclear and nucleolar fluorescence intensity
laser strength = 5%, laser output = 100%, detector gain = 700, pinhole = 3µm
Nucleus / Nucleolus
RPL15A-GST-EGFP / 158±11.2 / 80±9.5 / 253±1.0
RPL15A-m-pNLS1-GST-EGFP / 139±11.3 / 51±9.1 / 198±12.2
RPL15A-m-pNLS1, 2-GST-EGFP / 183±15.8 / 27±13.0 / 112±4.5
RPL15A-m-pNLS3-GST-EGFP / 188±14 / 62±4.0 / 162±2.1
Supplemental Table 10. Fluorescence intensities of RPS8A-GST-EGFP and RPS8A- m-pNLS2-GST-EGFP. n = 6, r = 3.
Fluorescent fusion protein / Cytoplasmic fluorescence intensitylaser strength = 10%, laser output = 100%, detector gain = 900, pinhole = 3µm / Nuclear and nucleolar fluorescence intensity
laser strength = 10%, laser output = 100%, detector gain = 900, pinhole = 3µm
Nucleus / Nucleolus
RPS8A-GST-EGFP / 73±1.9 / 45±2.1 / 213±4.1
RPS8A-m-pNLS2-GST-EGFP / 54±7.2 / 41±4.2 / 64±2.1
1
Supplemental Table 11. Oligos used for functional analysis of pNLS/pNoLSs (restriction sites are in bold and underlined)
Name of oligos / Sequence / Restriction siteSV40 NLS-1*-Sense / AATTC ATG CCC AAG AAG AAG CGG AAG GTG A
/ EcoRI
SV40 NLS-Antisense / AGCTT CAC CTT CCG CTT CTT CTT GGG CAT G / HindIII
SV40 NLS-2*-Sense / GATCC CCC AAG AAG AAG CGG AAG GTG A / BamHI
RPL23aA-4 pNoLSs (pNoLS 2, 3, 5, 6)-Sense / AATTC ATG AAG AAA GAC AAA AAG ATT AGG AAG AAA ATT AAG AAA AAG G / EcoRI
RPL23aA-4 pNoLSs (pNoLS 2, 3, 5, 6)-Antisense / GATCC CTT TTT CTT AAT TTT CTT CCT AAT CTT TTT GTC TTT CTT CAT G / BamHI
RPL23aA-4 pNoLSs+pRBS** (pNoLS 2, 3, 5, 6)-Sense / AATTC ATG AAG AAA GAC AAA AAG ATT AGG AAG AAA ATT AAG AAA AAG AAA AAG GCT TAC GTG AGG CTT G / EcoRI
RPL23aA-4 pNoLSs+pRBS** (pNoLS 2, 3, 5, 6)-Antisense / GATCC AAG CCT CAC GTA AGC CTT TTT CTT TTT CTT AAT TTT CTT CCT AAT CTT TTT GTC TTT CTT CAT G / BamHI
RPL23aA-8 pNoLSs-Sense / AATTC ATG AAG AAA GCT GAT AAG GCC TTG AAA AAG AAA GAC AAA AAG ATT AGG AAG
AAA AAG AAA ATT AAG AAA AAG AAA AAG AAA AAG G / EcoRI
RPL23aA-8 pNoLSs-Antisense / GATCC CTT TTT CTT TTT CTT TTT CTT AAT TTT CTT TTT CTT CCT AAT CTT TTT GTC TTT CTT TTT CAA GGC CTT ATC AGC TTT CTT CAT G / BamHI
RPL23aA-8 pNoLSs+pRBS-Sense / AATTC ATG AAG AAA GAT AAG TTG AAA AAG AAA GAC AAA AAG ATT AGG AAG AAA AAG AAA ATT AAG AAA AAG AAA AAG AAA AAG GCT TAC GTG AGG CTT G / EcoRI
RPL23aA-8 pNoLSs+pRBS-Antisense / GATCC AAG CCT CAC GTA AGC CTT TTT CTT TTT CTT TTT CTT AAT TTT CTT TTT CTT CCT AAT CTT TTT GTC TTT CTT TTT CAA CTT ATC TTT CTT CAT G / BamHI
RPL15A-pNLSs-sense / AATTC ATG AGG AAG AAA AAG GCT CGT AGA CGT GTG AGA CGT G / EcoRI
RPL15A-pNLSs-antisense / GATCC ACG TCT CAC ACG TCT ACG AGC CTT TTT CTT CCT CAT G / BamHI
RPS8A-pNLSs-sense / AATTC ATG AAG AGG CGT AGG AAG AAA CGA AAG G / EcoRI
RPS8A-pNLSs-antisense / GATCC CTT TCG TTT CTT CCT ACG CCT CTT CAT G / BamHI
* SV40 NLS-1 was used to generate SV40 NLS-EGFP (control) and SV40 NLS-2 was used to generate pNLS/pNoLSs-SV40 NLS-EGFP construct. SV40 NLS-Antisense oligo was used for both constructs.
** pRBS = putative rRNA binding site
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