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1 Extensive cargo identification reveals distinct biological roles of the 12 pathways 2 3 Makoto Kimura1,*, Yuriko Morinaka1, Kenichiro Imai2,3, Shingo Kose1, Paul Horton2,3, and Naoko 4 Imamoto1,* 5 6 1Cellular Dynamics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan 7 2Artificial Intelligence Research Center, and 3Biotechnology Research Institute for Drug Discovery, 8 National Institute of Advanced Industrial Science and Technology (AIST), AIST Tokyo Waterfront 9 BIO-IT Research Building, 2-4-7 Aomi, Koto-ku, Tokyo, 135-0064, Japan 10 11 *For correspondence: [email protected] (M.K.); [email protected] (N.I.) 12 13 Editorial correspondence: Naoko Imamoto 14

1 15 Abstract 16 Vast numbers of are transported into and out of the nuclei by approximately 20 species of 17 importin-β family nucleocytoplasmic transport receptors. However, the significance of the multiple 18 parallel transport pathways that the receptors constitute is poorly understood because only limited 19 numbers of cargo proteins have been reported. Here, we identified cargo proteins specific to the 12 20 species of import receptors with a high-throughput method that employs stable isotope 21 labeling with amino acids in cell culture, an in vitro reconstituted transport system, and quantitative 22 mass spectrometry. The identified cargoes illuminated the manner of cargo allocation to the 23 receptors. The redundancies of the receptors vary widely depending on the cargo . Cargoes 24 of the same are functionally related to one another, and the predominant protein groups in 25 the cargo cohorts differ among the receptors. Thus, the receptors are linked to distinct biological 26 processes by the nature of their cargoes. 27

2 28 Introduction 29 In interphase cells, proteins and migrate into and out of the nuclei through the central 30 channels of the nuclear pore complexes (NPC) embedded in the nuclear envelope. These nuclear 31 pores are lined with FG-repeat domains that constitute a permeability barrier, and only 32 macromolecules that reversibly interact with the FG-repeats can permeate this barrier (Schmidt and 33 Gorlich, 2016). One such group of proteins, the importin (Imp)-β family proteins, are 34 nucleocytoplasmic transport receptors (NTRs) that primarily carry nuclear proteins and small RNAs 35 as their cargoes through the nuclear pores, although non-importin family NTRs also act depending 36 on the cargo and physiological conditions (Kose et al., 2012; Lu et al., 2014; Weberruss et al., 2013). 37 The encodes twenty species of Imp-β family NTRs, of which ten [Imp-β, 38 transportin (Trn)-1, -2, -SR (-3), Imp-4, -5 (RanBP5), -7, -8, -9, and -11] are nuclear import 39 receptors, seven [exportin (Exp)-1 (CRM1), -2 (CAS/CSE1L), -5, -6, -7, -t, and RanBP17] are 40 export receptors, two (Imp-13 and Exp-4) are bi-directional receptors, and the function of RanBP6 41 is undetermined (Kimura and Imamoto, 2014). These NTRs constitute multiple parallel transport 42 pathways. The basic mechanism of directional transport was elucidated in the early years (Gorlich 43 and Kutay, 1999), but even today, the number of NTR-specific cargoes that has been reported is 44 surprisingly small, hindering a biological understanding of the nucleocytoplasmic transport system. 45 NTRs are thought to transport specific cohorts of cargoes by binding to specific sites on 46 those cargoes (Chook and Suel, 2011), but the consensus structures of the NTR-binding sites have 47 been established for only a few NTRs (Soniat and Chook, 2015) as follows: the classical nuclear 48 localization signal (cNLS) for the Imp-α family adapters, which connects Imp-β and cargoes (Lange 49 et al., 2007); PY-NLS for Trn-1 and -2 (Lee et al., 2006; Suel et al., 2008); the nuclear export signal 50 (NES) for Exp-1 (Hutten and Kehlenbach, 2007); the SR-rich domain that binds to Trn-SR 51 (Kataoka et al., 1999; Maertens et al., 2014); and Lys-rich NLS (IK-NLS) for Kap121p 52 (Imp-5 homolog; Kobayashi and Matsuura, 2013; Kobayashi et al., 2015). The β-like 53 importin-binding (BIB) domain is another NTR- (Jakel and Gorlich, 1998), but its 54 consensus sequence and NTR specificity remain obscure. Among the NTRs, Imp-β exclusively uses 55 one of the seven species of the Imp-α family of proteins as an adapter for cargo binding, and many 56 Imp-α/β cargoes have been reported, although Imp-β also directly binds to cargoes (Goldfarb et al., 57 2004). Among the import receptors, Trn-1 and its closest homolog Trn-2 have the second-highest

3 58 number of cargoes reported thus far, and the PY-NLS motif has been defined, although in some 59 cases the motif is difficult to recognize because of sequence diversity and structural disorder is 60 another requisite (Soniat and Chook, 2015 and 2016). For the cargoes of other NTRs, the consensus 61 structures of NTR-binding sites have hardly been derived because only limited numbers of cargoes 62 have been reported, including Imp-β-direct cargoes. 63 There are many reports on the differential spatiotemporal expression of Imp-β family 64 NTRs, including tissue specificities in (Quan et al., 2008), developmental or spermatogenic 65 stage specificities in mice (Major et al., 2011; Quan et al., 2008), and tissue or response specificities 66 in plants (Huang et al., 2010). Expression regulation is not only transcriptional but also 67 miRNA-mediated (Li et al., 2013; Szczyrba et al., 2013) or locally translationally mediated (Perry 68 and Fainzilber, 2009). Additionally, the NTRs are functionally regulated by protein modifications 69 (Wang et al., 2009), inhibitory factors (Lieu et al., 2014), and specific anchorings (Makhnevych et 70 al., 2003). These nucleocytoplasmic transport regulations must significantly influence cellular 71 physiology, and their significance may be elucidated if the affected cargoes can be specified. Indeed, 72 in previous studies, NTR regulations have been linked to cellular responses through the functions of 73 specific cargoes. For example, in prostate cells treated with a cinnamaldehyde derivative, the 74 expression of Imp-7 and the factor Egr1 are induced, and the Egr1 imported by Imp-7 75 activates apoptotic transcription (Kang et al., 2013). In another example, when the nuclear 76 import of some ribosomal proteins (RPs) is inhibited by the repression of Imp-7 expression, other 77 unassembled RPs restrain the negative regulator of and thereby activate p53 to inhibit 78 (Golomb et al., 2012). Additionally, the inhibition of Trn-2 by the caspase-generated 79 HuR (ELAVL1) fragment is crucial for the cytoplasmic retention of full-length HuR, which induces 80 myogenesis (Beauchamp et al., 2010) or staurosporine-induced (von Roretz et al., 2011). 81 In many other studies, mutations of particular NTR in model organisms, including yeast, flies, 82 and plants, have resulted in defects in specific biological processes (Kimura and Imamoto, 2014). 83 Thus, each NTR has its own inherent biological significance. However, the details of the molecular 84 processes are largely uncharacterized because the responsible cargoes have not been identified. If 85 we could identify more cargoes, further studies of cellular regulation by nucleocytoplasmic 86 transport would be possible. 87 We previously established a method for identifying the cargoes of a nuclear import

4 88 receptor called SILAC-Tp (Kimura et al., 2013a; Kimura et al., 2013b; Kimura et al., 2014). 89 SILAC-Tp employs stable isotope labeling with amino acids in cell culture (SILAC) (Ong et al., 90 2002), an in vitro reconstituted nuclear transport system (Adam et al., 1990), and quantitative mass 91 spectrometry. A recent advancement of the Orbitrap mass spectrometer drastically increased the 92 identified and quantified protein numbers, and this advancement has been successfully applied to 93 other cargo identification methods (Kirli et al., 2015; Thakar et al., 2013). Here, we utilized this 94 advancement for the SILAC-Tp method and identified import cargoes of all 12 NTRs, of which 10 95 are import and 2 are bi-directional receptors. Our results illustrate the basic framework and the 96 biological significance of the nucleocytoplasmic transport pathways. 97 98 Results and Discussion

99 SILAC-Tp effectively identifies cargoes 100 SILAC-Tp employs an in vitro nuclear transport system, and all 12 NTRs import their reported 101 specific cargoes in this system (Figure 1–figure supplement 1C). The transport system consists of 102 permeabilized HeLa cells labeled with “heavy” amino acids by SILAC, unlabeled HeLa nuclear 103 extract depleted of Imp-β family NTRs and RCC1, unlabeled HeLa cytosolic extract depleted of 104 Imp-β family NTRs, one species of recombinant NTR, p10/NTF2, and an ATP regeneration system. 105 Unlabeled “light” proteins in the nuclear extract are imported into the nuclei of the permeabilized 106 cells. Simultaneously, a control reaction without the NTR is performed. Next, the proteins are 107 extracted from the nuclei and identified and quantified by LC-MS/MS. The recipient nuclei contain 108 both the imported and endogenous proteins, and the ratio of the imported to the endogenous fraction 109 of a protein is calculated as the unlabeled/labeled or light/heavy (L/H) ratio. The quotient of the L/H

110 ratios with the NTR (+NTR) and without it (control), i.e., (L/H+NTR)/(L/HCtl), of a protein is defined 111 as the +NTR/Ctl value and is used as the index for cargo potentiality. 112 In one run of SILAC-Tp (control or +NTR), approximately 2,500 to 4,000 proteins were 113 identified, and the L/H ratios of 1,700 to 3,100 proteins were quantified. To calculate the +NTR/Ctl 114 value, one protein has to be quantified in both the control and +NTR reactions, and we discarded

115 L/H+NTR values that lacked the counterpart L/HCtl values. We performed three replicates of 116 SILAC-Tp for each of the 12 NTRs. In the three replicates, 1,235 to 1,671 proteins were assigned 117 with +NTR/Ctl values three times, and 364 to 502 proteins were assigned only twice

5 118 (Supplementary file 1). We did not consider proteins with single +NTR/Ctl values, although a 119 protein with only a single but high +NTR/Ctl value may still be a cargo (see below). To normalize

120 the index values of the three replicates, the Z-scores of the log2(+NTR/Ctl) were calculated within 121 each replicate (Figure 1–figure supplement 2A and 2B). Ranking the proteins that have three 122 +NTR/Ctl values by the median of the three Z-scores may reasonably sort the candidate cargoes. 123 However, if the lower Z-score of a protein with only two +NTR/Ctl values is higher than the 124 median Z-scores of those candidate cargoes, the protein may also be a candidate cargo. Thus, we 125 ranked the proteins by the second (the lower of the two or the middle of the three) Z-scores, and 126 termed the result the 2nd-Z-ranking (Supplementary file 1). 127 To define the border that separates candidate cargoes from other proteins in the 128 2nd-Z-ranking, we first reviewed the distribution of reported Trn-1 cargoes in the Trn-1 129 2nd-Z-ranking because many Trn-1 cargoes have been reported. For an unbiased evaluation, we 130 employed the lists of cargoes consolidated by other researchers (Chook and Suel, 2011). 131 Twenty-seven reported cargoes were included in the 2nd-Z-ranking (totaling 1,649 proteins; 132 Supplementary file 1, Trn-1 “Report and feature”). We calculated the reported cargo rates (to serve 133 as a proxy for precision), recall, and Fisher’s exact test p-values for rank cutoffs in increments of 134 1%. Computing reported cargo rate requires deciding which candidate cargoes should be considered 135 as false positives. Since a gold standard set of definitely non-cargo proteins is not available, it is not 136 clear which previously unreported cargoes should be counted as false positives, and which, if any, 137 should be discarded as unclear. Therefore we estimated reported cargo rates in two ways, (i) treating 138 all of the 1622 proteins not reported as cargoes as negative examples (Figure 1-figure supplement 139 3A and Figure 1-source data file 1A); and (ii) discarding proteins with undetermined or nuclear 140 subcellular localization according to Uniprot annotation, and treating the remaining 259 non-nuclear 141 proteins as negative examples (Figure 1-figure supplement 3A and Figure 1-source data file 1B). In 142 the former case, the reported cargo rate corresponds to a lower bound on the precision, and even in 143 the latter case the reported cargo rates are expected to underestimate precision, because almost 144 certainly some of the proteins that we exclude as unclear are in fact true cargoes. 145 To select cargoes with high sensitivity, we employed the cutoff of 15% that yields a high 146 recall of 0.741 (Figure 1-figure supplement 3B and Figure 1-source data file 1A and 1B; recall is 147 not affected by the assumptions of negative examples). Among the 27 reported cargoes, 20 cargoes

6 148 were ranked in the top 15% (247 proteins; p=5.39 × 10-12 by Fisher’s exact test), and the others were 149 dispersed in the lower ranks (Figures 1A and 2A; Figure 1–figure supplement 2C and 2E). 150 We examined the direct binding of Trn-1 to a subset of proteins in the 2nd-Z-ranking 151 using a bead halo assay (Patel and Rexach, 2008) (Supplementary file 2) in which the binding of 152 GFP-fusion proteins to GST-Trn-1 on glutathione-Sepharose beads was observed by fluorescence 153 microscopy. If RanGTP (a Q69L GTP-fixed mutant) (Bischoff and Ponstingl, 1995) inhibits the 154 protein–Trn-1 binding, the functionality of the binding is verified. For all the bead halo assays in 155 this work, we principally selected well-characterized proteins that have not been reported as cargoes 156 from (i) proteins ranked high (within the top 15% in the 2nd-Z-ranking or 4% in the 3rd-Z-ranking, 157 see below), around presumptive cutoffs (within about top 15–25% in the 2nd-Z-ranking), or lower 158 and (ii) highly ranked proteins that are suspected as indirect cargoes or false positives based on their 159 well-known features, e.g., S100A6 or EEF1A2 (see the legend of Supplementary file 2). The 160 negative rate of the bead halo assays should be higher than the true overall false positive rate of the 161 SILAC-Tp, because proteins in (ii) are selected preferentially. Seventeen novel candidate cargoes in 162 the top 266 (top 16%) bound to Trn-1, and RanGTP inhibited the binding (Figure 1A; 163 Supplementary file 1, Trn-1 “Direct binding”; Supplementary file 2). Although the assays were not 164 comprehensive, many of the highly ranked proteins are bona fide Trn-1 cargoes. The highly ranked 165 proteins that did not bind to Trn-1 in the assays are still candidate indirect cargoes that may form 166 complexes with other proteins that directly bind to Trn-1 (see the case of POLE3 for Imp-13 below). 167 As an example of a protein with only a single but high Z-score (+NTR/Ctl value), DIMT1 bound to 168 Trn-1 (DHRS4 with Imp-β is another example), but we did not consider such proteins. 169 Because many reported Trn-1 cargoes carry PY-NLSs, we examined the distribution of 170 PY-NLS motif-containing proteins in the 2nd-Z-ranking (Figure 1B). The percentages of PY-NLS 171 motif-containing proteins within a window width of 50 positions were higher in the range of the top 172 200, indicating a higher rate of PY-NLS motif-containing proteins within the top 250 (top 15%). 173 The reported Trn-1 cargoes were similarly distributed in the Trn-2 2nd-Z-ranking (Supplementary 174 file 1, Trn-2 “Report or feature”). Because Trn-1 and -2 share nearly the same reported cargoes 175 (Twyffels et al., 2014), this result demonstrates the reproducibility of the SILAC-Tp method. Based 176 on these evaluations, we assumed that the proteins in the top 15% (247 proteins) of the 177 2nd-Z-ranking are candidate cargoes with high sensitivity (0.741) and termed them the 2nd-Z-15%

7 178 cargoes. 179 Next, we examined whether the cutoff employed for Trn-1 is applicable to Imp-13 and 180 Trn-SR whose 2nd-Z-rankings include several reported cargoes. The Imp-13 2nd-Z-ranking 181 (totaling 2,060 proteins) includes eight reported cargoes (Supplementary file 1, Imp-13), and seven 182 of these are ranked in the top 244 (top 12%; p=2.83 × 10-7; Figure 2B; Figures 2–figure supplement 183 1A and 2A). In bead halo assays for a subset of the ranked proteins, 24 novel candidate cargoes in 184 the top 326 (top 16%) bound directly to Imp-13, and RanGTP inhibited the binding (Figure 2-figure 185 supplement 2A; Supplementary file 1, Imp-13; Supplementary file 2). One component of a reported 186 cargo complex, i.e., POLE3, did not bind to Imp-13, but its binding partner CHRAC1 (Walker et al., 187 2009) did. Thus, the binding partners of the direct cargoes are also ranked high. Many reported 188 Trn-SR cargoes are SR-domain proteins (Chook and Suel, 2011), and they can be grouped into 189 either SR-rich splicing factors (SFs) or other SR-domain proteins. The Trn-SR 2nd-Z-ranking 190 (totaling 2,021 proteins) contains three reported cargoes (Supplementary file 1, Trn-SR), and they 191 are ranked in the top 55 (top 3%; p=1.91 × 10-5; Figure 2C; Figure 2–figure supplement 2B). The 192 2nd-Z-ranking contains seven SR-rich SFs other than the reported SFs, and five of these are ranked 193 in the top 90 (top 4%; p=7.61 × 10-18). The 2nd-Z-ranking also contains another four proteins that 194 are annotated with “RS-domain” in UniProt, and three of these are ranked in the top 202 (top 10%; 195 p=3.65 × 10-3). Finally, in bead halo assays for a subset, 11 novel candidate cargoes in the top 237 196 (top 12%) bound directly to Trn-SR, and RanGTP inhibited the binding (Figure 2-figure supplement 197 2B; Supplementary file 1, Trn-SR; Supplementary file 2). Hence, the 2nd-Z-15% cargoes could also 198 be defined for Imp-13 (309 proteins) and Trn-SR (302 proteins), and we applied this cutoff to the 199 other NTRs that have few reported cargoes. The 2nd-Z-15% cargoes of the 12 NTRs are presented 200 in Supplementary file 3. Some of the 2nd-Z-15% cargoes with low numbers of L/H counts showed 201 deviation in Z-scores or L/H ratios in the three replicates of SILAC-Tp (Supplementary file 1), and 202 an example of their quantitation qualities is presented in Supplementary file 4. 203 Exceptionally, Imp-β uses Imp-α as an adaptor for cargo binding, and the cytosolic 204 extract used for the transport system contained endogenous Imp-α. Four Imp-αs were found in the 205 Imp-β 2nd-Z-ranking (totaling 2,027 proteins), and three of these are in the 2nd-Z-15% cargoes 206 (p=1.19 × 10-2; Supplementary file 1, Imp-β; Supplementary file 3). Thus, the Imp-β candidate 207 cargoes must include both Imp-β-direct and Imp-α-dependent cargoes. Indeed, 31 proteins in the top

8 208 276 (top 14%) bound directly to Imp-α, -β, or both in the bead halo assays (Supplementary file 1, 209 Imp-β; Figure 2–figure supplement 3; Supplementary file 2). The border for the Imp-β candidate 210 cargoes can be relaxed because Imp-β imports more cargoes than other NTRs with the help of 211 Imp-α. Indeed, in the bead halo assays, many proteins in the top 35% of the 2nd-Z-ranking bound to 212 Imp-α, although most of the proteins that bound directly to Imp-β were ranked in the top 259 (13%). 213 Here, we employed the Imp-β 2nd-Z-15% cargoes (303 proteins) to enable equal comparisons with 214 the cargoes of other NTRs. 215 216 Cargo selection with higher specificity 217 Deviation of the LC-MS/MS quantification within the three replicates complicates cargo selection. 218 However, the Z-scores of the highly ranked reported cargoes were reasonably high in all the three 219 replicates possibly because many of the reported cargoes are abundant proteins that seldom produce 220 outliers in quantification (Figure 1–figure supplement 2; Figure 2–figure supplement 1). To select 221 proteins that have high Z-scores in all the three replicates, we next ranked the proteins that had three 222 +NTR/Ctl values by the third (lowest) Z-scores (3rd-Z-ranking). The reported cargo rates, recall, 223 and p-values were calculated in 1% rank increments under two assumptions similarly to the case of 224 2nd-Z-ranking (Figure 1-figure supplement 3A and 3B and Figure 1-source data file 1C and 1D). 225 The reported cargo rate calculated under the assumption that proteins annotated with non-nuclear 226 localization (178 proteins) are negative examples is as high as 0.85 at the cutoff of top 4% (Figure 227 1-figure supplement 3A and Figure 1-source data file 1D). The Trn-1 3rd-Z-ranking (totaling 1,235 228 proteins) included 25 reported cargoes, and 17 of these were ranked in the top 37 (top 3%; p=1.67 × 229 10-22; Figures 1A and 2A; Figure 1–figure supplement 2D and 2F; Supplementary file 1). Seven 230 proteins in the top 47 (top 4%) were novel Trn-1-direct cargoes that were verified in the bead halo 231 assays (Figures 1A and 2A; Supplementary files 1 and 2). The percentage of PY-NLS 232 motif-containing proteins within a window width of 50 positions was highest at the first position 233 (Figure 1B), indicating that PY-NLS motif-containing proteins are concentrated in the top 50 (top 234 4%). Thus, most of the proteins that ranked in the top 4% (49 proteins) of the 3rd-Z-ranking are 235 highly reliable cargoes, and we termed these proteins the 3rd-Z-4% cargoes. In a comparison 236 between the Trn-1 2nd-Z-15% and 3rd-Z-4% cargoes, most of the 3rd-Z-4% cargoes were also 237 2nd-Z-15% cargoes (Figure 1A). Some reported or newly identified cargoes in the 2nd-Z-15%

9 238 cargoes were ranked lower in the 3rd-Z-ranking due to the deviations in the 3rd Z-scores. 239 In the Imp-13 3rd-Z-ranking (totaling 1,671 proteins), seven proteins were reported 240 cargoes, and six of these were ranked in the top 58 (top 3%; p=9.20 × 10-9; Figure 2B; Figure 2– 241 figure supplements 1B and 2A; Supplementary file 1). Additionally, the 3rd-Z-4% cargoes (66 242 proteins) included eight novel cargoes that directly bound to Imp-13 (Figure 2B; Figure 2–figure 243 supplement 2A; Supplementary files 1 and 2). In the Trn-SR 3rd-Z-ranking (totaling 1,591 proteins), 244 both of the two reported cargoes were ranked in the top 18 (top 1%; p=1.21 × 10-4), four of the five 245 other SR-rich SFs were in the top 45 (top 3%; p=2.74 × 10-6), one of the three SR-domain proteins 246 (other than the SR-rich SFs) was ranked 63rd (top 4%; p=0.11), and six novel cargoes within the top 247 4% (63 proteins) bound directly to Trn-SR (Figure 2C; Figure 2–figure supplement 2B; 248 Supplementary files 1 and 2). In cases of both Imp-13 and Trn-SR, the proteins were replaced 249 between the 2nd- and 3rd-Z-rankings in a manner similar to the case for Trn-1. We concluded that 250 the 3rd-Z-4% criteria is highly specific including few false positives, albeit at the cost of losing 251 many genuine cargoes. Hence, we employed the 3rd-Z-4% cargoes mainly for the characterization 252 of the identified cargoes, whereas the 2nd-Z-ranking was used for the evaluation of the import 253 efficiencies of the expected cargoes. The 3rd-Z-4% cargoes of the 12 NTRs are presented in Figure 254 3. 255 256 Redundancy in the import pathways 257 A total of 468 proteins were identified as 3rd-Z-4% cargoes of the 12 NTRs, and 332 of these are 258 unique to one NTR, which clearly reflects the division of roles among the NTRs (Supplementary 259 file 5B). Another 136 proteins were shared by two to seven NTRs, and the mean number of shared 260 cargoes between two NTRs was 4.8. In the maximum-likelihood phylogenetic tree of the 12 NTRs 261 (Figure 4A), Trn-1 and -2 (84% sequence identity) are paired most closely, and Imp-7 and -8 (65% 262 identity) are the second-most closely paired. These paired NTRs share 28 and 19 cargoes, 263 respectively, and they are paired similarly in a hierarchical clustering based on the cargo profiles 264 (Figure 4B and 4C). The other NTRs that were paired weakly in the phylogenetic tree, namely, 265 Imp-13 and Trn-SR (23% identity), Imp-4 and -5 (22% identity), and Imp-9 and -11 (19% identity), 266 did not form the same pairs when clustering by their cargoes. Thus, the NTR–cargo interactions are 267 conserved only within the highly homologous NTRs. The 2nd-Z-15% cargoes included as many as

10 268 1,416 proteins in total, 827 of which are shared by two to 12 NTRs, and 589 are unique to one NTR 269 (Supplementary file 5A and 5D). Imp-7 and -8 share the largest number (162) among the 270 2nd-Z-15% cargoes, but Trn-1 and -2 share no more than the other pairs. Of the 247 Trn-1 and 246 271 Trn-2 2nd-Z-15% cargoes, 69 are shared, and 36 of these are ranked within the top 50 in either 272 ranking. Thus, Trn-1 and -2 still share many highly ranked cargoes but few lower ranked cargoes 273 within the top 15%. The import efficiency of a cargo may differ between Trn-1 and -2, and only one 274 of Trn-1 or -2 may import inefficient cargoes that are ranked lower. 275 276 Division of roles among the NTRs 277 Because NTR-dependent transport is regulated, a cargo cohort of an NTR must be imported 278 simultaneously and act cooperatively. To explore the roles of the NTR cargoes, the 3rd-Z-4% and 279 2nd-Z-15% cargoes of each NTR were analyzed for enrichment of (GO) terms 280 (Gene Ontology Consortium, 2015) using g:Profiler (Reimand et al., 2016). For all the 281 combinations of a GO term and an NTR, the number of cargoes annotated with the term and the 282 significance (p-values according to g:SCS) of the term enrichment are listed (Supplementary files 283 6B, 6C, 7, and 8). Depending on the hierarchy of the GO terms, the terms are significantly 284 annotated (p<0.05) to the cargo cohorts of none to 12 of the NTRs. Broader terms with smaller term 285 depths are linked to more NTRs, whereas more defined terms with larger term depths are linked to 286 fewer NTRs. Indeed, all 12 of the NTRs are linked to many broad terms, although the cargo 287 numbers and the significances vary widely. Because similar terms were listed redundantly, we 288 selected representative GO terms from those enriched significantly (p<0.05) for the 3rd-Z-4% 289 cargoes of the 12 NTRs and tabulated the correspondences between the cargoes and the annotated 290 terms (Supplementary file 9). To compare the GO terms that are specifically linked to each NTR, 291 we listed the terms that are enriched significantly for the cargoes of four or fewer NTRs (Figures 5 292 and 6). Here again we extracted the representative terms to decrease the size of the list. The selected 293 terms for the 3rd-Z-4% cargoes plainly exhibit the roles of the cargo cohorts. For example, 294 significant numbers of Imp-4, -7 and Exp-4 cargoes are annotated with DNA recombination or DNA 295 conformation (geometric) change (Figure 5; Supplementary file 9), which are terms for biological 296 processes (BPs). These cargoes are also annotated with chromatin, which is a term for cellular 297 component (CC; Figure 6A; Supplementary file 9), and DNA binding, which is for molecular

11 298 function (MF; Figure 6B; Supplementary file 9), all related to DNA recombination and DNA 299 conformation change. For another example, the Trn-SR cargoes are significantly annotated with a 300 range of terms for BPs that are related to cell division or nuclear division and terms for CCs that 301 include condensed , kinetochore, spindle, and . Similarly, most of the 302 examined NTRs are linked to terms for BPs via the 3rd-Z-4% cargoes (Figure 5) as follows: Imp-β, 303 -4, -7, and Trn-SR are linked to chromatin or chromosome organization; Imp-β, -4, and -13 are 304 linked to DNA repair; Imp-β is linked to mRNA capping; Trn-SR and Exp-4 are linked to mRNA 305 ; Trn-1 and -2 are linked to mRNA stabilization; Trn-2, -SR, Imp-5, -9, and Exp-4 306 are linked to biogenesis or rRNA processing; Trn-SR is linked to , 307 modification, ubiquitination, and catabolic process; and Imp-4 and -7 are linked to apoptosis. The 308 NTRs are also consistently linked to the terms for CCs and MFs (Figure 6) as follows: Trn-SR, 309 Imp-5, -9, and Exp-4 are linked to Cajal body; Imp-β is linked to cap-binding complex; and Trn-SR 310 is linked to pre-mRNA binding, snoRNA binding, and RNA activity. 311 Nearly twice as many GO terms were annotated to the 2nd-Z-15% cargoes. The 312 correspondences between the 2nd-Z-15% cargoes of the 12 NTRs and selected representative GO 313 terms enriched significantly for them are tabulated in Supplementary file 10. The excerpted list of 314 terms enriched significantly for the cargoes of four or fewer NTRs contains terms partially different 315 from those in Figures 5 and 6 (Supplementary file 6A), but many of the NTRs are still linked to 316 terms similar to those of the 3rd-Z-4% cargoes. For example, Imp-β and Trn-SR are linked to terms 317 related to cell or nuclear division by the 3rd-Z-4% cargoes (Figure 5) and to partially different terms 318 that are still related to cell or nuclear division by the 2nd-Z-15% cargoes (Supplementary file 6A). 319 Additionally, Imp-4 is linked to terms related to DNA structure regulation, DNA repair, and 320 apoptosis in both lists. Similarly, most of the examined NTRs are linked in both lists to similar 321 terms that are related to any of the following: chromatin organization, chromosome organization, 322 DNA repair, ribosome biogenesis, protein modification, cell division, nuclear division, and 323 apoptosis. Thus, we regard the 3rd-Z-4% list as a core table of the cargo roles. Naturally, the 324 2nd-Z-15% cargoes linked the NTRs to additional terms (Supplementary file 6A) as follows: Imp-β, 325 -4, and Trn-SR are linked to DNA-dependent DNA replication; Trn-1 and Imp-7 are linked to gene 326 silencing by RNA; Imp-β, -7, and -13 are linked to rRNA transcription; Imp-β and Trn-SR are 327 linked to protein methylation; Trn-SR is linked to protein peptidyl-prolyl isomerization; Trn-2,

12 328 Imp-4, and -8 are linked to circadian rhythm; Imp-β, -4, -13, and Trn-SR are linked to terms for CCs 329 and MFs that are related to RNA (RNAP) II transcription; and subsets of the NTRs are 330 linked to varying terms that are related to differentiation, development, and response. As an 331 important result, we have illustrated the general framework of the division of roles among the NTRs 332 for the first time, in which one NTR is linked to many BPs and conversely each broadly defined BP 333 is supported by many NTRs, but each closely defined BP is supported by a restricted number of 334 NTRs. One typical example is the allocation of mRNA processing factors (see below). 335 336 Allocation of mRNA processing factors to the NTRs 337 Some of the GO terms related to mRNA processing were specifically linked to four or fewer NTRs 338 by the 3rd-Z-4% and 2nd-Z-15% cargoes (Figure 5; Supplementary file 6A). However, many other 339 terms related to mRNA processing were linked to more NTRs, and conversely, all of the NTRs were 340 implicated in mRNA processing. The 2nd- and 3rd-Z-rankings for the 12 NTRs included 275 and 341 242 proteins, respectively, that were annotated with mRNA processing (Supplementary file 6B and 342 6C). To see the allocation of these proteins to the NTRs, the ranks of these proteins are arranged in a 343 table (Supplementary file 11A). The 2nd- and 3rd-Z-rankings revealed similar results. As 344 summarized for the 2nd-Z-ranking (Figure 7), particular groups of the mRNA-processing factors 345 are allocated to specific NTRs, showing that each NTR is linked to distinct reactions in mRNA 346 processing: the proteins related to mRNA capping are allocated to Imp-β almost exclusively; 347 hnRNP A0 is allocated to Trn-1, -2, Imp-4, -11, and others; hnRNP A1, A2B1, A3, D, F, H1-3, and 348 M are allocated to Trn-1 and -2, and additionally Imp-9 and Exp-4; hnRNP U-like 1 are allocated to 349 Imp-7, -8, and -9; SR-rich SFs are primarily allocated to Trn-SR and secondarily to Imp-7, -8, and 350 -9; SFs 3A and B are allocated to Imp-4, -7, -8, -9, -11, and Exp-4; PQ-rich SF is allocated to Imp-4, 351 -7, -8, and Exp-4; snRNP A-C is allocated to Imp-4, -7, -8, and Exp-4; junction complex (EJC) 352 components are exclusively allocated to Imp-11 and -13; cleavage and polyadenylation specificity 353 factor (CPSF) 1 is allocated to Imp-7 and -9; CPSF5 (NUDT21), 6, and 7 are less specifically 354 allocated to other NTRs; general IIF is allocated to Imp-β, -4, and Trn-SR; and 355 RNAP II associating factors are allocated to Trn-SR and separately to other NTRs. Thus, the NTRs 356 import distinctive subsets of mRNA processing factors. In the 2nd-Z-ranking, the SR-rich SFs were 357 not allocated to Imp-5, but they were identified as the Imp-5 3rd-Z-4% cargoes. Thus, subsets of

13 358 proteins involved in a broadly defined BP, e.g., mRNA processing, are allocated to different NTRs, 359 in a manner representative of role division among the NTRs. 360 361 Allocation of ribosomal proteins to the NTRs 362 Ribosomal proteins (RPs) migrate into the nuclei for ribosome assembly, but the NTRs responsible 363 for import have been determined for only a few of RPs (Chook and Suel, 2011). The 3rd-Z-4% 364 cargoes include 15 RPs (Supplementary files 1, 5C, and 11B). To see the allocations of all of the 365 RPs to the NTRs, the ranks of the RPs in the 2nd-Z-rankings are arranged in a table (Figure 8). 366 Because the +NTR/Ctl values were obtained for most of the RPs in the three SILAC-Tp replicates, 367 the 2nd Z-scores are the median Z-scores in most cases, and they should fairly reflect the import 368 efficiencies. Half of the RPs are included in the 2nd-Z-15% cargoes of one to five NTRs, and most 369 of the RPs are ranked in the top 30% in the 2nd-Z-rankings of additional NTRs. Surprisingly, most 370 RPs, especially the 60S subunit proteins, are ranked in the top 50% of most of the 2nd-Z-rankings, 371 and few RPs are ranked lower. These findings imply that most of the RPs are allocated to multiple 372 NTRs, but the import efficiencies vary depending on the NTR. Indeed, several RPs are reported 373 cargoes of multiple NTRs (Jakel and Gorlich, 1998; Jakel et al., 2002). Imp-7, -8, and -9 primarily 374 import RPs, Imp-11 and Exp-4 secondarily import RPs, and all other NTRs also contribute to the 375 import of RPs to some extent. Among the highly homologous NTR pairs, Trn-2 and Imp-8 import 376 RPs more efficiently than Trn-1 and Imp-7, respectively, which indicates that RP import is one of 377 the roles shared unequally by similar NTRs. This differentiation is clearer in the 3rd-Z-rankings 378 (Supplementary file 11B). 379 380 Allocation of transcription factors to the NTRs 381 Sequence-specific DNA-binding transcription factors (annotated with “transcription factor activity, 382 sequence specific DNA binding” in GO) play pivotal roles in many cellular processes, but they are 383 not significantly enriched in the 3rd-Z-4% cargoes of any NTR (Supplementary file 6B). 384 Transcription cofactors (annotated with “transcription factor activity, protein binding”), which may 385 engage in gene-specific transcription, are significantly enriched in the 3rd-Z-4% cargoes of only 386 three NTRs (Figure 6B; Supplementary file 6B). Nonetheless, transcription factors (sequence 387 specific DNA binding) are enriched in the Imp-β 2nd-Z-15% cargoes, and cofactors (protein

14 388 binding) are enriched in the 2nd-Z-15% cargoes of ten NTRs (Supplementary file 6C). Additionally, 389 some transcription factors and cofactors are included in the 2nd-Z-15% cargoes, albeit not enriched. 390 Thus, the 2nd-Z-15% cargoes of each NTR include 17 to 36 transcription factors or cofactors as 391 listed at the bottom of Supplementary file 11D. We performed GO analyses (term type, BP) for 392 these transcription factors and cofactors (Supplementary file 11C and 11D). The annotated terms 393 may reflect both direct transcription regulation activities and indirect effects via transcription. The 394 proteins annotated with modification are enriched in the Imp-β and -13 cargoes, and the 395 term may reflect their direct functions. The cargoes of several NTRs annotated with varying types 396 of signaling may act as cofactors in receptor-regulated transcription. In contrast, 397 many of the transcription factors and cofactors identified as cargoes are annotated differently with 398 various terms related to cell proliferation, development, rhythmic processes, or apoptosis and may 399 act on these processes via transcriptional regulation. Thus, the NTRs import transcription factors 400 and cofactors that work in distinct cellular processes. 401 402 Characterizations of the cargoes of individual NTRs 403 The GO analyses elucidated the characteristics of the NTR-specific cargoes, but the terms are 404 annotated to not only the central players but also many indirect participants in biological processes. 405 Here, we primarily discuss the roles of the notable 3rd-Z-4% cargoes of each NTR and supplement 406 this information with references to the 2nd-Z-15% cargoes. To make our points clear, we classified 407 the 3rd-Z-4% and 2nd-Z-15% cargoes by their characteristics and their allocations to each NTR are 408 presented in Supplementary file 5C and 5E. We describe the features of the Imp-13 and Trn-SR 409 cargoes first, because it includes the discussion on an export cargo or SR-domains. Biological 410 functions linked to NTRs by the natures of their cargoes need to be verified by further experiments. 411 412 Imp-13 cargoes 413 Several Imp-13 cargoes have previously been reported, and our SILAC-Tp clearly reproduced the 414 reported import specificities. Nuclear transcription factor Y subunits β (NFYB) and γ (NFYC) have 415 been reported to be Imp-13 specific cargoes, whereas subunit α (NFYA), which has a BIB-like 416 sequence, has been reported to bind to multiple NTRs (Kahle et al., 2005). NFYB is ranked 1st in 417 both the Imp-13 2nd- and 3rd-Z-rankings, and NFYC is a 2nd-Z-15% cargo (Figure 3;

15 418 Supplementary files 1 and 3). Additionally, we identified NFYA as a cargo of multiple NTRs. 419 Interestingly, a subunit of the general transcription factor TFIIA (GTF2A2) that interacts with 420 NFYA (Rolland et al., 2014) is also a highly ranked Imp-13 3rd-Z-4% cargo. We could not identify 421 the Imp-13 reported cargo (Tao et al., 2006) in our MS, but proteins that 422 may interact with nuclear receptors, e.g., thyroid -associated protein 3 (THRAP3) 423 (Ito et al., 1999), RNA-binding protein 14 (RBM14) (Iwasaki et al., 2001), and transcription 424 activator BRG1 (SMARCA4) (Dai et al., 2008), were identified as Imp-13 2nd-Z-15% cargoes. 425 THRAP3 interacts with EJC (Lee et al., 2010), whose subunits, RNA-binding protein RBM8A and 426 mago nashi homolog MAGOH, are well-characterized Imp-13 cargoes (Mingot et al., 2001). 427 RBM8A and MAGOH are highly ranked Imp-13 3rd-Z-4% cargoes, which were not identified as 428 cargoes of the other NTRs with the exception of Trn-1. However, another EJC subunit, i.e., 429 4A-III (EIF4A3) (Shibuya et al., 2004), was identified as an Imp-11 430 3rd-Z-4% cargo. Thus, the EJC subunits are imported through different pathways. A 431 well-characterized Imp-13 cargo, SUMO-conjugating UBC9 (UBE2I) (Mingot et al., 2001), 432 was identified as a 3rd-Z-4% cargo, and SUMO2 and SUMO3 were also identified as 3rd-Z-4% 433 cargoes. Components of the chromatin accessibility complex CHRAC15 (CHRAC1) and DNA 434 polymerase ε subunit 3 (POLE3) are also Imp-13 reported cargoes (Walker et al., 2009), and they 435 are highly ranked 3rd-Z-4% cargoes. In the GO analysis, Imp-13 was linked to chromatin 436 modification by the 2nd-Z-15% cargoes (Supplementary file 6A). Nucleolar complex protein 2 437 homolog (NOC2L) is ranked 10th in both the 2nd and 3rd Z-scores, and -specific demethylase 438 2A (KDM2A) is ranked 2nd in the 2nd Z-score. The Imp-13 2nd-Z-15% cargoes include many 439 -related proteins that are involved in chromatin remodeling and transcription (Oma and Harata, 440 2011; Yoo et al., 2007). 441 Surprisingly, a reported Imp-13 export cargo eIF1A (EIF1AX; Mingot et al., 2001) was 442 identified as a 2nd-Z-15% cargo (ranked 84th and 164th by the 2nd and 3rd Z-score, respectively; 443 Supplementary files 1 and 3). If a protein endogenous to the permeabilized cell nuclei is exported

444 preferentially in the +NTR in vitro transport reaction, the (L/H+NTR)/(L/HCtl) value will be raised and 445 the protein will be ranked high. However, it cannot be generalized because we have only one 446 example. Most of the highly ranked Imp-13 cargoes must be import cargoes, because in all the bead 447 halo assays where the cargoes bound to Imp-13 RanGTP inhibited the binding (Supplementary file

16 448 2). 449 450 Trn-SR cargoes 451 The reported Trn-SR cargoes include SR-rich splicing factors (SFs) that coordinate transcription 452 elongation, mRNA splicing, and mRNA export (Zhong et al., 2009). Here, we found that proteins 453 engaging in these processes are also Trn-SR cargoes. The Trn-SR 3rd-Z-4% cargoes include the 454 RNA polymerase (RNAP) II elongation factors NELFE and PAF1 (a subunit of the Paf1 complex, 455 PAF1C), DDX and DHX family RNA , and the THO complex subunit THOC1 as well as 456 SR-rich SFs. The 2nd-Z-15% cargoes additionally include PAF1C subunits CTR9, CDC73, and 457 LEO, FACT complex subunits SSRP1 and SPT16, additional DDX and DHX family helicases, 458 and THOC6 and THOC3 (Supplementary file 5C and 5E). Trn-SR bound to NELFE, CDC73, 459 DDX5, and DDX27 in the bead halo assays (Figure 3, Supplementary files 1, 2, and 3). 460 Peptidyl-prolyl cis-trans , which are contained in human (Wahl et al., 2009), 461 were also identified as 3rd-Z-4% and 2nd-Z-15% cargoes. DnaJ homologs were also identified as 462 3rd-Z-4% and 2nd-Z-15% cargoes, although the component DNAJC8 (Zhou et al., 463 2002) was not. The 3rd-Z-4% cargoes also include proteins related to nuclear division or 464 chromosome segregation, the Ser/Thr protein PLK1, dual specificity TTK, 465 G2/M-specific cyclin-B1 (CCNB1), cyclin-dependent kinase (CDK) 2, protein FAM83D, and 466 1 light intermediate chain 1 (DYNC1LI1) in addition to proteins related to histone 467 or deacetylation including histone deacetylase complex subunit SAP18 and 468 SAGA-associated factor 29 homolog CCDC101. Indeed, in the bead halo assays, Trn-SR bound to 469 SAP18 and CCDC101 (Figure 3; Supplementary files 1, 2, and 3). Additionally, the 2nd-Z-15% 470 cargoes include many proteins that are related to nucleosome or chromatin regulation. Thus, the 471 Trn-SR cargoes are involved in chromosome regulation in addition to the coordination of 472 transcription elongation, mRNA splicing, and mRNA export. 473 Surprisingly, SR-rich SFs, which have been assumed to be Trn-SR-specific cargoes, were 474 also identified as cargoes of other NTRs (Figure 7). To determine the allocation of the other 475 SR-domain proteins to the NTRs, we here analyzed the distribution of SRSRSR hexa-peptide 476 sequences in the 3rd-Z-4% cargoes (Supplementary file 11E). Imp-5, -7, -8, and Exp-4 as well as 477 Trn-SR may be the specific NTRs for proteins with the hexa-peptide, most of which are nuclear

17 478 proteins. The hexa-peptide-containing proteins other than the SR-rich SFs are primarily included in 479 the Imp-5 and Exp-4 cargoes. 480 481 Imp-β cargoes 482 The Imp-β cargoes play roles in DNA synthesis and repair and chromatin regulation. The Imp-β 483 3rd-Z-4% cargoes include DNA polymerase δ subunits (POLD2 and 3) and mismatch repair 484 endonuclease PMS2 (Supplementary file 5C). Additionally, the Imp-β 2nd-Z-15% cargoes include 485 PCNA-associated factor KIAA0101 and DNA-(apurinic or apyrimidinic site) (APEX1) 486 (Supplementary file 5E). These proteins act in DNA synthesis or repair. The notable 3rd-Z-4% 487 cargoes related to chromatin regulation include high-mobility group (HMG) proteins, histone 488 acetyltransferase complex NuA4 subunit MRGBP, SWI/SNF-related regulator of chromatin 489 SMARCE1, Spindlin-1 (SPIN1), chromodomain-helicase CHD8, and lymphoid-specific helicase 490 HELLS. Additionally, the 2nd-Z-15% cargoes include the NuA4 subunit MORF4L2, SWI/SNF 491 complex subunit SMARCC2, chromatin assembly factor 1 subunit CHAF1B, polycomb protein 492 EED, and sister chromatid cohesion protein PDS5B. Chromatin remodeling by some of these 493 factors is closely related to transcription. The 3rd-Z-4% cargoes include general transcription factor 494 TFIIF (GTF2F1 and 2), TFIIH subunit MAT1, and TBP-associating factor TAF15, and the 495 2nd-Z-15% cargoes include TFIIH subunit cyclin-H (CCNH) and complex subunit 496 MED15. The sequence-specific transcription factors and cofactors are described above. mRNA 497 capping factors are Imp-β cargoes as described. Thus, many Imp-β cargoes are related to the initial 498 stage of . 499 500 Trn-1 and -2 cargoes 501 The transcription factor ATF1 was ranked 1st in both the 2nd- and 3rd-Z-rankings of the Trn-1 and 502 -2 but was ranked low for the other NTRs (Figure 3; Supplementary files 3 and 5). As described, 503 many of the cargoes that ranked higher in the Trn-1 and -2 2nd- and 3rd-Z-rankings (e.g., hnRNPs) 504 are shared by Trn-1 and -2, but ribosomal proteins (RPs) are included only in the Trn-2 3rd-Z-4% 505 cargoes. Additional divergences can be observed between their 2nd-Z-15% cargoes. As expected, 506 their cargoes include many mRNA processing factors, but among them are preferentially 507 included in the Trn-2 2nd-Z-15% cargoes (Figure 7). Actin and actin-related proteins (ARPs), which

18 508 play roles in chromatin remodeling and transcription (Visa and Percipalle, 2010; Yoo et al., 2007), 509 proteins related to nuclear division, and tRNA are preferentially Trn-1 cargoes, whereas 510 proteins related to DNA repair and HMG proteins are preferentially Trn-2 cargoes (Supplementary 511 file 5E). 512 513 Imp-4 cargoes 514 In the GO analysis, Imp-4 was linked to DNA metabolic processes, chromosome organization, and 515 related terms (Figures 5 and 6). Consistently, subunit 5 (RFC5) and HMG 516 proteins are Imp-4 3rd-Z-4% cargoes, and the 2nd-Z-15% cargoes include DNA polymerase α 517 subunit POLA1, DNA I (LIG1), DNA I (TOP1), SWI/SNF complex subunit 518 SMARCC2, the SWI/SNF-related chromatin regulator SMARCA5, nucleosome remodeling factor 519 subunit BPTF, and FACT complex subunit SPT16 (Supplementary file 5C and 5E). The 520 participation of the Imp-4 cargoes in chromatin organization is supported by a report that Imp-4 521 binds to the histone chaperon complex (Tagami et al., 2004), although the subunits were not 522 identified in our MS. Imp-4 was also linked to in the GO analysis. The Imp-4 3rd-Z-4% 523 cargoes include the regulator of chromosome condensation RCC1 and the Ser/Thr protein kinase 524 PLK1, and the 2nd-Z-15% cargoes include the sister chromatid cohesion protein PDS5 homolog 525 PDS5B. Imp-4 was also linked to programed cell death or apoptosis in the GO analysis. The 526 representative related 3rd-Z-4% cargoes are the death-promoting transcriptional repressor BCLAF1 527 and the tumor suppressor ARF (CDKN2A), and the 2nd-Z-15% cargoes are ribosomal L1 528 domain-containing protein 1 (RSL1D1) and apoptosis-inducing factor 1 (AIFM1). 529 530 Imp-5 cargoes 531 Few characteristics are unique to the Imp-5 3rd-Z-4% cargo cohort. However, this cohort includes 532 proteins related to ribosome biogenesis, such as rRNA 2'-O-methyltransferase (FBL), 533 H/ACA ribonucleoprotein complex subunit 1 (GAR1), and the ribosome biogenesis protein BOP1. 534 This group also includes proteins related to nucleosome or chromatin organization, including 535 spindlin-1 (SPIN1), protein DEK, the methyl-CpG-binding domain protein MBD2, and the paired 536 amphipathic helix protein SIN3B (Supplementary file 5C). SR-rich SFs are also included as 537 described. The Imp-5 2nd-Z-15% cargoes include many ARPs, proteins related to spindle

19 538 organization or -based processes, and several CDKs (Supplementary file 5E). Thus, a 539 portion of the Imp-5 cargoes may be involved in cytokinesis. A number of translation initiation 540 factors (eIFs) and elongation factors, many of which are annotated with nuclear localization 541 (Supplementary file 1), are also among the Imp-5 2nd-Z-15% cargoes. 542 543 Imp-7 and -8 cargoes 544 The cognate NTRs Imp-7 and -8 share many 3rd-Z-4% and 2nd-Z-15% cargoes (Figure 4; 545 Supplementary file 5A). The major cargoes of these NTRs are a range of mRNA SFs, but by the 3rd 546 Z-scores, snRNPs were identified only as Imp-7 and not Im-8 cargoes (Supplementary file 5C). 547 Additional divergences can be observed between the Imp-7 and -8 cargoes (Supplementary file 5C 548 and 5E). HMG proteins were identified only as Imp-7 3rd-Z-4% and 2nd-Z-15% cargoes, whereas 549 more RPs were identified as Imp-8 cargoes. Proteins related to cell cycle regulation, the mitotic 550 checkpoint protein BUB3, cell division cycle 5-like protein (CDC5L), and CDK12, are included in 551 the Imp-7 3rd-Z-4% cargoes, and the Ser/Thr protein kinase PLK1 is a 2nd-Z-15% cargo, but these 552 proteins are not Imp-8 cargoes. Many eIFs are Imp-8 but not Imp-7 2nd-Z-15% cargoes. 553 554 Imp-9 cargoes 555 The Imp-9 cargoes include many RPs and mRNA SFs. Proteins that are important for DNA 556 packaging or nucleosome organization were also identified as Imp-9 cargoes (Supplementary file 557 5C and 5E). Histone H2A.Z, which is located in specific regions on chromosome (Weber and 558 Henikoff, 2014), is ranked 1st and 3rd in the 3rd and 2nd Z-scores, respectively. Additionally, the 559 linker histone H1 (H1F0), histone-lysine N-methyltransferase 2A (KMT2A), and the SPT16 and 560 SSRP1 subunits of the FACT complex, which regulates histone H2A.Z (Jeronimo et al., 2015), were 561 also identified as 3rd-Z-4% cargoes. Among the Imp-9 2nd-Z-15% cargoes, other , DNA 562 topoisomerase I (TOP1) and IIα (TOP2A), HMG proteins, SWI/SNF-related matrix-associated 563 actin-dependent regulator of chromatin subfamily E member 1 (SMARCE1), and scaffold 564 attachment factor B1 (SAFB) are included. 565 566 Imp-11 cargoes 567 Imp-11 was linked to developmental processes in the GO analysis (Figure 5; Supplementary file

20 568 6A), and few proteins with typical nuclear functions, such as DNA replication, nucleosome 569 organization, and transcription, were found among the Imp-11 3rd-Z-4% cargoes (Supplementary 570 file 5C). The Imp-11 2nd-Z-15% cargoes include several proteins related to nuclear division, such 571 as Pogo transposable element with ZNF domain (POGZ), α-endosulfine (ENSA), CDK regulatory 572 subunit 2 (CKS2), and the Ser/Thr protein kinase NEK7 (Supplementary file 5E). Many ARPs, 573 and their related factors, tRNA ligases, and mRNA SFs are also in the Imp-11 2nd-Z-15% 574 cargoes. 575 576 Exp-4 cargoes 577 The subunits of RNAP II elongation factors and mRNA processing factors are the representative 578 Exp-4 cargoes, although they are also cargoes of several other NTRs (Supplementary file 5C and 579 5E). PAF1C subunit parafibromin (CDC73) is an Exp-4 3rd-Z-4% cargo, and other PAF1C subunits, 580 i.e., PAF1 and RTF1, FACT complex subunits, i.e., SSRP1 and SPT16 (SUPT16H), and elongation 581 complex protein 2 (ELP2) are 2nd-Z-15% cargoes. A variety of mRNA processing factors, including 582 3’-end processing factors and THO complex subunits, are also Exp-4 3rd-Z-4% and 2nd-Z-15% 583 cargoes. Thus, the factors that act in processes from transcription elongation to mRNA export are 584 included in the Exp-4 cargoes. As discussed for another bi-directional NTR Imp-13, the possibility 585 cannot be denied that the identified Exp-4 candidate cargoes include export cargoes. 586 587 Seemingly non-nuclear proteins 588 A number of nucleoporins (NUPs), which are the components of the NPC, were identified as 589 cargoes. Increasing evidence demonstrates that the import of NUPs through NPCs is important for 590 gene expression (Burns and Wente, 2014). Moreover, many mitochondrial proteins are highly 591 ranked. These proteins preferentially localize to the mitochondria due to chaperon-regulated or 592 cotranslational mechanisms in vivo and might interact with NTRs in the in vitro transport system. 593 The transport system contains cytosolic extract and unlabeled (light) mitochondrial proteins in it

594 could be imported if they interact with NTRs. The (L/H+NTR)/(L/HCtl) values of them can be 595 calculated, because LC-MS/MS can quantify low levels of labeled (heavy) proteins whether they 596 are endogenous to the recipient nuclei or residual after washing. Thus, mitochondrial proteins with

597 high (L/H+NTR)/(L/HCtl) values are imported proteins even if the import is fortuitous. Nuclear

21 598 localization is annotated to many mitochondrial proteins (Supplementary file 1), and actual nuclear 599 localization is possible as in the cases of AIFM1 and ATFS-1 (Nargund et al., 2012; Susin et al., 600 1999). As was the case with the high-throughput cargo identification of the export receptor Exp-1 601 (CRM1) (Kirli et al., 2015), our method identified other seemingly cytoplasmic proteins as cargoes. 602 We did not detect direct binding between the NTRs and some of these cytoplasmic proteins, e.g., 603 Ras-related Rab family proteins and S100 proteins, in the bead halo assays (Supplementary file 2), 604 but nuclear import by indirect binding is still possible. 605 606 Here, we have presented the first complete picture of nuclear import via the 12 importin pathways. 607 The 12 pathways must serve distinct roles because the NTRs are linked to different cellular 608 processes by their cargoes. However, the cargoes are intricately allocated to the NTRs, and each 609 NTR is linked to multiple cellular processes. The biological functions of NTRs designated in this 610 work should be further clarified in future experiments. 611 We used HeLa nuclear extract as the cargo source, but it might not reconstitute all NTR– 612 cargo interactions precisely because proteins in the nuclear extract might have different 613 modifications or binding partners from those in where NTRs bind to cargoes in vivo. 614 Some reported cargoes were ranked lower in the 2nd- and 3rd-Z-ranking, and it might be 615 attributable to these differences of protein states. Alternatively, the transport capacity of our in vitro 616 transport system might not be enough to identify all the cargoes, especially those with low transport 617 efficiency. To reach a definitive conclusion, experiments in vivo might be needed. 618 We could not find any novel motifs that may serve as NTR-binding sites on the identified 619 cargoes using the ungapped motif search method of MEME (Bailey and Elkan, 1994). A more 620 extensive search for such motifs and higher order structures using alternative methods is currently 621 underway. 622 623 Materials and Methods

624 SILAC-Tp 625 SILAC-Tp has previously been described in detail (Kimura et al., 2014), but we provide a brief 626 description here. HeLa-S3 cytosolic and nuclear extracts were depleted of Imp-β family NTRs with 627 phenyl-Sepharose (GE healthcare), and the nuclear extract was subsequently depleted of RCC1 with

22 628 a -affinity method and concentrated. The extracts were dialyzed against transport buffer (TB, 20 629 mM HEPES–KOH (pH 7.3), 110 mM KOAc, 2 mM MgOAc, 5 mM NaOAc, 0.5 mM EGTA, 2 mM 630 DTT, and 1 μg/mL each of aprotinin, pepstatin A, and leupeptin). Adherent HeLa-S3 cells were 13 13 631 labeled with u- C6 Lys and u- C6 Arg by SILAC (Ong et al., 2002) and seeded onto a glass plate. 632 After rinsing in ice cold TB, the cells were permeabilized with 40 μg/mL digitonin in TB for 5 min 633 on ice and then rinsed again. The permeabilized cells were pretreated with 4 μM RanGDP and an 634 ATP regeneration system in TB for 20 min at 30°C to remove the residual Imp-β family NTRs and 635 then rinsed. The cells were incubated in transport mixture (50% cytosolic extract, 10% nuclear 636 extract, 1 μM p10/NTF2, and ATP regeneration system in TB) with (+NTR) or without (Ctl) 0.3–0.7 637 μM of one NTR for 20 min at 30°C for the import reaction. (The NTR concentrations were 638 optimized using the recombinant cargoes presented in Figure 1-figure supplement 1C.) After rinsing, 639 the cells were incubated in extract mixture (50% cytosolic extract and ATP regeneration system in 640 TB) for 20 min at 30°C and rinsed with NaCl-TB (TB containing 110 mM NaCl instead of KOAc) 641 to remove the nonspecifically binding proteins. To extract the proteins, the cells were suspended in

642 nuclear buffer (20 mM Tris–HCl, pH 8.0, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 2 mM 643 DTT, and 1 μg/mL each of aprotinin, pepstatin A, and leupeptin), sonicated, and centrifuged. 644 Actually, the transport reactions for two NTRs were simultaneously performed with one 645 control reaction and triplicated. The simultaneously processed NTRs were Imp-β and Imp-13, Trn-1 646 and -2, Imp-7 and -8, Imp-9 and -11, and Imp-5 and Trn-SR, and the reactions for Imp-4 and Exp-4 647 were performed individually with controls. 648 649 Peptide analysis by LC-MS/MS 650 After the in vitro transport reaction, 25 μg each of the extracted proteins was concentrated by 651 acetone precipitation, reduced with DTT, and alkylated with iodoacetamide. The proteins were 652 digested with trypsin and Lys-C (enzyme/substrate ≈ 1/50) for 16 h at 37°C. The

653 peptides were evaporated to dryness, dissolved in Solvent-1 (0.1% TFA and 15% CH3CN), and 654 fractionated on EmporeTM Cation Exchange-SR (3M, Maplewood, Minnesota). For the fractionation, 655 the support was stacked manually inside the tapered end of a micropipette tip, the tip was fixed into 656 the punched lid of a microtube, and the liquids were run by centrifugation (Wisniewski et al., 2009). 657 The resin was sequentially washed by ethanol and Solvent-1 containing 500 mM ammonium acetate

23 658 and equilibrated with Solvent-1, and the peptides were then applied. After washing in Solvent-1, the 659 peptides were eluted stepwise by Solvent-1 containing 125, 250, and 500 mM ammonium acetate

660 and Solvent-2 (5% NH4OH, 30% methanol, and 15% CH3CN). The eluates were evaporated to

661 dryness, and the peptides were dissolved in 0.1% TFA and 2% CH3CN. 662 The peptides were applied to a liquid chromatograph (LC) (EASY-nLC 1000; Thermo 663 Fisher Scientific, Waltham, Massachusetts) coupled to a Q Exactive hybrid quadrupole-Orbitrap 664 mass spectrometer (Thermo Fisher Scientific) with a nanospray source in positive mode. The 665 LC was performed on a NANO-HPLC capillary column C18 (75 μm x 150 mm, 3 μm particle size,

666 Nikkyo Technos, Tokyo). The peptides were eluted with a 100-min 0–30% CH3CN gradient and a 667 subsequent 20-min 30–65% gradient in the presence of 0.1% formic acid at a flow rate of 300 668 nL/min. The Q Exactive-MS was operated in the top-10 data-dependent scan mode. The parameters 669 for the Q Exactive operation were as follows: spray voltage, 2.3 kV; capillary temperature, 45°C; 670 mass range (m/z), 350-1800; and normalized collision energy, 28%. The raw data were acquired 671 with Xcalibur (RRID:SCR_014593; ver. 2.2 SP1). 672 673 Protein identification and quantitation 674 The MS and MS/MS data were searched against the Swiss-Prot database (2014_07–2016_01) using 675 Proteome Discoverer (RRID:SCR_014477; ver. 1.4, Thermo Fisher Scientific) with the MASCOT 676 search engine software (RRID:SCR_014322; ver. 2.4.1, Matrix Science, London). The search 677 parameters were as follows: taxonomy, Homo sapiens; enzyme, trypsin; static modifications, 678 carbamidomethyl (Cys); dynamic modifications, oxidation (Met); precursor mass tolerance, ± 6 679 ppm; fragment mass tolerance, ± 20 mDa; maximum missed cleavages, 1; and quantitation, SILAC 680 (R6, K6). The proteins were considered identified when their false discovery rates were less than 681 5%. The SILAC L/H ratios were also calculated by Proteome Discoverer (ver. 1.4) with the default 682 setting: show the raw quan values, false; minimum quan value threshold, 0; replace missing quan 683 values with minimum intensity, false; use single-peak quan channels, false; apply quan value 684 corrections, true; reject all quan values if not all quan channels are present, false; fold change 685 threshold for up-/down-regulation, 1.5; maximum allowed fold change, 100; use ratios above 686 maximum allowed fold change for quantification, false; percent co-isolation excluding peptides 687 from quantification, 100; protein quantification, use only unique peptides; experimental bias, none.

24 688 Proteins with L/H count ≥1 were included in further analysis. The L/H counts are shown in 689 Supplementary file 1. To access the mass spectrometry data, see below. 690 From the SILAC quantitation values of the control and +NTR reactions, the +NTR/Ctl =

691 (L/H+NTR)/(L/HCtl) ratio of each protein was calculated, and the Z-score of the log2(+NTR/Ctl) of 692 each protein was calculated within each replicate. 693 Z-score=(X-μ)/ σ

694 where X is log2(+NTR/Ctl)=log2[(L/H+NTR)/(L/HCtl)] of each protein, μ is the mean of X, and σ is 695 the standard deviation of X. 696 697 Reported cargo rate and recall 698 To calculate reported cargo rate (a lower bound on precision) and recall (sensitivity), we used the 27 699 and 25 reported cargoes of Trn-1 as the positive examples of the 2nd- and 3rd-Z-rankings, 700 respectively. We do not have explicit labeling of negative examples. Most likely some portion of the 701 proteins not reported as cargoes are genuine cargoes, but it is difficult to estimate that portion. 702 Therefore, as a rough guide we tallied statistics under two simple assumptions: (i) that all proteins 703 not reported as cargoes should be treated as negative examples, and (ii) that in the proteins not 704 reported as cargoes, proteins annotated in Uniprot (RRID:SCR_002380) as having non-nuclear 705 subcellular localization should be treated as negative examples and the other proteins excluded from 706 the analysis (treated as neither positive nor negative). The first definition yielded 1622 and 1210 707 negative examples in the 2nd- and 3rd-Z-ranking, respectively, and the second definition 259 and 708 178 in the 2nd- and 3rd-Z-ranking, respectively. Since the first definition is maximally pessimistic, 709 it allows estimation of an upper bound on the rate of false positives, while the second definition is 710 more optimistic. 711 Reported cargo rate(i)=p(i)/[p(i)+n(i)] 712 Recall(i)=p(i)/P 713 where p(i) denotes the number of previously reported cargoes (a lower bound on the number of 714 positive examples) and n(i) denotes the number of negative examples in the top i%; while P denotes 715 the total number of previously reported cargoes. 716 717 Gene Ontology analysis

25 718 GO (RRID:SCR_002811) analyses were performed using g:Profiler (RRID:SCR_006809; 719 r1488-1536_e83_eg30) (Reimand et al., 2016). The search parameters were the following: organism, 720 Homo sapiens; significance threshold, g:SCS; statistical domain size, all known genes; GO version, 721 GO direct 2015-12-09 to 2016-01-21, releases/2015-12-08. 722 723 Phylogenetic analysis of the 12 Imp-β family NTRs 724 The phylogeny was inferred by maximum likelihood using RAxML (RRID:SCR_006086; ver. 725 8.1.17) (Stamatakis, 2006) with 1000 bootstrap replicates and the LG model with 726 gamma-distributed rate variation. The sequences were aligned using Clustal Omega 727 (RRID:SCR_001591; ver. 1.2.0) (Sievers et al., 2011) with the default parameters, and the resulting 728 multiple alignments were trimmed using trimAl (ver. 1.2) (Capella-Gutierrez et al., 2009) in 729 gappyout mode. 730 731 Hierarchical clustering of the 11 Imp-β family NTRs based on the degree of overlap of the 732 3rd-Z-4% cargoes 733 We performed a hierarchical clustering of the Imp-β family NTRs based on their cargo profile 734 similarities using Ward's method with Euclidean distance as implemented in the software R 735 (RRID:SCR_001905; R Development Core Team, 2012). Here, we omitted Imp-β because its 736 cargoes include many Imp-α-dependent indirect cargoes. To define a cargo profile for each NTR, 737 we first defined a set of cargoes by merging the 3rd-Z-4% cargoes of the 11 NTRs other than Imp-β, 738 which yielded a total of 426 cargoes. We then defined length 426 binary vectors for each NTR with 739 a 1 for each cargo in the top 4% list and a 0 otherwise and input these 11 vectors into R to perform 740 the clustering. 741 742 Bead halo assay 743 The proteins and extracts were prepared as described (Kimura et al., 2013). The 744 bead halo assays (Supplementary file 2) were performed as described (Patel and Rexach, 2008). 745 Briefly, GST or GST-NTR was immobilized on glutathione-Sepharose (GE healthcare), and mixed 746 with an extract of E. coli expressing a GFP-fusion protein in EHBN buffer (10 mM EDTA, 0.5% 747 1,6-hexanediol, 10 mg/mL bovine serum albumin, and 125 mM NaCl), and the binding was

26 748 observed by fluorescent microscopy. The GTP-fixed mutant of Ran Q69L-Ran, which inhibits 749 specific NTR–cargo interactions, was added to determine the specificity of the binding. The 750 expression and degradation levels of the GFP-fusion proteins were analyzed, and the concentrations 751 of GFP-moieties were quantified by triplicate quantitative Western blotting of the extracts with an 752 anti-GFP . Because the GFP-moiety weakly bound to GST-Trn-1, GST-Trn-2, and 753 GST-Trn-SR in the bead halo assay, the concentrations of the GFP-fusion proteins and GFP 754 (control) were equalized, and images were acquired and processed under identical condition. In 755 contrast, because the GFP-moiety does not bind to GST-Imp-13, GST-Imp-β, or GST-Imp-α, the 756 control reaction mixture for these NTRs contained higher concentration of GFP than any other 757 GFP-fusion proteins. Three images (GST, GST-NTR, and GST-NTR + Q69L-Ran) for each 758 GFP-fusion protein were acquired under identical conditions, and the background intensities and 759 dynamic ranges were equalized. 760 761 Cell line 762 HeLa-S3 (RRID:CVCL_0058; mycoplasma, not detected) was obtained from Dr. Fumio Hanaoka 763 (RIKEN). 764 765 766 See Supplementary file 12B.. 767 768 GFP-fusion proteins used for in vitro transport 769 The GFP-fusion proteins used in Figure 1–figure supplement 1C were prepared as described 770 (Kimura et al., 2013). For accessions and references, see Supplementary file 12B. The cDNA 771 (pF1KB9652) was from Kazusa DNA Res. Inst. (Kisarazu, Japan), and the others were cloned from 772 a HeLa cDNA library (SuperScript™, Life Technology) by PCR. 773 774 Database deposition 775 The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium 776 (RRID:SCR_004055; http://www.proteomexchange.org/) via the PRIDE (RRID:SCR_003411; 777 Vizcaino et al., 2016) partner repository with the dataset identifier PXD004655.

27 778 The .msf and .raw data files of each experiment summarized in Supplementary file 1 are 779 listed in Supplementary file 12A. The protein and peptide quantitation results can be seen by 780 opening .msf files by Proteome Discoverer software. To see spectra and chromatograms, .msf files 781 and corresponding .raw files must be in the same local directory. A demo version of Proteome 782 Discoverer can be downloaded at the Thermo Scientific omics software portal site 783 (https://portal.thermo-brims.com/). 784 785 Acknowledgements 786 We thank Martin Beck, Alessandro Ori, and Kentaro Tomii for helpful discussion, Masahiro Oka for 787 a supporting experiment, Shoko Motohashi and Ai Watanabe for technical assistance, and Hiroshi 788 Mamada for a plasmid. We also thank the Support Unit for Bio-Material Analysis, RIKEN BSI 789 Research Resource Center, especially Kaori Otsuki, Masaya Usui, and Aya Abe for MS analysis. 790 This work was supported by JSPS KAKENHI Grant Number 15K07064 and a RIKEN FY2014 791 Incentive Research Project to M.K., KAKENHI Grant Numbers 26251021, 26116526, and 792 15H05929 to N.I., and the Platform Project for Supporting in Drug Discovery and Life Science 793 Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from the Japan 794 Agency for Medical Research and Development (AMED) to K.I.. 795 796 Competing interests 797 The authors declare that no competing interests exist. 798 799 References

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1168 Figure 1. SILAC-Tp effectively sorts Trn-1 cargoes. 1169 (A) Z-scores in the Trn-1 2nd- and 3rd-Z-rankings. The 2nd (left) and 3rd (right) Z-scores are 1170 presented for the top 250 proteins in the Trn-1 2nd- and 3rd-Z-rankings, respectively. The total 1171 number of ranked (quantified) proteins and the number of previously reported cargoes included in 1172 the ranking are indicated at the bottom. The magenta bars represent previously reported cargoes. 1173 The blue and dark gray bars represent the proteins that did and did not bind directly to Trn-1, 1174 respectively, in the bead halo assays (Supplementary file 2). Identical proteins marked by the colors 1175 are connected by lines. Proteins that carry PY-NLS motifs are indicated by green bars. 1176 (B) Distribution of PY-NLS motif-containing proteins in the rankings. The percentage of the 1177 proteins carrying PY-NLS motifs in 50 consecutively aligned proteins is presented along with the 1178 2nd- and 3rd-Z-rankings (left and right, respectively). For example, the top 50 proteins in the 1179 2nd-Z-ranking include 19 (38%) PY-NLS motif-containing proteins, and thus the value at position 1

42 1180 is 38%. Two types of PY-NLS motifs, basic and hydrophobic, are defined as presented at the 1181 bottom. 1182 The following figure supplements and source data file are available for figure 1: 1183 Figure supplement 1. SILAC-Tp experimental system. 1184 Figure supplement 2. Trn-1 cargoes are effectively sorted by the 2nd or 3rd Z-scores in three 1185 replicates of SILAC-Tp. 1186 Figure supplement 3. The reported cargo rates and recall of the Trn-1 2nd- and 3rd-Z-ranking. 1187 Source data file 1. Statistical analysis of reported cargoes in the Trn-1 2nd- and 3rd-Z-ranking. 1188 1189 Figure 1–figure supplement 1. SILAC-Tp experimental system. 1190 (A) Methods outline. 1191 For details, see the Materials and Methods. This figure was originally published by Kimura et al. 1192 (2013a). 1193 (B) Depletion of the Imp-β family NTRs from the cytosolic and nuclear extracts. 1194 The cytosolic and nuclear extracts before and after the NTR depletion were analyzed by Western 1195 blotting with antibodies specific for each NTR. Asterisk, no band was detected. For Imp-11, the 1196 lower band indicated by the arrowhead was removed. For the antibodies, see Supplementary file 1197 12B.. 1198 (C) In vitro transport of specific cargoes. 1199 The reported cargoes were expressed as GFP-fusion proteins and purified. Then, the cargoes were 1200 added to the in vitro transport system with (+) or without (–) each specific NTR. The import was 1201 observed by fluorescence microscopy. IBB, Imp-β-binding domain of Imp-α. For the protein 1202 accessions, see Supplementary file 12B. 1203 1204 Figure 1–figure supplement 2. Trn-1 cargoes are effectively sorted by the 2nd or 3rd Z-scores 1205 in three replicates of SILAC-Tp.

1206 (A) Frequency distributions of the log2[(L/H+NTR)/(L/HCtl)] = log2(+NTR/Ctl) in three replicates of 1207 SILAC-Tp with Trn-1.

1208 (B) Frequency distributions of the Z-scores of log2[(L/H+NTR)/(L/HCtl)] in three replicates of 1209 SILAC-Tp with Trn-1. The Z-score of each protein was calculated as Z=(X-μ)/σ, where X is

43 1210 log2[(L/H+NTR)/(L/HCtl)]of a protein, μ is the mean of X in one replicate, and σ is the standard 1211 deviation of X. 1212 (C) Distribution of the three Z-scores in the Trn-1 2nd-Z-ranking. 1213 The Trn-1 2nd-Z-ranking consists of 1,649 proteins. Both of the two or all three of the Z-scores are 1214 plotted against the rank by the 2nd Z-score. The Z-scores in one replicate are presented as circles 1215 with the same color (blue, green, or violet), and the diamonds drawn in dark blue, dark green, and 1216 dark violet represent the Z-scores of reported cargoes in the respective replicates. 1217 (D) Distribution of the three Z-scores in the Trn-1 3rd-Z-ranking. 1218 The Trn-1 3rd-Z-ranking consists of 1,235 proteins. All three of the Z-scores are plotted against the 1219 rank by the 3rd Z-score. The marker colors are the same as those in (C). 1220 (E) Z-scores of the top 200 proteins in the Trn-1 2nd-Z-ranking. Close up of the top 200 in (C). 1221 (F) Z-scores of the top 200 proteins in the Trn-1 3rd-Z-ranking. Close up of the top 200 in (D). 1222 1223 Figure 1–figure supplement 3. Reported cargo rates and recall of the Trn-1 2nd- and 1224 3rd-Z-ranking. 1225 (A) Rates of reported cargoes in the Trn-1 2nd- and 3rd-Z-percentile rankings. 1226 The reported cargo rates in the top 1% to 50% ranks are shown under the two definitions of 1227 negative examples: unreported, proteins not reported as cargoes; and non-nuclear, proteins 1228 annotated with non-nuclear localization. 1229 (B) Recall by ranks of the Trn-1 2nd- and 3rd-Z-percentile rankings. 1230 The recall of reported cargoes in the top 1% to 50% ranks are indicated. 1231 See also Figure 1-source data file 1. 1232 1233 Figure 1-source data file 1. Statistical analysis of reported cargoes in the Trn-1 2nd- and 1234 3rd-Z-ranking. 1235 (A, B) Reported cargo rates, recall, and p-values of the Trn-1 2nd-Z-percentile ranking. 1236 Reported cargoes and proteins that have not been reported as cargoes (unreported candidate 1237 cargoes) in the top 1% to 20% ranks were counted in 1% rank increments, and the reported cargo 1238 rates (a lower bound on precision), recall (sensitivity), and p-values were calculated using two 1239 definitions of negative examples: (i) assuming any protein not reported as a cargo is negative (A), or

44 1240 (ii) assuming only those annotated to have non-nuclear localization in Uniprot to be negative (B). 1241 p-values were calculated by Fisher's exact test. The row marked in cyan corresponds to the 1242 2nd-Z-15% cargoes. 1243 (C, D) Reported cargo rates, recall, and p-values of the Trn-1 3rd-Z-percentile ranking. 1244 Reported cargo rates, recall, and p-values of the top 1% to 20% ranks were calculated similarly 1245 using two definitions of negative examples. The row marked in magenta corresponds to the 1246 3rd-Z-4% cargoes. 1247 See also Figure 1-figure supplement 3. 1248 1249 Figure 2. Trn-1, Imp-13, and Trn-SR cargo rankings. 1250 (A–C) The top 100 proteins in the Trn-1 (A), Imp-13 (B), and Trn-SR (C) 2nd- and 3rd-Z-rankings 1251 (left and right, respectively). Magenta, reported cargoes; blue, proteins bound directly to the NTR in 1252 the bead halo assays (Supplementary file 2); orange in (C), SR-rich SFs that have not been reported; 1253 and green in (C), other RS (SR)-domain proteins. Identical proteins marked by the colors are 1254 connected by lines. 1255 The following figure supplements are available for figure 2: 1256 Figure supplement 1. Imp-13 cargoes are effectively sorted by the 2nd or 3rd Z-scores in three 1257 replicates of SILAC-Tp. 1258 Figure supplement 2. SILAC-Tp effectively sorts Imp-13 and Trn-SR cargoes. 1259 Figure supplement 3. Imp-β cargo ranking and Z-scores in the 2nd- and 3rd-Z-rankings. 1260 1261 Figure 2–figure supplement 1. Imp-13 cargoes are effectively sorted by the 2nd or 3rd 1262 Z-scores in three replicates of SILAC-Tp. 1263 (A) Distribution of the three Z-scores in the Imp-13 2nd-Z-ranking. 1264 The Imp-13 2nd-Z-ranking consists of 2,060 proteins. Both of the two or all three of the Z-scores 1265 are plotted against the rank by the 2nd Z-score. The Z-scores in one replicate are presented as 1266 circles with the same color (blue, green, or violet), and the diamonds drawn in dark blue, dark green, 1267 and dark violet represent the Z-scores of reported cargoes in the respective replicates. Z-scores of 1268 the top 200 proteins are presented. 1269 (B) Distribution of the three Z-scores in the Imp-13 3rd-Z-ranking.

45 1270 The Imp-13 3rd-Z-ranking consists of 1,671 proteins. All three of the Z-scores are plotted against 1271 the rank by the 3rd Z-score. The marker colors are the same as those in (A). Z-scores of the top 200 1272 proteins are presented. 1273 1274 Figure 2–figure supplement 2. SILAC-Tp effectively sorts Imp-13 and Trn-SR cargoes. 1275 (A) Z-scores in the Imp-13 2nd- and 3rd-Z-rankings. 1276 The 2nd (left) and 3rd (right) Z-scores are presented for the top 250 proteins in the Imp-13 2nd- and 1277 3rd-Z-rankings, respectively. The total number of the ranked (quantified) proteins and the number 1278 of the previously reported cargoes included in the ranking are indicated at the bottom. The magenta 1279 bars represent the reported cargoes. The blue and dark gray bars represent proteins that did and did 1280 not bind directly to Imp-13, respectively, in the bead halo assays (Supplementary file 2). Identical 1281 proteins marked by the colors are connected by lines. 1282 (B) Z-scores in the Trn-SR 2nd- and 3rd-Z-rankings. 1283 The Z-scores in the Trn-SR 2nd- and 3rd-Z-rankings are presented as in (A). The orange and green 1284 bars represent SR-rich splicing factors that have not been reported and other RS-domain proteins, 1285 respectively. 1286 1287 Figure 2–figure supplement 3. Imp-β cargo ranking and Z-scores in the 2nd- and 1288 3rd-Z-rankings. 1289 (A) The top 100 proteins in the Imp-β 2nd- and 3rd-Z-rankings (left and right, respectively). 1290 Blue indicates proteins bound directly to Imp-β or both Imp-α and -β, and light blue indicates 1291 proteins bound directly to Imp-α but not Imp-β in the bead halo assays (Supplementary file 2). 1292 Yellow highlight indicates Imp-α. Identical proteins marked by the colors are connected by lines. 1293 (B) Z-scores in the Imp-β 2nd- and 3rd-Z-rankings. 1294 The 2nd (left) and 3rd (right) Z-scores are presented for the top 250 proteins in the Imp-β 2nd- and 1295 3rd-Z-rankings, respectively. The total number of the ranked proteins and the number of previously 1296 reported Imp-β direct cargoes that are included in the ranking are indicated at the bottom. Bar 1297 colors: blue, proteins bound directly to Imp-β or both Imp-α and -β in the bead halo assays; light 1298 blue, proteins bound directly to Imp-α but not Imp-β; dark gray; proteins that did not bind to either 1299 Imp-α or Imp-β; and yellow, Imp-α.

46 1300 1301 Figure 3. 3rd-Z-4% cargoes of the 12 NTRs. 1302 The 3rd-Z-4% cargoes of each NTR are listed by the gene names in the 3rd-Z-rank orders. The 1303 ranks by the 2nd Z-scores are also shown. The 3rd-Z-4% and 2nd-Z-15% cargoes are indicated by 1304 cyan in the rank columns. Colors in the gene name columns: magenta, reported cargoes; blue, 1305 cargoes bound directly to the NTR in the bead halo assays (Supplementary file 2); light blue, 1306 cargoes bond directly to Imp-α but not to Imp-β; gray, proteins that did not bind to the NTRs; and 1307 yellow, Imp-α. For the 2nd-Z-15% cargoes, see Supplementary file 3. 1308 1309 Figure 4. Phylogenetic tree and cargo profile hierarchical clustering of the Imp-β family 1310 import receptors. 1311 (A) Phylogenetic tree of the 12 Imp-β family import receptors with the bootstrap values. Scale bar 1312 indicates substitutions per site. 1313 (B) A hierarchical clustering dendrogram of the same NTRs (except Imp-β) based on the similarities 1314 of their 3rd-Z-4% cargo profiles. Imp-β was excluded because Imp-α connects to Imp-β and many 1315 of the identified cargoes. The scale indicates the intercluster distance. 1316 (C) The numbers of 3rd-Z-4% cargoes shared by two NTRs. For the 2nd-Z-15% cargoes, see 1317 Supplementary file 5A. 1318 1319 Figure 5. GO term (Biological Process) enrichments of the 3rd-Z-4% cargoes. 1320 The 3rd-Z-4% cargoes were analyzed for GO term (term type, Biological Process) enrichment. The 1321 significantly enriched terms (p<0.05, cyan) in the 3rd-Z-4% cargoes of four or fewer NTRs were 1322 selected, and a representative term for each group of highly similar is presented with their p-values 1323 and the numbers (#) of cargoes annotated with them. Total No. denotes the number of proteins 1324 annotated with each term in the database. Related terms are bundled in the same color. This table 1325 was extracted from Supplementary file 6B. All the GO terms annotated to the 3rd-Z-4% cargoes are 1326 listed in Supplementary file 7. The correspondence between each 3rd-Z-4% cargo and GO term is 1327 summarized in Supplementary file 9. For the 2nd-Z-15% cargoes, see Supplementary file 6A, 8, and 1328 10. 1329

47 1330 Figure 6. GO term (Cellular Component and Molecular Function) enrichments of the 1331 3rd-Z-4% cargoes. 1332 The 3rd-Z-4% cargoes were analyzed, and the results are presented in a format similar to that of 1333 Figure 5. 1334 (A) Term type, Cellular Component. 1335 (B) Term type, Molecular Function. 1336 These tables were extracted from Supplementary file 6B. All the GO terms annotated to the 1337 3rd-Z-4% cargoes are listed in Supplementary file 7. The correspondence between each 3rd-Z-4% 1338 cargo and GO term is summarized in Supplementary file 9. For the 2nd-Z-15% cargoes, see 1339 Supplementary file 6A, 8, and 10. 1340 1341 Figure 7. mRNA processing factors in the 2nd-Z-rankings. 1342 The ranks of the mRNA processing factors in the 2nd-Z-rankings of the 12 NTRs are presented. The 1343 color scale is set by percentile rank as indicated. The 2nd-Z-rankings of the 12 NTRs include 275 1344 proteins in total that are annotated with mRNA processing in GO. Of these, 69 were selected and 1345 are presented. For other factors and the 3rd-Z-rankings, see Supplementary file 11A. 1346 1347 Figure 8. Ribosomal proteins in the 2nd-Z-rankings. 1348 The ranks of the ribosomal proteins in the 2nd-Z-rankings of the 12 NTRs are presented. The color 1349 scale is set by the percentile rank as indicated. For the 3rd-Z-rankings, see Supplementary file 11B. 1350 1351 Additional files 1352 · Supplementary file 1. Results of SILAC-Tp. 1353 · Supplementary file 2. NTR–cargo direct binding. 1354 · Supplementary file 3. The 2nd-Z-15% cargoes of the 12 NTRs. 1355 · Supplementary file 4. Example of extracted ion chromatograms of peptides. 1356 · Supplementary file 5. Redundancy of NTRs: Cargoes shared by NTRs. 1357 · Supplementary file 6. GO term enrichments of the identified cargoes. 1358 · Supplementary file 7. The 3rd-Z-4% cargoes annotated with GO terms. 1359 · Supplementary file 8. The 2nd-Z-15% cargoes annotated with GO terms.

48 1360 · Supplementary file 9. Correspondences between the 3rd-Z-4% cargoes and GO terms. 1361 · Supplementary file 10. Correspondences between the 2nd-Z-15% cargoes and GO terms. 1362 · Supplementary file 11. mRNA processing factors, ribosomal proteins, and transcription factors. 1363 · Supplementary file 12. A. MS data files. 1364 B. GFP-fusion proteins used for in vitro transport and antibodies. 1365 1366 Legends for supplementary files

1367 Supplementary file 1. Results of SILAC-Tp. 1368 Each sheet contains the result of SILAC-Tp with one of the 12 NTRs. The proteins that exhibited

1369 +NTR/Ctl = (L/H+NTR)/(L/HCtl) values at least once in the three replicates (Experiments 1–3) are

1370 listed with the log2[(L/H+NTR)/(L/HCtl)] values. The 2nd and 3rd Z-scores and the ranks according to 1371 those scores are also presented. The 2nd-Z-15% and 3rd-Z-4% cargoes are indicated in cyan. The 1372 LC-MS/MS quantitation data for each replicate (Experiments 1–3) are also included. Light/Heavy, 1373 the median of quantified L/H values; Light/Heavy count, the number of quantified values; 1374 Light/Heavy variability, coefficient-of-variation for log-normal distributed data. 1375 aReport: The proteins listed by Chook and Süel (2011) are regarded as reported cargoes, and the 1376 references are provided in the main text. For Imp-β, only direct cargoes are listed. For Trn-2, the 1377 reported Trn-1 cargoes are listed. 1378 bDirect Binding: The results of the bead halo assays (Supplementary file 2) are summarized. ++ or +, 1379 positive; ± or –, negative. 1380 cGO Nucleus: Annotated with "nucleus" in Gene Ontology (term type, cellular component). 1381 The rows can be sorted into preferable orders with Excel. 1382 To access the mass spectra, chromatograms, or raw data, see Supplementary file 12A. 1383 Statistics

1384 For each experiment, the number of proteins assigned with log2[(L/H+NTR)/(L/HCtl)] values (proteins

1385 assigned with Z-scores), the mean and standard deviation (S.D.) of log2[(L/H+NTR)/(L/HCtl)] are 1386 listed. 1387 Z-score = (X-μ)/σ

1388 where X = log2[(L/H+NTR)/(L/HCtl)] of each protein, μ is the mean of X in one experiment, and σ is 1389 the S.D. of X.

49 1390 1391 Supplementary file 2. NTR–cargo direct binding. 1392 The direct binding of the candidate cargoes to the NTRs was analyzed by bead halo assay. From 1393 well-characterized proteins that have not been reported as cargoes, (i) proteins ranked high (within 1394 the top 15% in the 2nd-Z-rankings or 4% in the 3rd-Z-rankings), around presumptive cutoffs 1395 (within about top 15–25% in the 2nd-Z-rankings), or lower and (ii) highly ranked proteins that are 1396 suspected as indirect cargoes or false positives based on their well-known features, e.g., PMPCA, 1397 GALE, UAP1, NQO2, EEF1A2, RAB2A, RAB8A, S100A4, S100A6, S100A13, and S100P, were 1398 selected and analyzed. Proteins in (i) verify the cargo identification and cutoff setting, and proteins 1399 in (ii) serve for finding indirect cargoes and false positives. The negative rate of these bead halo 1400 assays should be higher than the true overall false positive rate of the SILAC-Tp, because proteins 1401 in (ii) were selected preferentially. GST or GST-NTR was attached to glutathione-Sepharose beads, 1402 mixed with an extract of E. coli expressing GFP or a GFP-fusion protein, and observed by 1403 fluorescence microscopy. Q69L-Ran, which inhibits the NTR–cargo functional binding, was 1404 added as appropriate. The contrast of the bead fluorescence between the GST and GST-NTR 1405 indicates the binding, and the inhibition of this binding by Q69L-Ran certifies the specificity of the 1406 binding; ++ or +, positive; ± or –, negative. 1407 Summary of the results (p2–5): The results are summarized in both 2nd- and 3rd-Z-rank order. 1408 The 2nd-Z-15% and 3rd-Z-4% cargoes are indicated by cyan, and positive binding (++ or +) is 1409 indicated by blue. 1410 Trn-1 (p6–8): The GFP-fusion proteins were divided into five groups (A–E) according to the 1411 expression levels. Because GFP binds weakly to Trn-1, the concentrations of GFP (control) and 1412 GFP-fusion proteins were equalized within each group, and the binding was observed in the same 1413 conditions. The images are comparable within a group. 1414 Trn-2 (p9): GFP weakly binds to Trn-2, and the concentrations of GFP and GFP-fusion proteins 1415 were equalized. The images are comparable. Proteins whose ranks differed substantially between 1416 the Trn-1 and Trn-2 Z-ranking were assayed. 1417 Imp-13 (p10–13): GFP does not bind to Imp-13, and GFP was added to the control mixture at the 1418 highest concentration. Three images (GST, GST-Imp-13, and GST-Imp13 + Q69L-Ran) for each 1419 GFP-fusion protein were acquired under identical conditions, and the background intensities and

50 1420 dynamic ranges were equalized. 1421 Trn-SR (p14–16): GFP weakly binds to Trn-SR, and the procedures were similar to those used for 1422 Trn-1. The GFP-fusion proteins were divided into four groups (A–D), and the images are 1423 comparable within a group. 1424 Imp-α/β (p17–20): GFP does not bind to Imp-α or -β, and the procedures were similar to those used

1425 for Imp-13. GST-Imp-α2 lacks the N-terminal Imp-β-binding domain. 1426 Western blotting (p21–22) 1427 The GFP-fusion proteins in the E. coli extracts were relatively quantified by Western blotting using 1428 an anti-GFP antibody (Roche). The extracts containing the amounts of protein (ng) indicated at the 1429 bottoms were loaded. The arrowheads indicate the expected full-length products. The GFP-moieties 1430 including those of the partial products were quantified by chemiluminescence. GFP was used as the 1431 standard. The Western blots were replicated more than three times. 1432 Accessions and sequences (p23–27) 1433 The cDNAs were cloned from a HeLa cDNA library by PCR. The accession numbers of the 1434 proteins are listed. If the sequence of a used protein is different from that in the database, the 1435 deleted, substituting, or inserted amino acids are indicated by the colors. The sequences that 1436 matched perfectly are not presented. 1437 1438 Supplementary file 3. The 2nd-Z-15% cargoes of the 12 NTRs. 1439 The 2nd-Z-15% cargoes of each NTR are listed by the gene names in the 2nd-Z-rank orders. The 1440 ranks by the 3rd Z-scores are also shown. Cyan in the rank columns indicates the 2nd-Z-15% and 1441 3rd-Z-4% cargoes. Colors in the gene name columns: magenta, reported cargoes; blue, cargoes 1442 bound directly to the NTR in the bead halo assays (Supplementary file 2); light blue, cargoes bound 1443 directly to Imp-α but not Imp-β; gray, proteins that did not bind to the NTRs; yellow, Imp-α; and 1444 green, reported export cargoes. For the 3rd-Z-4% cargoes, see Figure 3. 1445 1446 Supplementary file 4. Example of extracted ion chromatograms (EICs) of peptides. 1447 EICs of GLUD1 peptides in the SILAC-Tp with Trn-1 1448 As a general problem of high-throughput LC-MS/MS quantitation, quantitative values of proteins 1449 with fewer quantified peptides deviate among the replicates. Referring to EICs of the quantified

51 1450 peptides is useful to avoid misidentification of cargoes. For example, the L/H ratios of a Trn-1 1451 2nd-Z-15% cargo GLUD1 (P00367, ranked 110th and 342nd by the 2nd and 3rd Z-score, 1452 respectively) deviated largely in the three replicates of SILAC-Tp with Trn-1 (Supplementary file 1). 1453 Panels (A–H) show EICs of the indicated peptide (trypsin targets, K and R, are written in lower 1454 cases) in the three (three Ctl and there +Trn) experiments (some peptides were not identified in all 1455 the experiments). Magenta letters indicate the quantified peptides and L/H rations. 1456 In panel (A), the elution time of the peptide TAMkYNLGLDLr differs largely between 1457 the expriment-1 Ctl and experiment-2 +Trn-1, the peak shape of the experiment-2 +Trn-1 is 1458 irregular, and the L/H ratio of it is much higher than those of other peptides in +Trn-1 experiments 1459 (B and E). Thus, there is concern about misidentification. Because the L/H count of GLUD1 in the 1460 experiment-2 +Trn-1 is two (TAMkYNLGLDLr and NLNHVSYGr, A and B) and the L/H ratio of a 1461 protein is defined as the median, the L/H ratio of GLUD1 in the expriment-2 +Trn-1 is affected by 1462 the L/H ratio of TAMkYNLGLDLr. Exclusion of the L/H ratio of TAMkYNLGLDLr in the 1463 experiment-2 +Trn-1 lowers the Z-score rank of GLUD1 significantly. 1464 In panel (C), the chromatogram of the peptide HGGTIPIVPTAEFQDr in the 1465 experiment-1 Ctl has an irregular peak, and the L/H ratio of it is much higher than those of other 1466 peptides in the Ctl experiments (A–H). Thus, overlap with other peptide or other failures may be 1467 possible. However, the L/H count of GLUD1 in the experiment-1 Ctl is four (TAMkYNLGLDLr, 1468 HGGTIPIVPTAEFQDr, ALASLMTYk, and GASIVEDkLVEDLr) and the value of 1469 HGGTIPIVPTAEFQDr does not affect the median. (The L/H ratio of TAMkYNLGLDLr, whose 1470 EIC differ between the experiment-1 Ctl and expriment-2 +Trn in (A) as mentioned above, may 1471 affect the L/H ratio of GLUD1 in the experiment-1 Ctl, but we assumed that it is reliable.) 1472 As above, the L/H ratios of proteins with low L/H counts (Supplementary file 1) may be 1473 affected by LC-MS/MS artifacts, and misidentification can be avoided by referring to the EICs. All 1474 the EICs and MS spectra in this work can be accessed by downloading the mass spectrometry data 1475 and Proteome Discoverer software (see the Materials and Methods and Supplementary file 12A). 1476 1477 Supplementary file 5. Redundancy of NTRs: Cargoes shared by NTRs. 1478 A. The numbers of the 2nd-Z-15% cargoes shared by two NTRs 1479 For the 3rd-Z-4% cargoes, see Figure 4.

52 1480 B. Redundancy of the 3rd-Z-4% cargoes 1481 Many proteins are included in the 3rd-Z-4% cargoes of multiple NTRs. The ranks of these cargoes 1482 in the 3rd-Z-rankings for all 12 NTRs are presented. 1483 C. Relationships between the NTRs and the characteristics of their 3rd-Z-4% cargo proteins 1484 The 3rd-Z-4% cargoes are grouped according to their characteristics (functions or biological 1485 processes that the proteins act in), and their ranks are presented as in (B). To make our points clear, 1486 typical terms for the protein characteristics and typical proteins related to the terms have been 1487 selected with reference to Gene Ontology (GO) and UniProt. Thus, the terms in this sheet are 1488 slightly different from those in the databases, fewer proteins than annotated in the databases are 1489 grouped, and the list is redundant. For the complete linkages between the GO terms and the 1490 3rd-Z-4% cargoes, see Supplementary file 7. 1491 D. Redundancy of the 2nd-Z-15% cargoes 1492 The ranks of the 2nd-Z-15% cargoes are presented as in (B). 1493 E. Relationships between the NTRs and the characteristics of their 2rd-Z-15% cargo proteins 1494 The 2nd-Z-15% cargoes are grouped, and their ranks are presented in a manner similar to that in (C). 1495 For the complete linkages between the GO terms and the 2nd-Z-15% cargoes, see Supplementary 1496 file 8. 1497 1498 Supplementary file 6. GO term enrichments of the identified cargoes. 1499 A. Extraction of the GO term enrichments of the 2nd-Z-15% cargoes 1500 The 2nd-Z-15% cargoes were analyzed for GO term enrichment in (C). The terms that were 1501 significantly enriched (p<0.05, cyan) in the 2nd-Z-15% cargoes of four or fewer NTRs were 1502 selected, and terms that represent many similar terms are presented. With the p-values, the numbers 1503 (#) of cargoes annotated with each of the terms are presented. Total No. represents the number of 1504 proteins annotated with each term in the database. Related terms are bundled in the same color. For 1505 the 3rd-Z-4% cargoes, see Figures 5 and 6. The correspondences between each 2nd-Z-15% cargo 1506 and GO term are summarized in Supplementary file 10. All the GO terms annotated to the 1507 2nd-Z-15% cargoes are listed in Supplementary file 8. 1508 B. Full table of the GO term enrichments of the 3rd-Z-4% cargoes 1509 The 3rd-Z-4% cargoes were analyzed for GO term enrichment. For all combinations of GO terms

53 1510 and NTRs, the p-values for the term enrichments in the 3rd-Z-4% cargoes and the numbers (#) of 1511 cargoes annotated with the terms are presented. The numbers following “# in” are the total numbers 1512 of 3rd-Z-4% cargoes. Total No. represents the number of proteins annotated with each term in the 1513 database. Cyan, p<0.05. Figures 5 and 6 were extracted from this table. This table was derived from 1514 Supplementary file 7, and see Supplementary file 7 to retrieve the protein accessions. 1515 C. Full table of the GO term enrichments of the 2nd-Z-15% cargoes 1516 The 2nd-Z-15% cargoes were analyzed and are presented in a manner similar to that in (B). (A) was 1517 extracted from this table. This table was derived from Supplementary file 8, and see Supplementary 1518 file 8 to retrieve the protein accessions. 1519 1520 Supplementary file 7. The 3rd-Z-4% cargoes annotated with GO terms. 1521 With respect to each NTR, the accessions of the 3rd-Z-4% cargoes annotated with each GO term are 1522 listed. Cyan, significant term enrichment (p<0.05) in the 3rd-Z-4% cargoes of the NTR. Total No. 1523 represents the number of proteins annotated with each term in the database. Supplementary files 6B 1524 and 9 were derived from this table. For the 2nd-Z-15% cargoes, see Supplementary file 8. 1525 1526 Supplementary file 8. The 2nd-Z-15% cargoes annotated with GO terms. 1527 With respect to each NTR, the accessions of the 2nd-Z-15% cargoes annotated with each GO term 1528 are listed. Cyan, significant term enrichment (p<0.05) in the 2nd-Z-15% cargoes of the NTR. Total 1529 No. represents the number of proteins annotated with each term in the database. Supplementary files 1530 6C and 10 were derived from this table. For the 3nd-Z-4% cargoes, see Supplementary file 7. 1531 1532 Supplementary file 9. Correspondences between the 3rd-Z-4% cargoes and GO terms. 1533 Each sheet shows the correspondences between the 3rd-Z-4% cargoes of one NTR and selected GO 1534 terms. A term annotation to a cargo is indicated by “1” in the corresponding cell. Reported cargoes 1535 are indicated by magenta in the gene name cells, and the results of the bead halo assays 1536 (Supplementary file 2) are also indicated by colors in the gene name cells: blue, cargoes directly 1537 bound to the NTR; light blue, cargoes directly bound to Imp-α but not Imp-β; gray, proteins that did 1538 not bind to the NTR. GO terms that represent many similar terms were selected from the terms 1539 enriched significantly (p<0.05) for the 3rd-Z-4% cargoes of each NTR, and broadly defined terms

54 1540 were deselected. Magenta and orange in the term ID cells indicate terms that are significantly 1541 enriched for the cargoes of four or fewer NTRs, and of them magenta indicates the terms presented 1542 in Figures 5 and 6. Related GO terms are bundled in the same color, and different colors are used to 1543 distinguish the columns easily. The NTRs added in the transport reactions (white in the rank cells) 1544 were not analyzed. This table was derived from Supplementary file 7. For 2nd-Z-15% cargoes, see 1545 Supplementary file 10. 1546 1547 Supplementary file 10. Correspondences between the 2nd-Z-15% cargoes and GO terms. 1548 Each sheet shows the correspondences between the 2nd-Z-15% cargoes of one NTR and selected 1549 GO terms. A term annotation to a cargo is indicated by “1” in the corresponding cell. Reported 1550 cargoes are indicated by magenta in the gene name cells, and the results of the bead halo assays 1551 (Supplementary file 2) are also indicated by colors in the gene name cells: blue, cargoes directly 1552 bound to the NTR; light blue, cargoes directly bond to Imp-α but not Imp-β; gray, proteins that did 1553 not bind to the NTR. GO terms that represent many similar terms were selected from the terms 1554 enriched significantly (p<0.05) for the 2nd-Z-15% cargoes of each NTR, and broadly defined terms 1555 were deselected. Magenta and orange in the term ID cells indicate terms that are significantly 1556 enriched for the cargoes of four or fewer NTRs, and of them magenta indicates the terms presented 1557 in Supplementary file 6A. Related GO terms are bundled in the same color, and different colors are 1558 used to distinguish the columns easily. The NTRs added in the transport reactions (white in the rank 1559 cells) were not analyzed. This table was derived from Supplementary file 8. For 3rd-Z-4% cargoes, 1560 see Supplementary file 9. 1561 1562 Supplementary file 11. mRNA processing factors, ribosomal proteins, and transcription 1563 factors. 1564 A. Ranks of mRNA processing factors 1565 All of the 2nd-Z-15% and 3rd-Z-4% cargoes that are annotated with mRNA processing in Gene 1566 Ontology (GO) are listed with the ranks in the 2nd- and 3rd-Z-rankings. The color scale is set by 1567 percentile rank as indicated. Figure 7 was extracted from this table. 1568 B. The 3rd-Z-rankings of ribosomal proteins 1569 The ranks of the ribosomal proteins in the 3rd-Z-rankings of the 12 NTRs are presented. The color

55 1570 scale is set by percentile rank as indicated. For the 2nd-Z-rankings, see Figure 8. 1571 C. Extracts of the GO term enrichments of the transcription factors found in the 2nd-Z-15% cargoes 1572 Seventeen to 36 proteins in the 2nd-Z-15% cargoes of each NTR are annotated with "transcription 1573 factor activity, sequence-specific DNA binding" or "transcription factor activity, protein binding" in 1574 GO. The factors were analyzed for GO term enrichment (term type, biological process, BP) in (D). 1575 Typical terms that represent similar terms were extracted from (D). The p-value for the term 1576 enrichment and the number (#) of factors annotated with the term are presented. Total No. 1577 represents the number of proteins annotated with each term in the database. Cyan, p<0.05. Related 1578 terms are bundled in the same color. 1579 D. Full table of the GO term enrichments of the transcription factors found in the 2nd-Z-15% 1580 cargoes 1581 The 2nd-Z-15% cargoes that are annotated with "transcription factor activity, sequence-specific 1582 DNA binding" or "transcription factor activity, protein binding" in GO were analyzed for GO term 1583 enrichment (term type, BP). The analyzed transcription factors are listed at the bottom. For all of the 1584 combinations of GO terms and NTRs, the p-value for the term enrichment and the number (#) of 1585 transcription factors annotated with the term are presented. The numbers following “# in” are the 1586 total numbers of transcription factors in the 2nd-Z-15% cargoes. Total No. represents the number of 1587 proteins annotated with each term in the database. Cyan, p<0.05. (C) was extracted from this table. 1588 E. SRSRSR motif in the 3rd-Z-4% cargoes 1589 The 3rd-Z-4% cargoes that contain an "SRSRSR" hexa-peptide sequence were counted. 1590 1591 Supplementary file 12. 1592 A. MS data files. 1593 The mass spectrometry proteomics data (.msf and .raw files) have been deposited to the 1594 ProteomeXchange Consortium (http://www.proteomexchange.org/) with the dataset identifier 1595 PXD004655. The results of protein and peptide identification and quantitation are summarized in 1596 Supplementary file 1, and the .msf and .raw data files corresponding to each experiment in 1597 Supplementary file 1 are listed in this table. The quantitation results can be seen by opening .msf 1598 files by Proteome Discoverer software. To see spectra and chromatograms, .msf files and 1599 corresponding .raw files must be in the same local directory. A demo version of Proteome

56 1600 Discoverer can be downloaded at the Thermo Scientific omics software portal site 1601 (https://portal.thermo-brims.com/). 1602 B. GFP-fusion proteins used for in vitro transport and antibodies.

57 A B PY- PY- 2nd Z-score Rank 3rd Z-score NLS NLS Rate of PY-NLS proteins 5 4 3 2 1 0 -1 0 1 2 3 4 5 (% in 50 consecutive proteins) 1 Ranked by 2nd Z-score Ranked by 3rd Z-score Rank 10 40 30 20 10 0 0 10 20 30 40 50 1 20 100 30

40 200

50 300 60

400 70

80 500

90 600 100

110 700

120 800

130 900 140

150 1000

160 1100 170 1200 180

190 1300

200 1400 210 1500 220

230 1600

240 Basic PY-NLS: [Basic segment]X1–7[RKH]X2–6PY Basic segment: 5–17 aa Ratio of K, R or H >0.25 250 Both termini, K, R or H Reported Cargo: 27 Reported Cargo: 25 Quantified Protein: 1649 Quantified Protein: 1235 Hydrophobic PY-NLS: [FYKVRQP][GAS][VMPKSDE][VMSPQR]X [RKH]X PY Direct Binding: Blue, Positive; Dark Gray, Negative; Gray, Not Determined 3–15 0–9 A Heavy Light C SILAC Imp-β – + Imp-β – + u-13C6 Arg, u-13C6 Lys GFP-IBB-GST GFP-RPL23A Digitonin

Trn-1 – + Trn-2 – + Permeabilized Cells GFP-SNRPA1 GFP-ELAVL1

Nuclear Extract Trn-SR – + Imp-4 – + Import Import In vitro Transport ±NTR GFP-RBM4 GFP-RPS3A

Imp-5 – + Imp-7 – +

GFP-RPL23A GFP-HHEX Wash

Imp-8 – + Imp-9 – +

GFP-SRP19 GFP-PPP2R1A Protein Extraction

Imp-11 – + Imp-13 – +

Trypsin Digestion, LC-MS GFP-RPL12 GFP-UBE2I

Exp-4 – + B GFP-SOX2 Cytosolic Extract Nuclear Extract Depletion – + – + Imp-β Trn-1 Trn-SR Imp-4 Imp-5 Imp-7 Imp-9 Imp-11 * Imp-13 Exp-4 * A 500 B 500

r 400 r 400

300 300

200 200

100 100 Protein Numb e Protein Numb e

0 0 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 L/HTrn1 Z-score log2 L/HCtl C 10 8 6 4 2 score

- 0 Z -2 -4 -6 -8 10 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 Rank by 2nd Z-score D 6 5 4 3

2 1 score

- 0 Z -1 -2 -3 -4 -5 01 100 200 300 400 500 600 700 800 900 1000 1100 1200 Rank by 3rd Z-score

E 7 F 5 6 5 4 4 3 3 2 2 score score - - Z 1 Z 0 1 -1 0 -2 -3 -1 01 20 40 60 80 100 120 140 160 180 200 10 20 40 60 80 100 120 140 160 180 200 Rank by 2nd Z-score Rank by 3rd Z-score A B 1 1 (Negative examples) 0.9 0.9 3rd Z (Non-nuclear) 0.8 0.8 2nd Z (Non-nuclear) 3rd Z (Unreported) 0.7 0.7 2nd Z (Unreported) 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 Recall (Sensitivity) 2nd Z-score Reported Cargo Rate 0.2 0.2 0.1 0.1 3rd Z-score 0 0 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 % Rank 100%=1649 (2nd Z) 1235 (3rd Z) % Rank A Trn-1 B Imp-13 C Trn-SR Rank 2nd Z-score 3rd Z-score Rank 2nd Z-score 3rd Z-score Rank 2nd Z-score 3rd Z-score 1 ATF1 ATF1 1 NFYB NFYB 1 PMPCA SRRM2 2 KHDRBS1 KHDRBS1 2 KDM2A LRRC59 2 TNPO3 TRAP1 3 MRTO4 MRTO4 3 S100P GTF2A2 3 SRRM2 PHF6 4 HNRNPM HNRNPM 4 PRPSAP1 MAGOH 4 AIP SRSF6 5 TUBB3 BYSL 5 LRRC59 RBM8A 5 ERC1 SRSF8 6 HNRNPAB HNRNPA1 6 GTF2A2 CHRAC1 6 DCTN4 PLK1 7 HNRNPA1 HNRNPAB 7 UGGT1 POLE3 7 TRAP1 GOLIM4 8 HNRNPD ALYREF 8 MAGOH DDX27 8 SRSF2 AIP 9 BYSL HNRNPA3 9 RBM8A NFYA 9 THOC1 SRSF7 10 ALYREF HNRNPD 10 NOC2L NOC2L 10 PHF6 CNOT10 11 HNRNPA0 HNRNPA0 11 DHRS4 NQO2 11 PRPF38B SRSF2 12 QKI PABPN1 12 DDX27 SRSF8 12 KIF2C ZNF281 13 HNRNPA3 HEXIM1 13 CD3EAP TMEM43 13 ALDH18A1 DHX30 14 HEXIM1 EWSR1 14 UAP1 ARPC5 14 PAF1 ZC3H4 15 NUP205 HNRNPA2B1 15 GALE TXNDC17 15 ATP5E ACOT9 16 HNRNPA2B1 PQBP1 16 TPM2 SFPQ 16 SRSF6 DYNC1LI1 17 SLIRP ELAVL1 17 SHCBP1 CDC42BPB 17 ATAD2 SRGAP2 18 GOLGA2 ZNF787 18 PHF10 NAPRT1 18 SCAF8 SRSF1 19 EWSR1 HNRNPH2 19 CHRAC1 BOLA2 19 SRSF8 PLOD3 20 NFYA MACROD1 20 DIDO1 TIGAR 20 ACOT9 SUB1 21 PABPN1 FUS 21 OGDH RBM27 21 PLK1 PAF1 22 IVD PDLIM1 22 DDX23 COMT 22 GOLIM4 MAP7D1 23 TIMM13 UBQLN4 23 POLE3 RPL37 23 ITPR1 SAP18 24 PDLIM1 MAZ 24 CAPN1 SUMO2 24 KIF15 LSM14A 25 PBK HNRNPH1 25 ARHGEF7 RAVER1 25 SDHA NELFE 26 S100A6 PNN 26 AGPS UGGT1 26 SRSF7 CD2AP 27 EEF1A2 CLTA 27 ENAH MAP7 27 CCNB1 C9orf78 28 NOP56 TIGAR 28 UNC45A CAPN1 28 HADHA SLTM 29 CBX5 HNRNPF 29 PDHA1 SMARCE1 29 ZC3H4 CBX1 30 CARHSP1 PPP2R1B 30 PDCD11 ENOPH1 30 TARS2 WDR12 31 PQBP1 SAP18 31 AKAP13 ZYX 31 MLF2 CWF19L1 32 CPNE1 FAH 32 FDFT1 EEF2 32 XRN2 DBNL 33 AARS C5orf24 33 NFYA PRC1 33 CNOT10 DNAJB1 34 HNRNPH1 DNPEP 34 SRSF8 TIMM8A 34 PGRMC1 RBM12 35 SSBP1 EXOSC10 35 IPO4 WDR18 35 TTC9C TTK 36 DDT HNRPDL 36 MCCC2 CBS 36 DDX27 NAT10 37 COTL1 HIST1H4A 37 SLC9A3R1 PHF10 37 CBX1 DHX38 38 MT2A SMC3 38 UBE2O NAT10 38 ZNF281 UBR4 39 AKR1C1 FAHD1 39 MKI67 SUMO3 39 WBP11 DHX36 40 OLA1 DAZAP1 40 DYNC1LI1 GNPDA1 40 PDE3A DKC1 41 HNRNPH2 LONP1 41 PBK DBI 41 RPL7L1 ILKAP 42 EIF5A SMARCC1 42 TRMT10C BROX 42 POP4 CCNB1 43 LASP1 NOP56 43 CTTN EEFSEC 43 ADAR RPS6KA3 44 TMSB4X LRPAP1 44 METAP2 GOLGA4 44 ANXA3 TATDN1 45 ETFA HPD 45 CARHSP1 CLTC 45 SNX5 SRSF3 46 FUS MAGOH 46 ARPC1A IKBKAP 46 BLVRA PQBP1 47 TRIP13 BUD31 47 PELP1 G6PD 47 PYCR2 CDK2 48 NAP1L4 ARL6IP4 48 PDLIM5 GN=S100A13 48 AAAS DDX21 49 AKR1C3 MPST 49 TARS2 AHNAK 49 FUBP1 CSTF1 50 MCTS1 SUPT6H 50 YAP1 ZNF787 50 RBM25 DHX15 51 ELAVL1 PRPF38B 51 ATP6V1H GEMIN5 51 TK1 MPG 52 CMBL EXOSC5 52 SARS GRWD1 52 NADKD1 DNAJB4 53 ACTR2 BZW1 53 EIF4H SRSF2 53 PPIG CCDC101 54 GLRX3 NIT2 54 PDCD5 PPIE 54 CARHSP1 THOC1 55 ANXA11 NHP2 55 EDF1 SRSF3 55 SRSF1 PPP1CA 56 UQCRC2 CDC5L 56 EEF2 PELP1 56 RAVER1 RTFDC1 57 C5orf24 SHMT1 57 KTN1 TXLNG 57 EFHD2 FAM83D 58 PIR GRHPR 58 NNT UBE2I 58 SNX1 DDX5 59 ACTN1 EXOSC1 59 SH3BGRL APEH 59 EIF1AX PPIL1 60 EML4 POLR2I 60 NQO2 KRT8 60 MDC1 WDR3 61 CKS2 LACTB2 61 S100A4 LIN7C 61 DHX30 CPSF7 62 CKM WBP11 62 ENOPH1 UTP18 62 CPSF7 RAB8A 63 CNBP CHD4 63 ARPC5 PPP1R12A 63 SLC9A3R1 U2AF1 64 GFPT1 GSN 64 CLIC1 RPL11 64 COX4I1 HK2 65 PWP1 PGM2 65 CKM MARCKSL1 65 SAMHD1 LDB1 66 PFN1 TRNT1 66 AKR1C3 KRT17 66 SMN1 FTH1 67 STMN1 QDPR 67 AARS UFL1 67 ISOC1 ZRANB2 68 ACTN4 PPP6C 68 EIF5A HNRPDL 68 GTF2F1 SNX1 69 PRC1 ECM29 69 TUBB4B TXLNA 69 PES1 HMGB1 70 OTUB1 CTNNA1 70 AKR1C1 PABPC4 70 DHX38 CUL3 71 DSTN GDA 71 MYL12A SPAG9 71 SRGAP2 UBE2M 72 THOC1 PLIN3 72 PAWR ZC3HAV1 72 SRSF9 SEC13 73 BROX RBM8A 73 BOLA2 ARPC5L 73 SNX12 ACTR10 74 CLUH AIMP2 74 ARL6IP5 GSTM3 74 NAT10 PDHA1 75 CSRP1 COX17 75 TXLNA TXN 75 GBE1 REXO2 76 TPT1 NPM3 76 TIGAR IWS1 76 NUMA1 PPP5C 77 HINT1 RBMX 77 HN1 SMU1 77 NFKB1 EMG1 78 PDCD5 RANGAP1 78 CHMP4A OGDH 78 PLOD3 RAE1 79 NAP1L1 CDC73 79 REXO4 RALY 79 RTFDC1 CCDC6 80 MESDC2 TRMT1L 80 RAN RAB21 80 MRPL11 SIN3B 81 PSMD10 CDK11A 81 PLOD2 XRN2 81 DYNC1LI1 PRC1 82 SCLY SEC16A 82 CNBP GTF2F2 82 ANXA4 LSG1 83 CTPS1 CAND1 83 SRSF2 MYO1C 83 DDX21 ANXA4 84 COX17 PRDX5 84 EIF1AX HDAC3 84 MPST PPP1CC 85 ACTC1 TCEB2 85 IPO5 CWC15 85 DDX47 THOC3 86 ZNF787 TATDN1 86 NAPRT1 TPM3 86 CD2AP THYN1 87 PDLIM5 TBL1XR1 87 S100A6 NCBP2 87 SAP18 PFDN4 88 GBE1 EIF6 88 CSRP1 WRNIP1 88 ACO2 KIAA0101 89 SLC25A3 CIAPIN1 89 SUMF2 PSME4 89 GATAD2A PPIE 90 PCBP2 PRCC 90 EEF1A1 CLUH 90 SRSF3 HMGB3 91 TXN RARS 91 TAGLN2 RPL31 91 PPP1R12A CSNK1A1 92 CTTN MTA2 92 PLIN2 SNCG 92 CAMK2G SCFD1 93 PRPF6 NTPCR 93 PFN1 FLNB 93 PNN CKAP5 94 CDA MARS 94 ELAVL1 SRSF7 94 SMU1 FBL 95 HNRNPF AK2 95 BLVRA TUBG1 95 RAB13 SCRIB 96 MACROD1 HYOU1 96 STRAP PABPC1 96 NOP58 DBR1 97 MDN1 COA4 97 PXN DDX17 97 MED15 TPRKB 98 KPNA2 CNDP2 98 TMEM43 COA4 98 SUB1 FEN1 99 UBQLN4 SCLY 99 CDC42BPB PXN 99 DKC1 CSTF2 100 DBI PEPD 100 RBM27 ELAVL1 100 CWF19L1 FKBP2 8 7 A 7 B 6 6 5 5

4 3 4 2 3 score

1 score - - Z 0 Z 2 -1 -2 1 -3 -4 0 -5 -1 10 20 40 60 80 100 120 140 160 180 200 10 20 40 60 80 100 120 140 160 180 200 Rank by 2nd Z-score Rank by 3rd Z-score A Imp-13 B Trn-SR 2nd Z-score Rank 3rd Z-score 2nd Z-score Rank 3rd Z-score 6 5 4 3 2 1 0 -1 0 1 2 3 4 4 3 2 1 0 -1 0 1 2 3 1 1

10 10

20 20

30 30

40 40

50 50

60 60

70 70

80 80

90 90

100 100

110 110

120 120

130 130

140 140

150 150

160 160

170 170

180 180

190 190

200 200

210 210

220 220

230 230

240 240

250 250 Reported Cargo: 8 Reported Cargo: 7 Reported Cargo: 3 Reported Cargo: 2 Quantified Protein: 2060 Quantified Protein: 1671 SR-rich Splicing Factor: 7 SR-rich Splicing Factor: 5 RS-domain: 4 RS-domain: 3 Quantified Protein: 2021 Quantified Protein: 1591 Direct Binding: Blue, Positive; Dark Gray, Negative; Gray, Not Determined A Imp-β B Imp-β Rank 2nd Z-score 3rd Z-score 1 KPNB1 KPNB1 2nd Z-score Rank 3rd Z-score 2 RNMT RNMT 3 FAM103A1 FAM103A1 5 4 3 2 1 0 -1 0 1 2 3 4 4 KIFC1 KIFC1 5 TSR1 TSR1 1 6 PBRM1 LRRC59 7 EGFR NCBP2 8 GLUD1 NCBP1 10 9 MLF2 ALKBH5 10 H1F0 NUP214 11 POLR1E SRRT 20 12 RPN1 NUP88 13 ATP5B GTF2F1 14 HADHA HELLS 30 15 ATF7 API5 16 NTMT1 CHD8 17 NADKD1 POLD3 18 LONP1 RCC1 40 19 NCBP2 ZNF787 20 LRRC59 MYOF 21 NUP214 MTA1 50 22 HELLS SHMT2 23 MYOF ALYREF 24 DDX55 GTF2F2 60 25 NCBP1 CTCF 26 OGDH NOP9 27 ALKBH5 ZNF24 28 TRMT10C PMS2 70 29 KHSRP WDHD1 30 NUP88 MAZ 31 NFYA LDB1 80 32 UBR4 MNAT1 33 MTA1 ZNF146 34 OXCT1 TPX2 35 GTF2F1 OGDH 90 36 UQCRC1 CCDC86 37 SRRT POLR3C 38 GNL3L THYN1 100 39 PRPSAP1 SMARCE1 40 DSG2 POLD2 41 BAZ1B CYR61 110 42 HIST1H4A SPIN1 43 WDR43 CCDC58 44 DARS2 HMGA1 45 ZNF787 LMNA 120 46 RCC1 SLTM 47 GNS U2SURP 48 CYR61 TCERG1 130 49 ZNF691 PES1 50 C5orf24 HNRPLL 51 ESYT2 KPNA2 140 52 NQO2 HMGN3 53 API5 RBM27 54 CTSD KHSRP 55 NSUN5 SUMF2 150 56 CLK3 HNRPDL 57 RBM27 TAF15 58 CHD8 DKC1 160 59 GDI1 HNRNPUL2 60 WDHD1 C9orf78 61 GTF2F2 MRGBP 62 SNW1 HNRNPU 170 63 UAP1 OAT 64 GRSF1 ATP5A1 65 SMARCA4 RCC2 180 66 LDB1 MPLKIP 67 ZNF24 SNRNP40 68 DDX5 ITPR1 190 69 HSPD1 CCNH 70 POLD3 THOC1 71 NDUFAF4 FUBP1 72 PNISR HNRNPA1 200 73 NOP9 SMU1 74 KIF2C RPL37 75 OAT LARP4 210 76 BROX PTRF 77 NUP188 UTP14A 78 DDX27 ELAVL1 220 79 MRGBP SFPQ 80 POLD2 RSL1D1 81 KPNA2 REEP5 82 SHMT2 SARNP 230 83 ITPR1 TCOF1 84 ALYREF ISOC1 85 RBM39 PITRM1 240 86 SLTM DDX5 87 CHERP POLR3F 88 DECR1 DUS3L 250 89 SLC25A3 POLR1A 90 TCOF1 SNRNP70 Reported Direct Cargo: 1 Reported Direct Cargo: 1 91 CDC42BPB NT5DC1 Quantified Protein: 2027 Quantified Protein: 1614 92 GGCT ACIN1 93 DDX23 GATAD2A Yellow, Imp-α 94 CTCF ATP5B 95 PMS2 DHX36 Direct Binding: Blue, Bind to Imp-β; Light Blue, Bind to Imp-α but Not Imp-β; 96 CD3EAP FBL 97 SMARCE1 FUS Dark Gray, Negative; Gray, Not Determined 98 ANLN RAP1B 99 ILKAP CHCHD2P9 100 TPX2 GATAD1 Imp- Trn-1 Trn-2 Trn-SR Imp-4 Imp-5 Z-score Z-score Z-score Z-score Z-score Z-score Rank Gene Name Rank Gene Name Rank Gene Name Rank Gene Name Rank Gene Name Rank Gene Name 3rd 2nd 3rd 2nd 3rd 2nd 3rd 2nd 3rd 2nd 3rd 2nd 1 1 KPNB1 1 1 ATF1 1 1 ATF1 1 3 SRRM2 1 2 IPO4 1 1 IPO5 2 2 RNMT 2 2 KHDRBS1 2 10 ETFA 2 7 TRAP1 2 8 PFKL 2 11 NFYA 3 3 FAM103A1 3 3 MRTO4 3 4 KHDRBS1 3 10 PHF6 3 10 BMS1 3 17 DDX56 4 4 KIFC1 4 4 HNRNPM 4 5 HNRNPM 4 16 SRSF6 4 3 TFRC 4 42 HK2 5 5 TSR1 5 9 BYSL 5 9 MRTO4 5 19 SRSF8 5 18 MFAP1 5 26 TP53I3 6 20 LRRC59 6 7 HNRNPA1 6 7 HNRNPA1 6 21 PLK1 6 25 ALPP 6 27 BOP1 7 19 NCBP2 7 6 HNRNPAB 7 14 HNRNPA3 7 22 GOLIM4 7 32 FKBP10 7 6 THOC1 8 25 NCBP1 8 10 ALYREF 8 13 HNRNPA0 8 4 AIP 8 55 NELFE 8 132 SPIN1 9 27 ALKBH5 9 13 HNRNPA3 9 16 ALYREF 9 26 SRSF7 9 17 RBMX 9 146 FBL 10 21 NUP214 10 8 HNRNPD 10 12 HNRNPAB 10 33 CNOT10 10 31 SFPQ 10 117 C5orf24 11 37 SRRT 11 11 HNRNPA0 11 22 PABPN1 11 8 SRSF2 11 90 CALU 11 156 PDS5B 12 30 NUP88 12 21 PABPN1 12 8 HNRNPD 12 38 ZNF281 12 19 SLTM 12 189 RBM39 13 35 GTF2F1 13 14 HEXIM1 13 23 EWSR1 13 61 DHX30 13 88 ESYT1 13 52 LSM14A 14 22 HELLS 14 19 EWSR1 14 34 DECR1 14 29 ZC3H4 14 115 HNRNPUL2 14 165 ENOPH1 15 53 API5 15 16 HNRNPA2B1 15 19 HNRNPA2B1 15 20 ACOT9 15 42 PLK1 15 44 DIDO1 16 58 CHD8 16 31 PQBP1 16 69 UBTF 16 81 DYNC1LI1 16 89 CMAS 16 238 CDC42BPB 17 70 POLD3 17 51 ELAVL1 17 42 HNRNPH2 17 71 SRGAP2 17 12 PICALM 17 195 XRN2 18 46 RCC1 18 86 ZNF787 18 43 FUS 18 55 SRSF1 18 119 RCC1 18 175 U2AF1 19 45 ZNF787 19 41 HNRNPH2 19 29 HEXIM1 19 78 PLOD3 19 101 PRCC 19 131 MAP7D1 20 23 MYOF 20 96 MACROD1 20 31 PQBP1 20 98 SUB1 20 13 BCLAF1 20 150 SNX5 21 33 MTA1 21 46 FUS 21 37 ELAVL1 21 14 PAF1 21 111 PPIL3 21 278 DEK 22 82 SHMT2 22 24 PDLIM1 22 30 NOP56 22 130 MAP7D1 22 67 HSPA9 22 142 RAVER1 23 84 ALYREF 23 99 UBQLN4 23 40 HNRNPH1 23 87 SAP18 23 129 DHX9 23 94 MFAP1 24 61 GTF2F2 24 119 MAZ 24 91 CTR9 24 118 LSM14A 24 130 HMGB1 24 76 TUFM 25 94 CTCF 25 34 HNRNPH1 25 123 ATP2A2 25 123 NELFE 25 131 FTH1 25 22 NADKD1 26 73 NOP9 26 162 PNN 26 28 PRPF6 26 86 CD2AP 26 165 RAB7A 26 145 CPSF7 27 67 ZNF24 27 122 CLTA 27 17 UTP18 27 141 C9orf78 27 71 CHCHD2P9 27 51 PTPN1 28 95 PMS2 28 195 TIGAR 28 52 HNRNPF 28 101 SLTM 28 147 DNAJC8 28 308 GBE1 29 60 WDHD1 29 95 HNRNPF 29 45 APRT 29 37 CBX1 29 211 RPL36AL 29 119 GNB2 30 109 MAZ 30 182 PPP2R1B 30 206 ARL6IP4 30 223 WDR12 30 92 SHMT2 30 368 CD44 31 66 LDB1 31 206 SAP18 31 185 USP39 31 100 CWF19L1 31 163 ISOC1 31 184 PHF6 32 127 MNAT1 32 279 FAH 32 50 C5orf24 32 204 DBNL 32 180 ZMYM3 32 153 MBD2 33 144 ZNF146 33 57 C5orf24 33 65 MAZ 33 198 DNAJB1 33 83 H1FX 33 188 DDX46 34 100 TPX2 34 319 DNPEP 34 300 SAP18 34 159 RBM12 34 191 GOT2 34 226 ZNF146 35 26 OGDH 35 192 EXOSC10 35 105 BUD31 35 180 TTK 35 262 AQR 35 12 SNX12 36 149 CCDC86 36 134 HNRPDL 36 228 DAZAP1 36 74 NAT10 36 214 KIF1BP 36 420 GAR1 37 128 POLR3C 37 187 HIST1H4A 37 416 RPL32 37 70 DHX38 37 229 GTF2F2 37 418 LUC7L 38 119 THYN1 38 147 SMC3 38 450 RPL22 38 108 UBR4 38 246 PCID2 38 182 SNRNP27 39 97 SMARCE1 39 331 FAHD1 39 383 ATL3 39 295 DHX36 39 269 SCFD1 39 218 U2AF2 40 80 POLD2 40 233 DAZAP1 40 239 RAB9A 40 99 DKC1 40 79 PLIN2 40 326 MAZ 41 48 CYR61 41 276 LONP1 41 217 RAB7A 41 219 ILKAP 41 179 CDKN2A 41 114 PLOD3 42 155 SPIN1 42 378 SMARCC1 42 272 LACTB2 42 27 CCNB1 42 198 CWF19L1 42 380 SRSF7 43 121 CCDC58 43 28 NOP56 43 287 CTNND1 43 177 RPS6KA3 43 173 HMGB3 43 367 HSPA9 44 180 HMGA1 44 116 LRPAP1 44 144 ZYX 44 235 TATDN1 44 94 MATR3 44 370 ISOC1 45 130 LMNA 45 499 HPD 45 425 PRRC2A 45 90 SRSF3 45 37 EIF1AX 45 379 SRSF3 46 86 SLTM 46 388 MAGOH 46 85 HNRNPH3 46 150 PQBP1 46 207 HMGB2 46 449 RAB31 47 173 U2SURP 47 124 BUD31 47 491 RPL11 47 353 CDK2 47 272 POLR2L 47 510 SDHA 48 185 TCERG1 48 321 ARL6IP4 48 417 RPL23A 48 83 DDX21 48 123 RFC5 48 369 SRSF8 49 189 PES1 49 376 MPST 49 141 PNN 49 384 CSTF1 49 24 AKAP8 49 344 PLRG1 50 145 HNRPLL 50 255 ACSL4 50 406 DHX15 50 110 GPKOW 50 451 NUCKS1 51 81 KPNA2 51 161 MPG 51 514 DDX17 52 188 HMGN3 52 336 DNAJB4 52 479 LUC7L2 53 57 RBM27 53 416 CCDC101 53 137 HNRNPA0 54 29 KHSRP 54 9 THOC1 54 155 ACTR10 55 191 SUMF2 55 318 PPP1CA 55 306 RPL22 56 222 HNRPDL 56 79 RTFDC1 56 357 ARL6IP4 57 225 TAF15 57 275 FAM83D 57 271 UBE4B 58 263 DKC1 58 116 DDX5 58 275 PLIN2 59 141 HNRNPUL2 59 466 PPIL1 59 477 HNRNPL 60 219 C9orf78 60 266 WDR3 60 527 PRC1 61 79 MRGBP 61 62 CPSF7 61 500 SIN3B 62 123 HNRNPU 62 320 RAB8A 62 375 RBM26 63 75 OAT 63 202 U2AF1 63 466 ADAR 64 168 ATP5A1 Imp-7 Imp-8 Imp-9 Imp-11 Imp-13 Exp-4 Z-score Z-score Z-score Z-score Z-score Z-score Rank Gene Name Rank Gene Name Rank Gene Name Rank Gene Name Rank Gene Name Rank Gene Name 3rd 2nd 3rd 2nd 3rd 2nd 3rd 2nd 3rd 2nd 3rd 2nd 1 2 TRAP1 1 4 ZNF787 1 3 H2AFZ 1 9 RALY 1 1 NFYB 1 3 DDX39B 2 9 ZNF787 2 6 TFRC 2 1 ACAT1 2 12 TFRC 2 5 LRRC59 2 4 DDX39A 3 25 ERP44 3 17 HSPD1 3 13 CYR61 3 34 AKR1C1 3 6 GTF2A2 3 9 JUP 4 23 HMGB2 4 12 NOP56 4 16 RALY 4 24 EIF4A3 4 8 MAGOH 4 23 VIM 5 37 HIST1H4A 5 3 RALY 5 4 FUBP1 5 53 RBM25 5 9 RBM8A 5 25 XRN2 6 47 HNRNPUL1 6 34 MAZ 6 5 RBMX 6 11 ACO2 6 19 CHRAC1 6 8 TMPO 7 29 ZNF207 7 40 RPL11 7 44 GOT2 7 36 ALDH18A1 7 23 POLE3 7 24 HSPD1 8 51 ACADVL 8 42 RPL22 8 40 IVD 8 91 ACP1 8 12 DDX27 8 35 ALDH1B1 9 63 BUB3 9 28 GOT2 9 45 TFRC 9 43 ESYT2 9 33 NFYA 9 31 CS 10 16 VIM 10 7 HIST1H4A 10 18 CS 10 47 RBM39 10 10 NOC2L 10 67 TFRC 11 40 NUP155 11 36 SLC25A1 11 7 PRPF6 11 120 GLOD4 11 60 NQO2 11 7 SLTM 12 80 DIDO1 12 60 ZNF24 12 31 HSPA9 12 41 LRRFIP1 12 34 SRSF8 12 34 TOMM70A 13 53 HMGB3 13 25 NNT 13 9 HNRNPUL1 13 124 MACROD1 13 98 TMEM43 13 96 PSIP1 14 44 FKBP10 14 88 ARMC1 14 12 SUPT16H 14 140 CKAP5 14 63 ARPC5 14 13 NOP56 15 65 ZNF146 15 89 RAB14 15 84 BMS1 15 25 IL18 15 174 TXNDC17 15 75 ALYREF 16 72 GOT2 16 19 DDX21 16 75 ZNF787 16 173 RDX 16 143 SFPQ 16 82 HNRNPA0 17 83 ZNF24 17 67 RAB1A 17 55 KMT2A 17 185 MSN 17 99 CDC42BPB 17 120 GTF2I 18 70 SDHA 18 77 RBM3 18 78 MAZ 18 49 AFG3L2 18 86 NAPRT1 18 89 FAM32A 19 39 SHMT2 19 21 NFYA 19 47 SHMT2 19 127 LGALS1 19 73 BOLA2 19 98 ZNF638 20 102 H1FX 20 99 RAB2A 20 25 DHX30 20 116 TMA7 20 76 TIGAR 20 103 CPSF6 21 105 SRSF1 21 43 SNX27 21 151 SRRM2 21 177 U2AF1 21 100 RBM27 21 78 RBMX 22 68 SRSF2 22 61 RAB8A 22 146 SART1 22 143 NEDD8 22 111 COMT 22 105 SUMF2 23 115 LUC7L3 23 63 FKBP10 23 157 H1F0 23 62 HIST1H4A 23 130 RPL37 23 122 RRBP1 24 89 OXCT1 24 103 LGALS1 24 62 PDHB 24 172 DTYMK 24 118 SUMO2 24 115 FBL 25 86 SRSF7 25 78 RAB21 25 191 PRPF3 25 187 APRT 25 226 RAVER1 25 135 EXOSC1 26 173 PRPF6 26 10 SHMT2 26 130 WDR74 26 167 LANCL2 26 7 UGGT1 26 42 SFPQ 27 67 FXR1 27 106 GOLPH3 27 211 RPL27A 27 108 MRTO4 27 224 MAP7 27 134 THBS1 28 148 RSRC2 28 127 DDX3X 28 30 JUP 28 216 VPS45 28 24 CAPN1 28 136 RBM26 29 132 MAZ 29 101 RAB1B 29 131 ACAA2 29 221 NCOR2 29 234 SMARCE1 29 156 NONO 30 217 SF3B5 30 93 RAB10 30 23 DDX17 30 110 TBL1XR1 30 62 ENOPH1 30 155 LUC7L2 31 50 TUFM 31 125 HK1 31 81 RPS7 31 186 ARHGDIA 31 250 ZYX 31 62 CPSF7 32 166 CDC5L 32 146 THBS1 32 194 PPP1R10 32 255 RPL35A 32 56 EEF2 32 193 CDC73 33 146 ACIN1 33 29 CCAR1 33 229 SF3B5 33 162 CLINT1 33 132 PRC1 33 70 ARL6IP4 34 81 SRSF11 34 134 LUC7L3 34 92 ALYREF 34 121 ACYP1 34 279 TIMM8A 34 66 U2AF1 35 207 SNRPA1 35 148 U2AF1 35 166 WBSCR22 35 98 RAD23A 35 292 WDR18 35 93 RBM39 36 208 ARL6IP4 36 20 VIM 36 43 MYOF 36 215 TXNL1 36 172 CBS 36 214 ERH 37 183 SNRPB2 37 165 NUDT21 37 214 DDX24 37 101 GDI2 37 18 PHF10 37 61 WDR33 38 219 CDK12 38 149 ACIN1 38 149 YY1 38 189 KTN1 38 195 NAT10 38 99 GAR1 39 26 HSPD1 39 120 SRSF7 39 165 RPL23A 39 26 MYL12A 39 309 SUMO3 39 189 CD55 40 206 SF3A1 40 177 HNRNPH3 40 193 RPL35A 40 286 U2AF2 40 278 GNPDA1 40 153 MAP7 41 97 SLC27A2 41 191 ELAVL1 41 208 NUP98 41 3 JUP 41 157 DBI 41 170 SUMO1 42 155 HMGB1 42 39 U2SURP 42 284 LMNB1 42 52 LPP 42 228 BROX 42 39 HIST1H4A 43 204 ARGLU1 43 166 MYO1B 43 250 PPIB 43 70 CCBL2 43 377 EEFSEC 43 177 HNRNPA1 44 253 RAB31 44 197 ACSL4 44 170 CELF1 44 73 DDX46 44 148 GOLGA4 44 171 PTBP1 45 295 RPN1 45 128 U2AF2 45 205 RPL15 45 318 SERPINB6 45 379 CLTC 45 233 RAP1B 46 151 GNB1 46 62 RAB13 46 274 SSRP1 46 200 TBCB 46 324 IKBKAP 46 147 RAB35 47 140 IFI16 47 118 SRSF3 47 54 BCLAF1 47 223 RPL34 47 384 G6PD 47 146 SCAF11 48 117 SNRPA 48 217 SF3B4 48 156 SNRPA1 48 328 BTF3 48 189 S100A13 48 53 ARGLU1 49 165 HNRNPA3 49 196 MYO1C 49 73 CD55 49 304 RPL18A 49 412 AHNAK 49 92 SRSF3 50 177 SF3A2 50 193 G3BP1 50 262 RPL7A 50 85 RTF1 50 316 ZNF787 51 282 SF3B2 51 172 DDX6 51 88 BBX 51 153 GALK1 51 406 GEMIN5 52 22 NNT 52 50 HNRNPUL1 52 128 RPL36 52 349 NPEPPS 52 233 GRWD1 53 260 SNRPD3 53 147 SF3B1 53 326 RPL27 53 83 SRSF2 54 240 RAP1B 54 136 RPL30 54 453 PPIE 55 311 SF3B4 55 207 SF3B5 55 357 SRSF3 56 286 THBS1 56 69 RAB31 56 47 PELP1 57 222 SF3A3 57 153 EIF3L 57 215 TXLNG 58 228 SNRPB 58 91 FIS1 58 244 UBE2I 59 81 SRSF11 59 478 APEH 60 221 SUB1 60 425 KRT8 61 475 LIN7C 62 229 UTP18 63 231 PPP1R12A 64 401 RPL11 65 162 MARCKSL1 66 397 KRT17 A B 65.5

12

11 100 Imp-13 54.9 36.5 Trn-SR 62 10 0.1 Imp-13 79.8 Imp-11 Trn-SR

82.2 9 Exp-4 Exp-4 Imp-5 Imp-4 Imp-9 Imp-7 Imp-8 100 8 Trn-1 Trn-2 100 7 Imp-11 Imp-9 Imp-8 Imp-7 Imp-4 Imp-β

6 Trn-1 Trn-2 Imp-5

C Total in Trn-1 Trn-2 Trn-SR Imp-4 Imp-5 Imp-7 Imp-8 Imp-9 Imp-11 Imp-13 Exp-4 Unique 3rd-Z-4% Imp- 4 2 3 5 3 5 5 6 0 4 3 42 63 Trn-1 28 2 0 4 5 5 3 3 3 6 15 49 Trn-2 2 1 5 4 7 4 2 3 5 12 50 Trn-SR 4 10 4 6 2 1 4 4 36 63 Imp-4 4 7 4 7 1 1 4 25 49 Imp-5 8 8 3 4 7 11 23 62 Imp-7 19 8 1 2 7 22 58 Imp-8 8 6 4 8 24 60 Imp-9 4 1 6 28 53 Imp-11 0 5 39 52 Imp-13 3 46 66 Exp-4 20 49 Imp- Trn-1 Trn-2 Trn-SR Imp-4 Imp-5 Imp-7 Imp-8 Imp-9 Imp-11 Imp-13 Exp-4 Total Term ID Term Name p # p # p # p # p # p # p # p # p # p # p # p # No. GO:0006259 DNA Metabolic Process 8E-06 11 0.199 6 1 2 0.146 7 0.002 8 1 4 0.632 6 1 2 0.34 6 1 2 2E-04 10 8E-06 10 978 GO:0006310 DNA Recombination 0.503 4 1 1 1 2 0.008 5 1 1 0.019 5 1 2 1 1 1 1 1 3 0.008 5 298 GO:0006323 DNA Packaging 1 3 1 1 1 2 0.039 4 0.003 5 1 2 1 1 1 1 1 1 1 1 200 GO:0071103 DNA Conformation Change 0.312 4 1 1 1 2 1E-04 6 4E-04 6 0.244 4 1 1 1 1 1 1 1 1 263 GO:0032392 DNA Geometric Change 1 2 4E-04 4 0.061 3 1 2 63 GO:0010216 Maintenance of DNA Methylation 0.031 2 6 GO:0000737 DNA Catabolic Process, Endonucleolytic 0.002 3 0.436 2 1 1 23 GO:0006281 DNA Repair 0.002 7 1 3 1 2 0.007 6 1 1 1 4 1 1 1 3 1 2 0.043 6 0.111 5 519 GO:0006289 Nucleotide-Excision Repair 0.475 3 0.005 4 1 1 1 1 0.017 4 1 1 116 GO:0006284 Base-Excision Repair 0.043 3 1 1 1 2 1 1 52 GO:0006325 Chromatin Organization 6E-07 11 1 3 1 3 0.313 6 0.062 6 1 5 1 4 1 1 0.943 5 1 4 0.374 6 1 4 768 GO:0031497 Chromatin Assembly 1 3 1 1 1 1 0.017 4 0.035 4 1 1 1 1 1 1 1 1 1 1 162 GO:0016568 Chromatin Modification 2E-05 9 1 2 1 1 0.967 5 1 3 0.967 5 1 1 1 1 1 2 1 3 1 5 1 4 614 GO:0006338 Chromatin Remodeling 0.037 4 1 2 1 1 1 1 1 1 1 1 1 1 1 2 1 1 152 GO:0070828 Heterochromatin Organization 7E-04 3 1 1 14 GO:0051276 Chromosome Organization 4E-11 16 1 5 1 4 3E-06 12 2E-06 11 0.348 7 0.021 8 1 4 0.723 6 1 4 0.43 7 0.497 6 1126 GO:0032200 Organization 0.031 4 1 2 1 1 1 3 1 1 1 1 1 1 1 1 1 1 1 1 0.414 3 145 GO:0006368 Transcription Elongation from RNA Polymerase 1E-04 5 1 1 1 2 0.118 3 1 2 1 1 1 2 1 1 95 II GO:0016458 Gene Silencing 1E-05 7 1 2 1 2 1 2 1 1 1 2 1 2 1 2 1 3 1 1 1 1 244 GO:0034660 ncRNA Metabolic Process 3E-06 9 0.986 4 1 4 0.312 5 1 2 0.023 6 1 3 1 3 0.107 5 1 2 0.358 5 1 4 480 GO:0034470 ncRNA Processing 0.046 5 0.241 4 0.307 4 0.054 5 1 1 0.054 5 1 1 1 3 0.018 5 1 2 0.954 4 0.272 4 331 GO:0006370 7-Methylguanosine mRNA Capping 6E-12 7 0.616 2 33 GO:0000387 Spliceosomal snRNP Assembly 1 2 1 1 0.017 3 0.012 3 1 1 41 GO:0000389 mRNA 3'-Splice Site Recognition 3E-05 3 6 GO:0006378 mRNA Polyadenylation 1 1 0.018 3 1 1 1 1 0.007 3 37 GO:0098789 pre-mRNA Cleavage Required for 0.031 2 6 Polyadenylation GO:0000184 Nuclear-Transcribed mRNA Catabolic Process, 1 2 1 2 0.006 4 1 1 0.454 3 3E-10 8 0.008 4 0.021 4 123 Nonsense-Mediated Decay GO:0048255 mRNA Stabilization 1 1 0.005 3 0.006 3 1 1 1 1 1 1 1 1 34 GO:0010501 RNA Secondary Structure Unwinding 0.032 3 0.032 3 0.023 3 1 2 1 2 1 1 1 2 45 GO:0042254 Ribosome Biogenesis 0.203 4 0.064 4 0.003 5 4E-04 6 1 2 0.236 4 0.158 4 5E-08 8 0.1 4 0.26 4 0.073 4 235 GO:0006364 rRNA Processing 1 2 0.456 3 0.557 3 0.001 5 1 1 0.047 4 0.894 3 0.019 4 1 2 0.052 4 0.014 4 155 GO:0042273 Ribosomal Large Subunit Biogenesis 1 1 1 2 1 2 1 1 1 1 0.014 3 1 1 1 1 43 GO:0006412 Translation 1 3 0.333 5 1E-04 8 1 5 1 2 1 4 0.895 5 2E-06 10 1E-05 9 1 4 0.001 8 1 3 677 GO:0006413 Translational Initiation 1 2 1 1 0.006 5 1 1 1 1 1 1 0.014 5 2E-07 8 1 3 1 2 273 GO:0006414 Translational Elongation 0.062 4 1 2 1 1 1 3 3E-08 8 0.076 4 0.198 4 219 GO:0006415 Translational Termination 0.028 4 1 1 1 3 6E-09 8 0.973 3 1 3 179 GO:0006457 Protein Folding 1 1 0.012 5 1 3 1 1 0.162 4 1 2 1 2 1 2 1 2 1 1 240 GO:0061077 -Mediated Protein Folding 1 2 1 1 0.032 3 1 2 1 1 1 1 51 GO:0010608 Posttranscriptional Regulation of Gene 8E-04 7 0.003 6 0.068 5 0.017 6 1 3 1 3 1 3 0.01 6 1 1 1 1 0.277 5 0.902 4 454 Expression GO:0043412 Macromolecule Modification 0.024 15 1 7 1 5 2E-04 18 0.617 11 1 10 0.807 12 1 7 1 6 1 9 0.832 13 0.617 11 4139 GO:0036211 Protein Modification Process 1 11 1 6 1 4 0.004 16 0.42 11 1 9 0.538 12 1 6 1 6 1 9 0.542 13 1 9 3959 GO:0016567 Protein Ubiquitination 1 1 1 1 0.036 7 1 3 1 1 1 2 1 1 1 1 1 1 784 GO:0018205 Peptidyl-Lysine Modification 0.007 6 1 1 1 1 1 2 1 2 1 1 1 2 1 1 0.149 5 1 1 398 GO:0031145 Anaphase-Promoting Complex-Dependent 4E-04 5 1 2 1 2 120 Proteasomal -Dependent Protein Catabolic Process GO:0030163 Protein Catabolic Process 1 1 1 3 1 1 0.005 8 1 4 1 2 1 2 1 5 1 3 1 1 827 GO:0007049 Cell Cycle 5E-05 13 1 4 1 3 0.004 11 0.15 8 1 6 1 7 1 7 1 2 1 4 1 6 0.15 8 1796 GO:0000278 Mitotic Cell Cycle 2E-04 10 1 3 1 1 0.025 8 0.033 7 1 3 1 5 1 5 1 1 1 3 1 4 1 2 1038 GO:0051301 Cell Division 1 4 1 1 1 1 0.001 8 1 4 1 2 1 2 1 1 1 1 1 2 1 1 672 GO:0000075 Cell Cycle Checkpoint 5E-04 6 1 3 1 1 1 3 1 1 249 GO:0071173 Spindle Assembly Checkpoint 4E-04 4 1 2 1 2 46 GO:0007091 Metaphase/Anaphase Transition of Mitotic Cell Cycle 1E-03 4 1 2 1 2 59 GO:0007059 Chromosome Segregation 1 3 1 1 0.024 5 0.141 4 1 1 1 2 1 1 1 2 1 1 279 GO:0000280 Nuclear Division 0.042 6 1 1 0.004 7 1 4 1 1 1 2 1 1 1 3 550 GO:0007077 Mitotic Nuclear Envelope Disassembly 0.026 3 1 2 1 1 1 1 1 1 44 GO:0048856 Anatomical Structure Development 1 12 0.057 14 0.102 14 0.503 15 1 10 1 11 0.009 17 3E-06 22 1 12 1E-05 20 0.231 16 0.329 13 5272 GO:0048731 System Development 1 11 0.224 12 1 11 1 13 1 8 1 10 0.107 14 0.035 15 1 10 0.034 14 1 13 1 10 4482 GO:0048869 Cellular Developmental Process 1 8 0.461 11 0.735 11 1 12 0.025 13 1 6 1 11 7E-05 18 1 7 0.002 15 1 11 0.127 12 4112 GO:0030154 Cell Differentiation 1 8 1 10 1 10 1 12 0.336 11 1 6 1 11 2E-04 17 1 7 0.001 15 1 10 0.069 12 3859 GO:0001649 Osteoblast Differentiation 1 3 1 2 1 2 1 2 1 1 1 2 1 1 0.046 4 209 GO:0003012 Muscle System Process 1 1 1 1 1 2 1 1 1 3 1 1 1 2 1 2 1 1 0.031 5 398 GO:0012501 Programmed Cell Death 1 8 1 3 1 1 0.326 9 0.034 9 1 8 0.003 11 0.179 9 0.384 8 1 3 1 5 1 7 1921 GO:0097194 Execution Phase of Apoptosis 1 1 1 1 0.122 3 0.004 4 1 2 1 2 1 1 96 GO:0030262 Apoptotic Nuclear Changes 0.004 3 0.006 3 1 1 1 1 30 GO:0006309 Apoptotic DNA Fragmentation 7E-04 3 0.264 2 1 1 18 GO:0009607 Response to Biotic Stimulus 1 5 1 4 1 3 1 5 1 2 1 3 0.008 8 0.089 7 1 2 1 1 1 3 1 4 984 GO:0006974 Cellular Response to DNA Damage Stimulus 0.037 7 1 4 1 5 3E-05 9 1 3 1 5 1 1 1 4 1 3 0.5 6 0.007 7 811 GO:0006979 Response to Oxidative Stress 1 3 1 2 1 2 1 1 1 3 1 2 1 1 1 4 0.033 5 401 GO:0070887 Cellular Response to Chemical Stimulus 1 9 0.103 10 0.842 9 1 10 1 6 1 9 0.124 11 0.16 11 0.007 12 2E-06 16 1 9 0.003 12 2852 GO:0014070 Response to Organic Cyclic Compound 1 2 1 4 1 3 1 4 1 3 1 2 0.382 6 1 5 1 3 1E-04 9 1 4 1 4 890 GO:1901698 Response to Nitrogen Compound 1 1 1 3 1 4 1 5 1 5 1 6 0.019 8 1 2 1 3 1 1 1 5 1087 GO:1901700 Response to -Containing Compound 1 3 1 5 1 6 1 6 1 4 1 5 0.006 10 0.058 9 1 6 0.021 9 1 4 0.086 8 1660 GO:0010038 Response to Metal Ion 1 1 0.014 5 1 3 1 2 1 1 1 1 1 2 1 2 1 2 1 2 1 2 349 GO:0006952 Defense Response 1 4 1 2 1 3 1 3 1 7 1 5 0.018 10 0.144 9 1 3 1 3 1 3 1 6 1867 GO:0002376 Immune System Process 1 6 1 3 1 4 1 6 0.002 12 1 6 0.069 11 0.003 13 1 5 1 7 1 6 1 8 2673 GO:0032606 Type I Interferon Production 1 1 1 1 0.006 4 0.013 4 1 2 1 1 1 1 127 GO:0007165 Signal Transduction 1 11 1 10 1 10 0.009 19 1 9 0.479 16 0.036 17 1E-04 21 1 9 0.009 17 1 13 0.244 14 5865 GO:0007154 Cell Communication 1 12 1 12 1 10 0.008 20 1 11 0.119 18 0.03 18 2E-05 23 1 10 0.001 19 0.215 18 0.165 15 6420 No. in Top 4% 63 49 50 63 49 62 58 60 53 52 66 49 A Imp- Trn-1 Trn-2 Trn-SR Imp-4 Imp-5 Imp-7 Imp-8 Imp-9 Imp-11 Imp-13 Exp-4 Total Term ID Term Name p # p # p # p # p # p # p # p # p # p # p # p # No. GO:0008622 Epsilon DNA Polymerase Complex 0.024 2 5 GO:0044452 Nucleolar Part 1 2 1 1 0.033 3 0.076 3 1 1 0.076 3 1 2 1 1 3E-04 4 60 GO:0015030 Cajal Body 1 1 6E-04 4 6E-04 4 1 1 0.024 3 1 2 0.019 3 52 GO:0042382 Paraspeckles 1 1 1 1 1 1 1 1 1 1 2E-05 3 6 GO:0034399 Nuclear Periphery 1 1 1 2 1 1 1 1 0.249 3 1 1 1 1 0.31 3 1 1 1 2 0.006 4 122 GO:0005635 Nuclear Envelope 2E-05 8 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 3 1 1 0.172 5 1 2 410 GO:0000785 Chromatin 0.012 6 0.724 4 1 1 1 2 1E-04 7 1 3 1 3 1 2 0.073 5 1 2 1 3 0.003 6 442 GO:0000791 Euchromatin 1 1 0.005 3 1 1 31 GO:0098687 Chromosomal Region 1 3 1 2 2E-06 8 1 1 1 3 1 3 1 1 1 1 1 3 1 3 301 GO:0000793 Condensed 3 1 1 0.004 5 0.032 4 1 1 0.067 4 1 1 191 GO:0000776 Kinetochore 0.015 4 1 1 1 1 1 2 1 2 116 GO:0005819 Spindle 1 3 1 1 4E-05 7 1 1 0.455 4 1 2 1 2 1 2 279 GO:0005813 Centrosome 1 1 1 1 0.001 7 1 2 1 2 1 1 1 2 476 GO:0090575 RNA Polymerase II Transcription Factor 0.006 4 1 2 1 2 1 2 1 1 1 1 0.008 4 1 2 96 Complex GO:0016602 CCAAT-Binding Factor Complex 1 1 1 1 0.014 2 4 GO:0005845 mRNA Cap Binding Complex 1E-06 4 12 GO:0005732 Small Nucleolar Ribonucleoprotein Complex 1 1 1 1 1 1 1 1 0.441 2 1 1 0.001 3 20 GO:0030532 Small Nuclear Ribonucleoprotein Complex 1 1 1 2 1E-19 11 0.054 3 4E-06 5 1 1 60 GO:0097525 Spliceosomal snRNP Complex 1 1 1 2 3E-20 11 0.037 3 2E-06 5 1 1 53 GO:0005684 U2-Type Spliceosomal Complex 0.008 3 5E-10 6 0.006 3 1 1 1 1 29 GO:0089701 U2AF 1 1 0.002 2 0.002 2 0.001 2 1 1 2 GO:0005849 mRNA Complex 1 1 1 1 1 1 3E-04 3 14 GO:0044391 Ribosomal Subunit 0.019 4 1 1 1 1 1 3 3E-09 8 0.727 3 1 2 162 GO:0031428 Box C/D snoRNP Complex 1 1 1 1 1 1 1 1 0.018 2 6 GO:0005856 0.293 9 1 5 1 3 2E-04 13 1 5 1 7 1 4 1 6 1 5 0.001 11 0.014 11 1 4 1949 GO:0010494 Cytoplasmic Stress Granule 1 1 1 1 1 1 1 2 1 1 0.008 3 32 GO:0044445 Cytosolic Part 0.048 4 1 2 1 1 1 3 2E-08 8 1 3 1 2 205 GO:0005739 0.622 8 1 5 1 4 1 4 0.102 8 1 5 9E-04 11 1E-05 13 3E-04 11 0.003 10 0.966 8 0.102 8 1699 GO:0005783 Endoplasmic Reticulum 1 2 1 3 1 4 1 1 1 5 1 3 1 6 8E-04 11 1 1 1 2 1 4 1 5 1623 GO:0005768 Endosome 1 1 1 1 1 2 1 4 1 3 1 4 1 3 2E-08 12 1 1 1 2 1 1 1 3 779 GO:0005794 Golgi Apparatus 1 2 1 2 1 3 1 5 1 2 1 2 2E-04 11 1 3 1 3 1 5 1 2 1436 GO:0031410 Cytoplasmic Vesicle 1 1 1 2 1 2 1 4 1 5 1 3 1 4 3E-07 13 1 4 1 2 1 1 1 5 1232 GO:0005925 Focal Adhesion 1 2 1 1 1 2 1 1 1 4 1 1 0.004 6 0.002 6 0.039 5 0.008 6 1 2 383 GO:0005905 Coated Pit 1 1 1 2 1 1 1 2 1 1 1 1 1 1 0.033 3 62 GO:0043209 Myelin Sheath 1 1 1 2 1 3 0.047 4 1 2 1 2 0.032 4 1 1 1 1 175 No. in Top 4% 63 49 50 63 49 62 58 60 53 52 66 49 B Imp- Trn-1 Trn-2 Trn-SR Imp-4 Imp-5 Imp-7 Imp-8 Imp-9 Imp-11 Imp-13 Exp-4 Total Term ID Term Name p # p # p # p # p # p # p # p # p # p # p # p # No. GO:0034061 DNA Polymerase Activity 0.008 3 1 1 1 2 30 GO:0003690 Double-Stranded DNA Binding 0.023 7 1 4 1 4 1 2 0.62 5 1 5 0.153 6 1 3 1 4 1 2 0.62 5 752 GO:0003697 Single-Stranded DNA Binding 1 1 0.001 4 1 2 1 1 1 2 0.164 3 1 2 1 1 1 2 1 2 88 GO:0098847 Sequence-Specific Single Stranded DNA 0.039 2 0.046 2 1 1 9 Binding GO:0008301 DNA Binding, Bending 0.001 3 0.002 3 20 GO:0043566 Structure-Specific DNA Binding 0.289 3 1 1 1 1 0.002 4 0.226 3 0.162 3 1 1 98 GO:0000217 DNA Secondary Structure Binding 7E-04 3 0.001 3 1 1 18 GO:0000400 Four-Way Junction DNA Binding 5E-05 3 9E-05 3 1 1 8 GO:0097100 Supercoiled DNA Binding 0.007 2 0.011 2 1 1 4 GO:0003682 Chromatin Binding 8E-11 12 1 3 1 2 1 4 0.061 5 1 2 1 2 1 3 1E-05 8 1 1 1 4 0.924 4 457 GO:0042162 Telomeric DNA Binding 0.003 3 0.004 3 1 1 30 GO:0001047 Core Promoter Binding 1 3 1 1 1 2 1 2 1 2 1 2 1 2 0.91 3 0.019 4 1 2 0.514 3 156 GO:0000988 Transcription Factor Activity, Protein Binding 3E-04 8 0.012 6 0.227 5 0.006 7 1 1 1 2 1 2 1 2 1 4 1 3 1 4 1 2 587 GO:0003727 Single-Stranded RNA Binding 1 2 2E-08 6 4E-04 4 1 1 1 1 1 2 1 1 1 2 1 2 1 2 62 GO:0036002 Pre-mRNA Binding 1 1 1 1 0.004 3 1 1 1 1 1 1 1 1 1 1 1 1 23 GO:0071208 Histone Pre-mRNA DCP Binding 0.011 2 4 GO:0003730 mRNA 3'-UTR Binding 1 1 0.015 3 0.018 3 1 1 1 1 49 GO:0017091 AU-rich Element Binding 0.001 3 0.002 3 1 1 0.432 2 1 1 23 GO:0017069 snRNA Binding 1 1 1 1 1 1 1 1 3E-07 5 1 1 1 1 1 1 1 1 34 GO:0035614 snRNA Stem-Loop Binding 0.002 2 2 GO:0004004 ATP-Dependent RNA Helicase Activity 2E-07 6 1 1 0.102 3 0.001 4 0.05 3 1 2 1 1 1 2 66 GO:0030515 snoRNA Binding 1 1 1 1 1 1 0.005 3 1 1 0.471 2 0.323 2 24 GO:0051082 Unfolded Protein Binding 1 1 0.009 4 1 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1 102 GO:0019789 SUMO Activity 4E-04 3 11 GO:0019901 Protein Kinase Binding 1 1 1 2 1 3 0.046 6 1 2 1 3 1 2 1 3 1 1 1 3 1 4 1 3 543 GO:0003988 Acetyl-CoA C-Acyltransferase Activity 0.021 2 6 GO:0016887 ATPase Activity 1 4 1 1 1 1 0.008 6 1 1 1 3 1 2 1E-05 8 1 3 1 3 1 1 1 3 402 GO:0003924 GTPase Activity 1 3 1 1 1 1 1 3 0.126 4 2E-09 9 1 2 1 2 225 No. in Top 4% 63 49 50 63 49 62 58 60 53 52 66 49 Accession Major Feature Gene Name Rank by 2nd Z-score Imp- Trn-1 Trn-2 Trn-SR Imp-4 Imp-5 Imp-7 Imp-8 Imp-9 Imp-11 Imp-13 Exp-4 O43148 mRNA Capping RNMT 2 1432 1420 1906 358 327 1487 1066 687 1560 598 447 Q09161 Cap-Binding NCBP1 25 327 1037 1373 751 1466 906 1220 655 1248 594 1075 P52298 NCBP2 19 1416 1529 865 1510 602 Q13151 hnRNP HNRNPA0 247 11 13 145 50 137 92 422 105 7 558 82 P09651 HNRNPA1 245 7 7 285 364 776 323 438 171 773 641 177 P22626 HNRNPA2B1 301 16 19 382 555 902 336 420 160 711 531 123 P51991 HNRNPA3 634 13 14 531 260 823 165 163 182 405 987 210 P07910 HNRNPC 496 857 259 386 324 1140 337 570 117 563 676 229 Q14103 HNRNPD 851 8 8 566 623 1300 565 938 709 1237 1512 633 P52597 HNRNPF 1678 95 52 1702 382 211 268 230 608 1289 1251 304 P31943 HNRNPH1 438 34 40 948 171 723 233 325 243 628 646 202 P55795 HNRNPH2 741 41 42 1452 1126 534 363 280 302 802 573 208 P31942 HNRNPH3 373 167 85 753 708 686 221 177 177 394 404 184 P61978 HNRNPK 509 1130 331 351 611 919 459 569 318 982 580 315 P14866 HNRNPL 453 491 125 634 154 477 532 621 148 999 921 236 Q8WVV9 HNRPLL 145 1397 1430 1331 1281 774 320 542 479 1018 1056 212 P52272 HNRNPM 403 4 5 294 532 754 618 723 216 333 777 292 O60506 SYNCRIP 770 866 483 1123 503 508 435 529 575 808 628 693 O43390 HNRNPR 471 1421 133 374 1269 598 463 719 293 1236 674 388 Q00839 HNRNPU 123 837 205 395 415 1025 759 1247 475 1133 645 556 Q9BUJ2 HNRNPUL1 572 1600 1586 1503 354 1382 47 50 9 1253 1323 100 Q8IYB3 SR-Rich SRRM1 1440 1040 1977 863 1842 276 6 723 Q9UQ35 Splicing Factor SRRM2 202 203 147 3 1412 1463 249 54 151 803 1872 355 Q07955 SRSF1 1617 328 113 55 984 1159 105 84 238 490 786 145 Q01130 SRSF2 133 234 143 8 156 628 68 85 152 373 83 164 P84103 SRSF3 274 294 254 90 243 379 110 118 49 236 357 92 Q13243 SRSF5 1002 213 378 524 538 833 457 659 212 1504 644 379 Q13247 SRSF6 1964 212 279 16 740 537 288 446 124 1091 1728 287 Q16629 SRSF7 377 553 592 26 200 380 86 120 85 224 232 79 Q9BRL6 SRSF8 115 738 1244 19 369 34 Q05519 SRSF11 1194 342 162 1764 221 1199 81 81 93 338 1576 185 Q15459 Splicing Factor SF3A1 1621 442 366 1667 118 982 206 228 190 184 1313 150 Q15428 3A, B SF3A2 1623 402 320 1644 126 782 177 209 202 106 1414 133 Q12874 SF3A3 1650 375 291 1674 114 1021 222 243 231 227 1367 160 O75533 SF3B1 1660 343 273 1542 81 862 211 147 188 56 1326 84 Q13435 SF3B2 1638 432 276 1522 143 1000 282 206 178 214 1491 151 Q15393 SF3B3 1604 322 288 1516 172 999 316 352 192 71 1191 207 Q15427 SF3B4 1552 458 214 1566 215 1029 311 217 180 126 843 217 Q9BWJ5 SF3B5 1634 418 247 1478 265 1077 217 207 229 77 1317 188 Q9Y3B4 SF3B14 1663 336 246 1685 43 912 181 188 330 148 1483 131 Q01081 Splicing Factor U2AF1 190 154 201 202 329 175 99 148 48 177 1476 66 P26368 U2AF U2AF2 261 360 342 1293 220 218 111 128 76 286 1107 106 P23246 PQ-Rich SF SFPQ 111 219 1209 1890 31 1831 74 24 547 1542 143 42 P09012 snRNP A-F SNRPA 1030 444 400 218 459 1063 117 142 218 415 1887 361 P09661 SNRPA1 1463 338 316 1664 112 976 207 231 156 107 1475 141 P14678 SNRPB 764 333 222 1540 238 795 228 143 210 226 736 219 P08579 SNRPB2 1422 311 257 1603 84 1050 183 216 144 268 1510 85 P09234 SNRPC 354 555 393 915 411 825 130 170 272 310 710 91 P62314 SNRPD1 743 300 241 1271 166 896 359 441 196 481 1602 364 P62316 SNRPD2 1199 604 238 1646 284 1308 352 493 311 342 1084 227 P62318 SNRPD3 843 384 226 1523 549 861 260 162 217 282 631 256 P62304 SNRPE 1512 357 203 1399 230 948 285 293 241 396 1458 431 P62306 SNRPF 584 426 466 893 73 1191 171 266 223 240 562 329 Q9Y5S9 Exon Junction RBM8A 497 517 505 976 783 1857 1614 1052 281 109 9 570 P38919 Complex EIF4A3 629 289 392 256 394 998 535 852 490 24 737 681 P61326 MAGOH 1643 388 329 1183 484 1414 929 975 402 841 8 450 Q10570 Cleavage and CPSF1 1029 690 1105 1481 231 1950 48 1236 56 1586 1837 114 Q9P2I0 Polyadenylation CPSF2 825 578 351 309 1434 1326 917 1541 530 99 1856 1730 Q9UKF6 Specificity CPSF3 1104 1348 274 1367 1270 1666 1358 1664 663 1639 1359 O43809 Factor NUDT21 415 335 99 157 593 253 254 165 154 590 1478 254 Q16630 CPSF6 281 647 368 125 201 289 125 96 121 738 687 103 Q8N684 CPSF7 187 521 191 62 421 145 158 208 89 138 1136 62 P35269 General GTF2F1 35 587 192 68 64 685 224 846 416 466 210 309 P13984 Transcription GTF2F2 61 636 249 134 229 964 741 811 236 837 510 472 Q8N7H5 RNA Pol II PAF1 1901 886 1304 14 579 1478 1224 827 1657 33 Q6P1J9 Associating CDC73 1478 266 542 206 992 1355 484 1227 329 1635 1829 193 Q6PD62 Factor CTR9 106 1398 91 189 29 159 79 760 1509 324 Q8WVC0 LEO1 1248 1271 792 272 624 1570 574 1601 187 1546 1277 404 P18615 NELFE 911 1482 1117 123 55 737 757 1317 487 1199 1891 882 Color Scale: Top 4% 4–8% 8–15% Rank by 2nd Z-score Accession Gene Name Imp- Trn-1 Trn-2 Trn-SR Imp-4 Imp-5 Imp-7 Imp-8 Imp-9 Imp-11 Imp-13 Exp-4 P08865 RPSA 1112 1181 580 1143 442 599 568 436 520 514 805 640 P15880 RPS2 1279 860 840 992 1095 859 633 488 481 565 740 594 P23396 RPS3 1266 1058 645 1042 962 657 537 418 512 372 973 494 P61247 RPS3A 1127 1047 859 1096 672 745 548 413 351 455 827 475 P62701 RPS4X 1197 911 835 982 925 624 474 394 422 558 800 564 P46782 RPS5 1094 967 678 1095 725 769 478 252 338 502 798 458 P62753 RPS6 673 902 456 1173 590 829 507 502 395 522 791 369 P62081 RPS7 1103 1031 907 1127 889 655 511 431 81 588 747 560 P62241 RPS8 1161 1083 871 1020 927 691 530 464 424 503 691 489 P46781 RPS9 1147 1016 699 963 704 626 392 404 366 523 880 535 P46783 RPS10 924 1105 521 669 675 703 420 335 328 253 662 490 P62280 RPS11 1170 1086 743 735 842 555 490 390 369 587 751 448 P25398 RPS12 1110 1166 727 1033 1029 752 418 326 411 623 619 482 P62277 RPS13 1087 830 888 1140 812 693 516 429 420 521 916 456 P62263 RPS14 1089 1203 936 1248 1075 881 319 382 418 657 689 623 P62841 RPS15 1373 908 691 997 1002 487 510 449 583 953 936 612 P62244 RPS15A 1037 1263 862 1014 878 651 503 432 436 329 1020 546 P62249 RPS16 1045 1041 763 966 957 588 517 369 371 583 821 653

40S Subunit P0CW22 RPS17L 1210 1158 709 802 784 512 520 447 361 403 1018 441 P62269 RPS18 981 1089 674 869 911 583 467 391 333 729 977 521 P39019 RPS19 1122 1099 937 993 888 594 461 384 360 285 851 506 P60866 RPS20 1123 968 858 736 995 472 424 226 365 293 701 544 P63220 RPS21 1215 988 954 1036 718 653 704 340 444 549 502 614 P62266 RPS23 1219 957 776 785 756 475 305 297 348 516 634 345 P62847 RPS24 1017 1056 899 1073 890 652 496 409 290 484 779 486 P62851 RPS25 1111 1211 1052 925 563 812 450 313 525 428 1066 410 P62854 RPS26 975 1400 1428 885 1000 916 428 250 371 1242 P42677 RPS27 1051 997 684 811 616 595 514 405 428 610 446 631 P62979 RPS27A 710 451 1108 966 668 828 350 483 265 489 577 P62857 RPS28 828 1106 802 809 917 596 557 367 401 598 726 391 P62273 RPS29 1256 1064 1068 873 916 722 519 318 536 1056 702 365 P62861 FAU(RPS30) 1265 981 675 1487 908 990 528 320 403 150 695 407 P05388 RPLP0 1152 1066 662 1028 1146 711 690 821 564 731 1069 520 P05386 RPLP1 526 732 650 1030 373 913 817 678 594 416 323 503 P05387 RPLP2 908 733 476 766 929 666 575 481 686 260 302 401 P39023 RPL3 830 665 524 1047 455 800 332 160 184 366 824 161 P36578 RPL4 832 563 410 1247 633 891 317 302 207 459 758 246 P46777 RPL5 873 638 566 841 853 1053 830 843 624 612 889 778 Q02878 RPL6 701 562 478 1199 601 815 389 331 306 689 578 340 P18124 RPL7 816 618 457 1057 739 789 354 227 137 157 716 305 Q6DKI1 RPL7L1 1842 1483 41 P62424 RPL7A 929 775 582 1230 404 639 353 237 262 448 743 180 P62917 RPL8 728 731 481 1266 504 773 333 246 153 234 757 303 P32969 RPL9 1153 662 347 1334 368 806 318 195 265 595 870 291 P27635 RPL10 960 789 668 1177 974 809 399 311 263 489 712 420 P62906 RPL10A 808 621 360 1227 461 787 313 241 147 471 769 251 P62913 RPL11 793 712 491 1326 1123 710 497 40 201 375 401 377 P30050 RPL12 863 763 614 1208 305 814 338 321 254 314 845 286 P26373 RPL13 836 530 492 1197 275 775 244 185 189 323 759 247 P40429 RPL13A 737 940 375 1203 943 864 239 362 228 305 656 398 P50914 RPL14 814 1136 561 1204 290 749 299 154 240 573 897 362 P61313 RPL15 801 771 432 1246 556 820 388 244 205 360 841 353 P18621 RPL17 799 1004 706 1170 610 350 308 315 209 518 597 166 Q07020 RPL18 758 760 538 1229 369 971 248 368 269 422 754 314 Q02543 RPL18A 847 484 551 1400 1285 830 437 328 232 304 605 289 P84098 RPL19 1109 1118 651 1243 406 695 502 301 384 176 975 367 P46778 RPL21 818 883 507 1161 647 836 382 319 247 607 509 139 P35268 RPL22 729 714 450 474 515 306 52 42 347 1021 398 376 60S Subunit P62829 RPL23 817 703 535 897 668 842 536 448 323 718 836 514 P62750 RPL23A 1092 625 417 1146 844 807 162 222 165 458 753 358 P83731 RPL24 1035 963 718 1141 643 743 421 307 299 139 1001 381 P61254 RPL26 650 821 523 1160 664 636 192 229 320 547 649 279 Q9UNX3 RPL26L1 1337 1693 454 567 122 P61353 RPL27 705 654 550 1158 614 925 301 192 326 430 941 216 P46776 RPL27A 792 615 477 1151 295 901 306 274 211 105 599 182 P46779 RPL28 666 566 412 1325 203 603 409 346 342 806 555 198 P47914 RPL29 982 682 598 1219 660 660 303 235 346 615 847 294 P62888 RPL30 621 659 570 1252 760 570 273 136 173 370 808 220 P62899 RPL31 543 1026 590 1142 1104 811 567 286 376 379 568 336 P62910 RPL32 885 583 416 1324 301 926 381 199 252 1055 910 242 P49207 RPL34 1135 1410 350 1285 597 546 278 123 305 223 658 260 P42766 RPL35 731 508 356 1058 518 593 175 168 104 67 454 128 P18077 RPL35A 951 614 772 1366 814 840 385 219 193 255 813 102 Q9Y3U8 RPL36 1157 845 317 1231 125 961 414 395 128 239 823 230 P83881 RPL36A 1238 382 295 1511 Q969Q0 RPL36AL 1347 522 937 211 609 601 489 168 P61927 RPL37 309 1012 458 1638 130 474 P61513 RPL37A 1142 827 526 940 613 707 551 238 237 562 1075 162 P63173 RPL38 780 1052 409 1046 387 623 280 210 266 555 586 264

Color Scale: Top 15% 15-30% 30-50% >50%