Immunoprecipitation and Mass Spectrometry Defines an Extensive

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Available online at www.sciencedirect.com 121 122 123 124 125 126 www.elsevier.com/locate/brainres 127 128 129 Review 130 131 fi 132 Immunoprecipitation and mass spectrometry de nes 133 – 134 an extensive RBM45 protein protein interaction 135 Q2 136 network 137 138 a a,b a a c 139 Yang Li , Mahlon Collins , Jiyan An , Rachel Geiser , Tony Tegeler , c c c a,b,n 140 Q1 Kristine Tsantilas , Krystine Garcia , Patrick Pirrotte , Robert Bowser 141 aDivisions of Neurology and Neurobiology, Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, 142 Phoenix, AZ 85013, USA 143 bUniversity of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA 144 cCenter for Proteomics, TGen (Translational Genomics Research Institute), Phoenix, AZ 85004, USA 145 146 147 article info abstract 148 149 Article history: The pathological accumulation of RNA-binding proteins (RBPs) within inclusion bodies is a 150 Received 30 January 2016 hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration 151 Received in revised form (FTLD). RBP aggregation results in both toxic gain and loss of normal function. Determining 152 25 February 2016 the protein binding partners and normal functions of disease-associated RBPs is necessary 153 Accepted 28 February 2016 to fully understand molecular mechanisms of RBPs in disease. Herein, we characterized 154 the protein–protein interactions (PPIs) of RBM45, a RBP that localizes to inclusions in ALS/ 155 – fi Keywords: FTLD. Using immunoprecipitation coupled to mass spectrometry (IP MS), we identi ed 132 156 fi RBM45 proteins that speci cally interact with RBM45 within HEK293 cells. Select PPIs were 157 Immunoprecipitation validated by immunoblot and immunocytochemistry, demonstrating that RBM45 associ- 158 Mass spectrometry ates with a number of other RBPs primarily via RNA-dependent interactions in the nucleus. 159 Protein–protein interaction Analysis of the biological processes and pathways associated with RBM45-interacting 160 ALS proteins indicates enrichment for nuclear RNA processing/splicing via association with 161 hnRNP proteins and cytoplasmic RNA translation via eiF2 and eiF4 pathways. Moreover, 162 several other ALS-linked RBPs, including TDP-43, FUS, Matrin-3, and hnRNP-A1, interact 163 with RBM45, consistent with prior observations of these proteins within intracellular 164 inclusions in ALS/FTLD. Taken together, our results define a PPI network for RBM45, 165 suggest novel functions for this protein, and provide new insights into the contributions of 166 RBM45 to neurodegeneration in ALS/FTLD. 167 This article is part of a Special Issue entitled SI:RNA Metabolism in Disease. 168 & 2016 Published by Elsevier B.V. 169 170 171 172 173 181 174 182 n 183 175 Correspondence to: Gregory W. Fulton ALS and Neuromuscular Research Center, Barrow Neurological Institute, Phoenix, AZ 85013, 176 USA. 184 177 E-mail address: [email protected] (R. Bowser). 185 178 186 http://dx.doi.org/10.1016/j.brainres.2016.02.047 187 179 0006-8993/& 2016 Published by Elsevier B.V. 180 188

Please cite this article as: Li, Y., et al., Immunoprecipitation and mass spectrometry deﬁnes an extensive RBM45 protein– protein interaction network. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.02.047 BRES : 44759

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189 Contents 249 190 250 191 1. Introduction...... 2 251 192 2. Results ...... 3 252 193 2.1. Identification of the RBM45 interacting proteins in HEK-293 cells...... 3 253 194 2.2. Validation of select RBM45 PPIs ...... 3 254 195 2.3. RBM45 homo-oligomerization mediates association with a large number of proteins ...... 11 255 196 2.4. Gene ontology and pathway analysis ...... 12 256 197 2.5. Co-localization analysis ...... 13 257 198 3. Discussion ...... 14 258 199 4. Experimental procedure ...... 17 259 200 4.1. Cell culture and plasmid construction ...... 17 260 201 4.2. LC-MS/MS protein identification...... 17 261 202 4.2.1. Immunoprecipitation...... 17 262 203 4.2.2. Protein digestion ...... 17 263 204 4.2.3. LC–MS analysis ...... 17 264 205 4.3. Protein identification ...... 17 265 206 4.4. Bioinformatics, pathway analysis and gene ontology analysis ...... 18 266 207 4.5. Reciprocal immunoprecipitation ...... 18 267 208 4.6. Immunoblot ...... 18 268 209 4.7. Immunocytochemistry ...... 18 269 210 4.8. Microscopy, digital deconvolution, and co-localization analysis ...... 19 270 211 Author contributions ...... 19 271 212 Competing financial interests ...... 19 272 213 Acknowledgments ...... 19 273 214 Appendix A. Supplementary material...... 19 274 215 References ...... 19 275 216 276 217 277 218 278 219 279 220 1. Introduction addition to self-aggregation, these proteins are capable of 280 221 sequestering other proteins into aggregates/inclusions as a 281 222 The aggregation of RNA-binding proteins (RBPs) into inclu- consequence of the normal functional associations between 282 223 sion bodies is one of the most prevalent and well- these proteins. For example, proteomic analysis of TDP-43 283 224 characterized pathological findings in amyotrophic lateral aggregates showed deposition of stress granule proteins G3BP 284 225 sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). and PABPC1 as well as paraspeckle proteins PSF and NONO 285 226 The identification of cytoplasmic mis-localized TDP-43 (Dammer et al., 2012). Similar observations of paraspeckle 286 227 (Neumann et al., 2006), and later FUS (Kwiatkowski et al., proteins p54nrb and NONO in FUS-positive inclusions 287 228 2009; Vance et al., 2009), as primary components of ubiquiti- (Shelkovnikova et al., 2014) provide additional evidence in 288 – 229 nated inclusions in motor neurons and glia in these disorders support of this concept. Thus, understanding the protein 289 230 led to the “two-hit” hypothesis of RBP-mediated neurodegen- protein interactions (PPIs) of ALS-linked RBPs is a necessary 290 fi 231 eration. This model proposes that the pathological aggrega- step towards de ning the protein composition of inclusions 291 232 tion of RBPs confers toxicity by simultaneous gain of toxic in ALS/FTLD and new insight into mechanisms of disease. 292 233 function of the aggregates and the loss of normal functions Determining RBP PPIs is also essential for understanding 293 234 served by these proteins in regulating gene expression. the normal functions of RBPs, and how these functions may 294 235 Ample experimental evidence now exists in support of this be compromised as a result of RBP aggregation in ALS/FTLD. 295 236 model, with studies consistently finding that under- or over- Numerous RBP functions depend on the association of RBPs 296 237 expression of numerous RBPs is sufficient to induce neuronal with protein/nucleic acid complexes. For example, FUS is a 297 238 cell death in a variety of model systems (reviewed in Ling component of both nuclear gems, which participate in snRNP 298 239 et al. (2013)). biogenesis, and paraspeckles, which are involved in cellular 299 240 This model of RBP-mediated neurodegeneration depends, stress responses (Shelkovnikova et al., 2014; Yamazaki et al., 300 241 in part, on the ability of RBPs to self-associate and interact 2012). The expression of mutant FUS reduces levels of these 301 242 with other RBPs within protein aggregates. Many ALS-linked nuclear sub-structures, suggesting mechanisms by which 302 243 RBPs, including TDP-43, FUS, hnRNP-A1, and TAF15 are loss of normal FUS function contributes to cell death in 303 244 aggregation prone as a result of prion-like domains contained ALS/FTLD. In addition, many ALS/FTLD-linked RBPs also 304 245 within their protein sequence (Johnson et al., 2009; King et al., associate with cytoplasmic stress granules (Li et al., 2013), 305 246 2012). Mutations in the prion-like domain lead to familial and disease-associated mutations tend to promote the excess 306 247 forms of ALS/FTLD marked by the pathological aggregation of formation of these structures (Kim et al., 2013). While stress 307 248 the mutant protein (reviewed in Gitler and Shorter (2011)). In granules normally aid in the response to cellular stress by 308

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309 protecting mRNAs and shifting gene expression towards a biological functions, and contributions to neurodegeneration 369 310 stress response, excessive stress granule formation promotes in ALS/FTLD. 370 311 the formation of insoluble RBP aggregates that may be 371 312 precursors to inclusion bodies (Kim et al., 2013; Li et al., 372 313 2013; Vance et al., 2013). This can lead to loss of other normal 2. Results 373 314 functions, such as impaired P-body formation that occurs in 374 fi 315 response to mutant FUS sequestration in stress granules 2.1. Identi cation of the RBM45 interacting proteins in HEK- 375 316 (Takanashi and Yamaguchi, 2014). PPIs can be used to predict 293 cells 376 317 these and similar functional associations (Dammer et al., 377 – 318 2012). Defining RBP PPIs, therefore, helps uncover novel A schematic outline of the immunoprecipitation mass spec- 378 319 functions and candidate disease mechanisms related to these trometry procedure used in this study is shown in Fig. 1A. 379 320 multifunctional proteins. FLAG-RBM45 or empty vector was overexpressed in HEK293 380 321 Given the diversity of RBP functions, which includes cells and immunoprecipitated using whole cell lysates in 381 322 regulating transcription, RNA splicing/export, and miRNA triplicates. HA-tagged RBM45 was also included and used as a 382 323 biogenesis (Ling et al., 2013), a relatively high-throughput reference for the data analysis. HEK293 cells expressing 383 324 approach is preferable to identify candidate functions/bind- empty vector alone served as negative controls. Regular IP 384 325 ing partners for targeted validation. Immunoprecipitation and formaldehyde crosslinking IP were performed in parallel 385 326 coupled to mass spectrometry (IP–MS) offers tremendous to identify strongly and weakly RBM45-associated proteins 386 327 promise towards identifying large sets of RBP protein–protein separately (Fig. 1A). Immunoblot analysis of the immunopre- 387 328 interactions (PPIs) and associated biological processes/path- cipitated fractions showed that tagged-RBM45 was enriched 388 329 ways. The sensitivity of this approach can be further in the pulldown. In contrast, no RBM45 was detected in the 389 330 enhanced by the use of cross-linking methods, such as pulldown in the vector control or IgG pulldown (Supplemen- 390 331 treatment with small cross-linking agents or formaldehyde, tal Fig. 1). These data demonstrate that tagged-RBM45 can be 391 fi fi 332 to detect low-affinity protein interactions (Li et al., 2015; Nittis ef ciently and speci cally immunoprecipitated from cell 392 333 et al., 2010). This approach has previously been used to extracts. Co-immunoprecipitated proteins were then sepa- 393 334 identify proteins interacting with the ALS-linked Ewing Sar- rated using SDS-PAGE and stained (Fig. 1B). Coomassie stain- 394 335 coma (EWS) RBP (Pahlich et al., 2009), where interactions with ing of the gels loaded with RBM45-IP (sample 1, 3 and 5) 395 336 hnRNPs and FUS are consistent with roles of EWS in mRNA identified several bands that were not present in vector 396 337 splicing (Law et al., 2006) and inclusion formation in ALS/ (sample 2 and 4) or IgG controls (sample 6 and 7). 397 338 FTLD (Mackenzie and Neumann, 2012), respectively. Thus, IP– Immunoprecipitated proteins were gel extracted, trypsin 398 339 MS can identify multiple protein binding partners of a given digested, and identified by liquid chromatography tandem 399 340 target and this information can be used to predict novel mass spectroscopy (LC-MS/MS) (Fig. 1A, see Section 4). We 400 341 functions and roles in disease. identified 235 unique proteins with a protein False Discovery Q3 401 342 Here, we applied this approach to RBM45, a recently Rate equal or lower than 1%. We then applied a manual 402 343 characterized RNA-binding protein found in inclusions in thresholding approach and a probabilistic PPI prediction 403 344 ALS, FTLD, and Alzheimer's disease (AD) (Collins et al., algorithm (SAINTexpress) to compute the most likely asso- 404 345 2012). These inclusions are positive for TDP-43, and RBM45 ciations between each of these 235 proteins and RBM45, 405 346 physically interacts with TDP-43 and FUS in vitro (Li et al., yielding 132 high-confidence candidates (Fig. 1A). These 132 406 347 2015). RBM45 contains three RNA-recognition motifs (RRMs), candidate proteins were found in at least 2 out of the 3 FLAG- 407 348 a nuclear localization sequence (NLS), and a homo- IP triplicates and were at least 2-fold more abundant com- 408 349 oligomerization (HOA) domain that mediates self- pared to vector control, suggesting that they specifically 409 350 association of the protein, and can localize to cytoplasmic associate with RBM45 (Table 1 and Supplemental Tables 1– 410 351 stress granules (Bakkar et al., 2015; Li et al., 2015). The 3). Of these 132 proteins, 28 were found exclusively by 411 352 expression of RBM45 is developmentally regulated and the regular-IP (Supplemental Table 4), 68 were found exclusively 412 353 highest expression levels occur in the brain (Tamada et al., by crosslinking-IP (Supplemental Table 5), and 36 were found 413 354 2002). These properties make RBM45 a promising target for in both regular-IP and crosslinking-IP groups (Supplemental 414 355 continued studies of ALS/FTLD, though at present little is Table 6). Analysis of the average number of total spectrum 415 356 known about the function of RBM45. To delineate protein counts by different immunoprecipitation groups showed that 416 357 binding partners of RBM45 and putative biological functions in both regular IP and crosslinking IP, the proteins identified 417 358 of the protein, we used an IP–MS approach to comprehen- from empty vector groups were significantly lower than the 418 359 sively characterize RBM45 protein–protein interactions (PPIs). proteins identified from FLAG-/HA-IP groups, providing 419 360 We identified 132 RBM45 PPIs by IP–MS, including PPIs with further evidence of the specificity of the approach (Supple- 420 361 many RBPs. Our results were used to associate RBM45 with mental Fig. 2). 421 362 biological processes and pathways. These were primarily 422 363 related to nuclear mRNA processing and cytoplasmic RNA 2.2. Validation of select RBM45 PPIs 423 364 translation. Our IP–MS findings also indicate that RBM45 424 365 interacts with a number of ALS-linked proteins, including We have previously demonstrated that FLAG-RBM45 associ- 425 366 TDP-43, FUS, Matrin-3, hnRNP-A1, and hnRNP-A2/B1. Selected ates with ALS-linked proteins TDP-43 and FUS in HEK293 cells 426 367 PPIs were externally validated via complementary techni- by immunoblot (Li et al., 2015). As expected, both TDP-43 and 427 368 ques. Collectively, our results shed new light on RBM45 PPIs, FUS were detected in the current IP–MS study (Table 1 and 428

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429 489 430 490 431 491 432 492 433 493 434 494 435 495 436 496 437 497 438 498 439 499 440 500 441 501 442 502 443 503 444 504 445 505 446 506 447 507 448 508 449 509 450 510 451 511 452 512 453 513 454 514 455 515 456 516 457 517 458 518 459 519 460 520 461 521 462 522 Fig. 1 – Identification of RBM45 interacting proteins. (A) Diagram of the immunoprecipitation–mass spectrometry approach to 463 523 identify the RBM45 interacting proteins. (B) Triplicate immunoprecipitates from HEK293 cells stably expressing FLAG-RBM45, HA- 464 524 RBM45 or empty vector were separated by SDS-PAGE and stained with Coomassie blue to visualize proteins. Immunoprecipitations 465 525 with FLAG (sample 1, 2, 3, 4), HA (sample 5) antibody or IgG (sample 6, 7) were performed. Crosslinking IP(sample1,2)andregularIP 466 526 (sample 3, 4, 5) were performed in parallel. For crosslinking IP, live cells were treated with 0.1% formaldehyde to cross-link proteins 467 527 prior to cell lysis and immunoprecipitation. The crosslinking was reversed by heating in SDS-sample buffer prior to SDS-PAGE. The 468 528 proteins along the entire length of the gel were extracted (excluding the IgG-heavy chain and IgG-light chain that are denoted by red 469 529 *) and analyzed by LC/MS–MS. (C) Pie-chart representation of functional classes of RBM45 interacting proteins. (For interpretation of 470 530 the references to color in this figure legend, the reader is referred to the web version of this article.) 471 531 472 532 473 Supplemental Table 1). We next confirmed specific interac- control to further demonstrate the specificity of the observed 533 474 tions of several identified candidate proteins with RBM45 by interactions. As expected, we failed to detect association of 534 475 co-immunoprecipitation (co-IP) followed by immunoblot GAPDH and FLAG-RBM45 (Fig. 2A, bottom). Moreover, we 535 476 (Fig. 2A): hnRNP-L, hnRNP-A1, hnRNP-A2B1, Matrin-3, hnRNP performed reciprocal co-IP assays using whole cell lysate 536 477 537 -A3, and RBM14. All of these candidate proteins were in the from HEK293 cells expressing FLAG-RBM45 and antibodies Q4 478 538 top 20% highest interaction probability and abundance (spec- against the selected candidate proteins. The reciprocal co-IP 479 539 tral counts), and identified in both the regular and cross- analysis demonstrated that FLAG-RBM45 co-purified with 480 540 linking IP experiments (Table 1). We stably expressed FLAG- endogenous hnRNP-L, hnRNP-A1, Matrin-3 and RBM14 pro- 481 541 RBM45 in HEK293 cells and performed co-IP from whole cell 482 teins (Fig. 2B). Taken together, these data provide evidence of 542 – fi fi 483 lysates. To detect transient or weak interactions, the cells the validity of the IP MS approach and con rm speci c 543 484 were treated with formaldehyde to cross-link associated interactions of selected candidate proteins with RBM45. 544 485 proteins prior to co-IP analysis. Anti-FLAG co-IP experiments Since RBM45 contains three RRM domains, it may associate 545 486 demonstrated that all the proteins tested co-purified with with its interacting proteins through RNA–protein interactions. 546 487 FLAG-RBM45 but not with IgG (Fig. 2A). GAPDH, which was To determine if associations between RBM45 and the previously 547 488 not identified by mass spectrometry, was used as negative tested proteins are RNA-dependent, we used in-cell RNase 548

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549 609

550 RAN 610 551 611 552 612 553 613

554 Ext. 614 555 615 556 616 mTOR Telomere 557 617 558 618 559 619

560 p70S6K 620 561 621 562 EIF2 eIF4/ 622 563 623 564 624 565 625 11 11 11 11 11 566 626 567 627 . a 111 111 111 111 x 111 111 111 111 111 x 11 11 111 111 11xx 111 111 111 568 111 628 569 629 þ þ þþ 570 þþ 630 571 631 572 632 þþ þþ þþ þþ þþ þþ þþ þþ þþ þþ þþ þþ þþ þþ þþ þ þþþ þþþ þþþ þþ 573 þþ 633 574 634 þþ þ þ þ þ þ þ þ þ þ þ þ þþ þ þ þþ þþ þ þ þ þ 575 þ 635 576 636 577 637 Fold spectra increaseManual AvgP SAINTexpress SAINTexpress Canonical pathway þþþ þ þþþ þ þþþ þþ þþþ þþ þþþ þþ þþþ þþ þþþ þþ þþþ þþ þþþ þþ þþþ þþ þþþ þþ þþþ þþ þþ þþþ þþ þþþ þþ þþ þþþ þþ þþþ þþ þþþ þþ 578 þþþ þþ 638 579 639 580 640 581 641 582 642 27 25 99 583 50 643 Spectral count sum 207 113 111 584 644 585 645 586 646 587 647 588 648 589 649 590 650 591 651 592 652 593 653 594 654 595 655 596 656 ed RBM45 interacting proteins from manual analysis and SAINTexpress algorithm

597 ﬁ 657 598 658 599 659 600 660 601 661 602 662 603 663

604 Merged list of identi 664

605 – 665 606 666 607 667 Table 1 Proteins Q13148 TARDBP TAR DNA-binding protein 43 Accession Gene name DescriptionQ8IUH3 RBM45 RNA-binding protein 45 969 Regular XL Regular XL Regular XL Avg P14866 HNRNPL Heterogeneous nuclear ribonucleoprotein L Q15717 ELAVL1 ELAV-like protein 1 26 Q92841 DDX17 Probable ATP-dependent RNA helicase DDX17 25 P22626 HNRNPA2B1 Heterogeneous nuclear ribonucleoproteins A2/B1 Q96PK6 RBM14 RNA-binding protein 14 P43243 MATR3 Matrin-3 Q00839 HNRNPU Heterogeneous nuclear ribonucleoprotein U 107 P61978 HNRNPK Heterogeneous nuclear ribonucleoprotein K 105 P09651 HNRNPA1 Heterogeneous nuclear ribonucleoprotein A1 Q93008 USP9X Probable ubiquitin carboxyl-terminal hydrolase FAF-X 97 Q9NZI8 IGF2BP1 Insulin-like growth factor 2 mRNA-binding protein 1 63 Q8N163 CCAR2 Cell cycle and apoptosis regulator protein 2 62 P52272 HNRNPM Heterogeneous nuclear ribonucleoprotein M 60 Q08211 DHX9 ATP-dependent RNA helicase A 60 P08107Q13263 HSPA1A TRIM28 Heat shock 70 kDa protein Transcription 1A/1B intermediary factor 1-beta 55 58 P11940 PABPC1 Polyadenylate-binding protein 1 52 P51991 HNRNPA3 Heterogeneous nuclear ribonucleoprotein A3 P07910 HNRNPC Heterogeneous nuclear ribonucleoproteins C1/C2 48 P38159 RBMX RNA-binding motif protein, X chromosome 41 O43390 HNRNPR Heterogeneous nuclear ribonucleoprotein R 32 608 668

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669 729

670 RAN 730 671 731 672 732 673 733

674 Ext. 734 675 735 676 736 mTOR Telomere 677 737 678 738 679 739

680 p70S6K 740 681 741 682 EIF2 eIF4/ 742 683 743 684 744 685 745 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 686 746 687 747 11 111 11 11 111 111 688 11 748 689 749 þ þ þ þ þ þ þ þ þ þ þ þ þ þ 690 þ 750 691 751 692 752 þþ þþ 693 þþ 753 694 754 þþ þ þþ þ þþ þ þ þþ þ þþ þ þþ þ þþ þ þþ þ þþ þ þ þþ þ þ þþ þ þþ þ þþ þ þþ þ 695 þþ þ 755 696 756 697 757 Fold spectra increaseManual AvgP SAINTexpress SAINTexpress Canonical pathway þþþ þ þþþ þþ þþþ þ þþþ þ þþþ þþ þþþ þþ 698 þþþ þ 758 699 759 700 760 701 761 702 762 21 703 14 763 Spectral count sum 704 764 705 765 706 766 707 767 708 768 709 769 710 770 711 771 712 772 713 773 714 774 715 775 716 776 717 777 718 778 719 779 1 720 helicase DHX15 780 721 781 722 782

723 ) 783 724 784 725 785 continued 726 786 727 787 Proteins Accession Gene name DescriptionP23246 SFPQ Splicing factor, proline- and glutamine-rich 23 Regular XL Regular XL Regular XL Avg Q9Y2L1 DIS3 Exosome complex exonuclease RRP44 14 Q13310 PABPC4 Polyadenylate-binding protein 4 23 Q14103 HNRNPD Heterogeneous nuclear ribonucleoprotein D0 23 P01614 [Ig kappa chain V-II region Cum] 13 Q9NZB2 FAM120A Constitutive coactivator of PPAR-gamma-like protein P07437 TUBB Tubulin beta chain 12 P09874 PARP1 Poly [ADP-ribose] polymerase 1 20 P68104 EEF1A1 Elongation factor 1-alpha 1 20 P78347 GTF2I General transcription factor II-I 20 Q96PU8 QKI Protein quaking 20 O75643 SNRNP200 U5 small nuclear ribonucleoprotein 200 kDa helicase 19 Q15366 PCBP2 Poly(rC)-binding protein 2 19 Q92945 KHSRP Far upstream element-binding protein 2 19 Q99729 HNRNPAB Heterogeneous nuclear ribonucleoprotein A/B 19 O15042 U2SURP U2 snRNP-associated SURP motif-containing protein 18 P17844 DDX5 Probable ATP-dependent RNA helicase DDX5 18 P13639 EEF2 Elongation factor 2 16 P60709 ACTB Actin, cytoplasmic 1 16 P62269 HIST1H4A Histone H4 15 O00425 IGF2BP3 Insulin-like growth factor 2 mRNA-binding protein 3 14 O43143 DHX15 Pre-mRNA-splicing factor ATP-dependent RNA

728 Table 1 ( 788

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789 849 790 850 791 851 792 852 793 853 794 854 795 855 796 856 797 857 798 858 799 859 800 860 801 861 802 862 803 863 804 864 805 865 0.67 0.67 11 0.67 0.67 11 0.67 0.67 x 11 0.67 0.67 11 0.67 0.67 11 0.97 0.97 0.88 0.88 0.67 0.67 0.67 0.67 0.67 0.67 x 0.67 0.67 0.67 0.67 0.67 0.67 x 806 866 807 867 11 11 11 0.67 1 0.835 1 0.67 0.835 1 0.67 0.835 1 0.67 0.835 x x x 1 0.67 0.835 x 0.67 0.67 808 0.67 0.67 x x x 868 809 869 þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ 810 þ 870 811 871 812 872 þþ þþ þþ þþ 813 þþ 873 814 874 þþ þ þþ þ þþ þ þþ þ þþ þ þþ þ þþ þ þþ þ þþ þ þþ þ þþ þ þ þ þ þ þþ þ þþ þ þþ þ þþ þ þþ þ 815 þþ þ 875 816 876 817 877 þþþ þ þþþ þ þþþ þ þþþ þþþþ þþ þ þþþ þþ þþþ þþ þþþ þþ þþþ þþ þþþ þ 818 þþþ þ 878 819 879 820 880 821 881 822 882

823 12 883 824 884 825 885 826 886 827 887 828 888 829 889 830 890 831 891 832 892 833 893 834 894 835 895 836 896 837 897 838 898 839 899 840 900 841 901 842 902 843 903 844 904 845 905 846 906 847 907 P35637 FUS RNA-binding protein FUS P84090 ERH Enhancer of rudimentary homolog 6 Q7L2E3 DHX30 Putative ATP-dependent RNA helicase DHX30 12 Q6PKG0 LARP1 La-related protein 1 11 Q9Y6M1 IGF2BP2 Insulin-like growth factor 2 mRNA-binding protein 2 11 P62988 UBB Ubiquitin 10 P05388 RPLP0 60S acidic ribosomal protein P0 5 Q9UKM9 RALY RNA-binding protein Raly 10 P08238 HSP90AB1 Heat shock protein HSP 90-beta 5 P25205 MCM3 DNA replication licensing factor MCM3 8 P19338 NCL Nucleolin 5 Q99623 PHB2 Prohibitin-2 8 P49327 FASN Fatty acid synthase 5 O75688P62805 PPM1BP26599 HSPD1 PTBP1 Protein phosphatase 1B 60 kDa heat shock protein, Polypyrimidine mitochondrial tract-binding protein 1 15 20 64 Q13151 HNRNPA0 Heterogeneous nuclear ribonucleoprotein A0 20 P31943 HNRNPH1 Heterogeneous nuclear ribonucleoprotein H 16 P10809 RPS18 40S ribosomal protein S18 15 Q14974 KPNB1 Importin subunit beta-1 14 P63241 EIF5A Eukaryotic translation initiation factor 5A-1 11 P12956 XRCC6 X-ray repair cross-complementing protein 6 9 Q86U42 PABPN1 Polyadenylate-binding protein 2 9 O60814 HIST1H2BK Histone H2B type 1-K 7 P62701 RPS4X 40S ribosomal protein S4, X isoform 7 P04908 HIST1H2AB Histone H2A type 1-B/E 6 P55060 CSE1L Exportin-2 6 P62316 SNRPD2 Small nuclear ribonucleoprotein Sm D2 6 848 908

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909 969

910 RAN 970 911 971 912 972 913 973

914 Ext. 974 915 975 916 976 mTOR Telomere 917 977 918 978 919 979

920 p70S6K 980 921 981 922 EIF2 eIF4/ 982 923 983 924 984 925 985 0.67 0.67 0.67 0.67 0.33 0.33 0.67 0.67 0.33 0.33 0.67 0.67 0.67 0.67 0.67 0.67 0.67 0.67 0.66 0.66 926 986 927 987 0 1 0.5 0.33 0.33 0.66 0 0.33 x x x 0.33 1 0.665 1 0.33 0.665 1 0.33 0.665 1 0.33 0.665 x 0.66 0.67 0.665 0.97 0.33 0.65 0.65 0.65 x x x 0.67 0.33 0.5 928 1 0 0.5 988 929 989 þ þ þ þ þ þ þ þ 930 þ 990 931 991 932 992 þþ þþ þþ þþ þþ þþ þþ þþ þþ 933 þþ 993 934 994 þ þþ þ þþ þ þþ þ þþ þ þþ þ þþ þ þþ þ þþ þ þþ þ þ þ þ þ þ þþ þ þ þ 935 þ 995 936 996 937 997 þþ þþþ þþ Fold spectra increaseManual AvgP SAINTexpress SAINTexpress Canonical pathway þþþ þþ þþþ þþ þþþ þþ þþþ þþ þþþ þþ þþþ þþ þþþ þþ þþþ þþ 938 þþþ þþ 998 939 999 940 1000 941 1001 942 1002 4 943 5 1003 Spectral count sum 944 1004 945 1005 946 1006 947 1007 948 1008 949 1009 950 1010 951 1011 952 1012 953 1013 954 1014 955 1015 956 1016 957 1017 958 1018 959 1019 protein 960 component 1020 961 1021 962 1022

963 ) 1023 964 1024 965 1025 continued 966 1026 967 1027 Proteins P08779 KRT16 [Keratin, type I cytoskeletal 16] 13 P23396 RPS3 40S ribosomal protein S3 7 Accession Gene name DescriptionP52597 HNRNPF Heterogeneous nuclear ribonucleoprotein F 5 Regular XL Regular XL Regular XL Avg Q15233 NONO Non-POU domain-containing octamer-binding P62314 SNRPD1 Small nuclear ribonucleoprotein Sm D1 5 P62318 SNRPD3 Small nuclear ribonucleoprotein Sm D3 5 Q15365 PCBP1 Poly(rC)-binding protein 1 4 Q15029 EFTUD2 116 kDa U5 small nuclear ribonucleoprotein Q8IY67 RAVER1 Ribonucleoprotein PTB-binding 1 5 Q9Y2W1 THRAP3 Thyroid hormone receptor-associated protein 3 5 P15927 RPA2 Replication protein A 32 kDa subunit 4 Q12906 ILF3 Interleukin enhancer-binding factor 3 23 O60506 SYNCRIP Heterogeneous nuclear ribonucleoprotein Q 21 P68363 TUBA1B Tubulin alpha-1B chain 15 P52294 KPNA1 Importin subunit alpha-5 13 Q12905 ILF2 Interleukin enhancer-binding factor 2 11 P68032P01859 ACTC1 IGHG2 Actin, alpha cardiac muscle 1 [Ig gamma-2 chain C region] 7 10 P39019 RPS19 40S ribosomal protein S19 6 O14979 HNRNPDL Heterogeneous nuclear ribonucleoprotein D-like 14 P67809 YBX1 Nuclease-sensitive element-binding protein 1 12 P05141 SLC25A5 ADP/ATP translocase 2 9

968 Table 1 ( 1028

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1029 1089 1030 1090 1031 1091 1032 1092 1033 1093 1034 1094 1035 1095 1036 1096 1037 1097 1038 1098 1039 1099 1040 1100 1041 1101 1042 1102 1043 1103 1044 1104 1045 1105 0.33 0.33 00 0.33 0.33 00 0.33 0.33 00 0.33 0.33 00 0.33 0.33 00 0.33 0.33 0.33 0.33 0.33 0.33 00 00 xx x 00 1046 1106 1047 1107 00x 0.33 0.33 00 0.33 0.33 x x x 0.33 0.33 0.32 0.32 x x x 0.59 0 0.295 x 000 00x 00x 00xxx 00x 1048 00 1108 1049 1109 þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ 1050 þ 1110 1051 1111 1052 1112 þþ 1053 þþ 1113 1054 1114 þþ þ þþ þ þþ þ þþ þ þþ þ þþ þ þþ þ þþ þ þþ þ þþ þ þþ þ þþ þ þ þ þþ þ þþ þ þþ þ 1055 þþ þ 1115 1056 1116 1057 1117 þþþ þ þþþ þ þþþ þ þþþ þ þþþ þ þþ þþþ þþþ þþ þþ þþþ þ þþþ þ 1058 þþþ þ 1118 1059 1119 1060 1120 1061 1121 1062 1122 3 1063 3 1123 1064 1124 1065 1125 1066 1126 1067 1127 1068 1128 1069 1129 1070 1130 1071 1131 1072 1132 1073 1133

1074 IV region JI]1134 2 – 1075 1135 1076 1136 1077 1137

1078 nger CCCH domain-containing protein 4 3 1138 ﬁ 1079 1139 mitochondrial 1080 protein 1 1140 1081 1141 1082 1142 1083 1143 1084 1144 1085 1145 1086 1146 1087 1147 Q99459 CDC5L Cell division cycle 5-like protein 4 Q99873 PRMT1 Protein arginine N-methyltransferase 1 4 Q16576 RBBP7 Histone-binding protein RBBP7 3 Q9BXP5 SRRT Serrate RNA effector molecule homolog 4 Q7Z5L9 IRF2BP2 Interferon regulatory factor 2-binding protein 2 3 P09429 HMGB1 High mobility group protein B1 3 Q9HAV4 XPO5 Exportin-5 3 P42704 LRPPRC Leucine-rich PPR motif-containing protein, P05386 RPLP1 60S acidic ribosomal protein P1 2 P06313 [Ig kappa chain V P43246 MSH2 DNA mismatch repair protein Msh2 3 P31942 HNRNPH3 Heterogeneous nuclear ribonucleoprotein H3 2 P62851 RPS25 40S ribosomal protein S25 3 Q04837 SSBP1 Single-stranded DNA-binding protein, mitochondrial 3 Q13838 DDX39B Spliceosome RNA helicase DDX39B 3 Q9BUJ2 HNRNPUL1 Heterogeneous nuclear ribonucleoprotein U-like Q9UPT8 ZC3H4 Zinc P46782 RPS5 40S ribosomal protein S5 5 P62913 RPL11 60S ribosomal protein L11 9 P06748 NPM1 Nucleophosmin 5 P26373 RPL13 60S ribosomal protein L13 4 P05387 RPLP2 60S acidic ribosomal protein P2 3 P14174 MIF Macrophage migration inhibitory factor 3 P46783 RPS10 40S ribosomal protein S10 3 P62263 RPS14 40S ribosomal protein S14 3 P62917 RPL8 60S ribosomal protein L8 3 P82673 MRPS35 28S ribosomal protein S35, mitochondrial 3 P84103 SRSF3 Serine/arginine-rich splicing factor 3 3 Q13283 G3BP1 Ras GTPase-activating protein-binding protein 1 3 1088 1148

10 brain research ] ( ]]]]) ]]]– ]]]

1149 1209

1150 RAN 1210 1151 1211 1152 1212 1153 1213

1154 Ext. 1214 1155 1215 1156 1216 mTOR Telomere 1157 1217 1000. Fold-change values are

1158 4 1218 1159 1219 þþþ 1160 p70S6K 1220

1161 edict high likelihood of interaction. Fold- 1221 1000, EIF2 eIF4/ – 1162 m. Candidate RBM45 protein interactors are 1222

1163 101 1223

1164 þþ 1224 1165 1225 00 xx x 00 xx x 00 00 00 00 100, –

1166 ed in the thresholding analysis. Bracketed proteins were 1226 ﬁ 2.5 1167 1227 þ 00xxx 00xxx 00xxx 00xxx 1168 00xx 1228 1169 1229 þ þ þ þ þ 1170 þ 1230 1171 1231 1172 1232

1173 ed by immunoblot in the current or in the previous study (Li et al., 2015). 1233 ﬁ 1174 1234 þ þþ þ þþ þ þþ þ þþ þ þþ þ þþ þ þþ þ 1175 þþ þ 1235 1176 1236 1177 1237 þþþ þþ Fold spectra increaseManual AvgP SAINTexpress SAINTexpress Canonical pathway þþþ þ þþþ þ þþþ þ þþþ þ 1178 þþþ þ 1238 1179 1239 1180 1240 1181 1241 1182 1242 cations (empty vector) are categorized as follows: 2 1183 ﬁ 1243 Spectral count sum 1184 1244 1185 1245 ve canonical pathways are highlighted (EIF2 Signaling (EIF2), Regulation of eIF4 and p7056K Signaling (eIF4/p7056K), mTOR

1186 ﬁ 1246 1187 1247 1188 1248 1189 1249 1190 1250 1191 1251 1192 1252 1193 1253

1194 cations against negative control puri 1254 ﬁ 1195 1255 1196 1256 1197 1257 1198 1258 1199 1259 protein 2 1000. 1200 1000. 1260 – 4

1201 100. 1261 – 1202 1262 )

1203 putative contaminant. 1263 ¼ KRT2DDX3Y [Keratin, type II cytoskeletal 2KRT5 epidermal] Cluster of ATP-dependent RNA helicase DDX3Y [Keratin, type II cytoskeletal 5] 4 41 2

1204 b b b 1264 fold change ed as putative contaminants in the CRAPome database (v1.1). Bolded proteins highlight interactions veri ed using thresholding analysis and probabilistic scoring of associations (SAINTexpress). Proteins are sorted by decreasing AvgP, higher values pr manual analysis only (all others were found in both methods). 1205 ¼ 1265 fold change 101 ﬁ ﬁ " continued ¼ Externally Validated. ¼ fold change 2.5 " 1206 ¼ 1266 ¼ " Gene identi provided in the Supplemental Material section. Associations with top Signaling (mTOR), Telomere Extension by Telomerase (Telomere Ext.), and RAN Signaling (RAN)). Starred proteins were only identi changes are computed from positive puri identi 1207 Proteins listed were detected in either regular IP or crosslinking IP. The proteins were detected by either manual analysis or SAINTexpress algorith 1267 þ þþ þþþ a b O15523 P13647 [Description] Bold " Proteins " " Accession Gene name DescriptionP38919 EIF4A3 Eukaryotic initiation factor 4A-III 2 Regular XL Regular XL Regular XL Avg P60842 EIF4A1 Eukaryotic initiation factor 4A-I 2 P60866 RPS20 40S ribosomal protein S20 2 P62081 RPS7 40S ribosomal protein S7 2 P62249 RPS16 40S ribosomal protein S16 2 P62277 RPS13 40S ribosomal protein S13 2 Q14498 RBM39 RNA-binding protein 39 2 Q1KMD3 HNRNPUL2 Heterogeneous nuclear ribonucleoprotein U-like Q93009 USP7 Ubiquitin carboxyl-terminal hydrolase 7 2 Q9BQ04 RBM4B RNA-binding protein 4B 2 Q9H074 PAIP1 Polyadenylate-binding protein-interacting protein 1 2 P35908

1208 Table 1 ( 1268

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1269 1329 1270 1330 1271 1331 1272 1332 1273 1333 1274 1334 1275 1335 1276 1336 1277 1337 1278 1338 1279 1339 1280 1340 1281 1341 1282 1342 1283 1343 1284 1344 1285 1345 1286 1346 1287 1347 1288 1348 1289 1349 1290 1350 1291 1351 1292 1352 1293 1353 1294 1354 1295 1355 1296 1356 1297 1357 1298 1358 1299 1359 1300 1360 1301 1361 1302 1362 1303 1363 1304 Fig. 2 – Verification of RBM45 interacting proteins. (A) Pull-down of selected proteins with FLAG-RBM45 expressed in HEK293 1364 1305 cells. Crosslinking immunoprecipitations with FLAG antibody or IgG were performed. The IP fractions were immunoblotted with 1365 1306 hnRNP-L, hnRNP-A1, hnRNP-A2B1, Matrin-3, hnRNP-A3 and RBM14 antibodies. The same IP fractions were also immunobloted 1366 1307 with FLAG (FLAG-RBM45) and GAPDH (negative IP control) antibodies. (B) Immunoprecipitations of endogenous candidate 1367 1308 proteins in FLAG-RBM45 expressing cells shows that FLAG-RBM45 co-purified with the tested endogenous proteins. The 1368 1309 endogenous candidate proteins were immunoprecipitated with hnRNP-L, hnRNP-A1, Matrin-3 or RBM14 antibody, while IgG 1369 1310 pull-down was used for IP control. The immunoblots were detected with FLAG antibody (FLAG-RBM45), tested endogenous 1370 1311 protein specific antibodies, and GAPDH antibody (negative IP control). Similar validation studies for TDP-43 and FUS were 1371 1312 previously reported (Li et al., 2015). (C) In-cell RNase treatment and crosslinking-IP were performed on cells expressing FLAG- 1372 1313 RBM45. The amount of hnRNP-L, hnRNP-A1, the lower-molecular-weight band of hnRNP-A2B1, Matrin-3 and RBM14 that co- 1373 1314 purified with FLAG-RBM45 was reduced upon the RNase treatment. (D) The RBM45-(Δ286-318) construct, i.e. the homo- 1374 1315 oligomerization assembly (HOA) domain deficient construct (Li et al., 2015), exhibits significantly reduced binding to the tested 1375 1316 candidate proteins when compared to full-length-RBM45. Full-length FLAG-RBM45 or FLAG-Δ(286-318) construct were 1376 1317 expressed in HEK293 cells. FLAG-IP was performed as described previously. Immunoblot analysis shows that all the tested 1377 1318 candidate proteins displayed reduced co-IP% with FLAG-Δ(286-318) as compared with the full-length FLAG-RBM45. 1378 1319 1379 1320 1380 1321 treatment prior to the cross-linking and anti-FLAG IP (Li et al., hnRNP-A2B1 was not affected by RNase treatment (Fig. 2C). This 1381 1322 2015). The in-cell RNase treatment significantly reduced the result suggests that many RBM45 PPIs are RNA-dependent. 1382 1323 amounts of the hnRNP-L, hnRNP-A1, Matrin-3 and RBM14 1383 1324 proteins that co-purified with FLAG-RBM45 (Fig. 2C). Interest- 2.3. RBM45 homo-oligomerization mediates association with 1384 1325 ingly, the amount of the lower-molecular-weight species (arrows, a large number of proteins 1385 1326 Fig. 2C) of the hnRNP-A2B1 protein that was co-purified with 1386 1327 FLAG-RBM45 reduced upon RNase treatment. However, the co- We previously reported that the RBM45 homo-oligomer 1387 1328 purified amount of the higher-molecular-weight species of assembly (HOA) domain mediates association with TDP-43 1388

12 brain research ] ( ]]]]) ]]]– ]]]

1389 and FUS (Li et al., 2015). We hypothesize that the HOA domain well as downstream processing events (“protein targeting to 1449 1390 serves as a general protein–protein interaction mediator. To ER”, “nonsense mediated mRNA decay”). Finally, terms unre- 1450 1391 test this hypothesis, we expressed FLAG-tagged constructs of lated to these phenomena and unconnected to any nodes 1451 1392 either the full-length RBM45 or the Δ(286-318) construct with included “apoptotic nuclear changes” and “telomere main- 1452 1393 the majority of the HOA domain deleted and incapable of tenance” (Fig. 3). 1453 1394 homo-oligomerization (Li et al., 2015). Anti-FLAG co-IP ana- To provide further insights into the biological processes 1454 1395 lysis showed that the tested candidate proteins co-purified identified by this approach, we took leading terms, those 1455 1396 efficiently with the FLAG-full-length RBM45. However, the terms with the highest number of associated proteins, from 1456 1397 FLAG-Δ(286-318) construct exhibited significantly reduced our results and visualized these terms with their associated 1457 1398 binding to all the tested candidate proteins (Fig. 2D). These proteins in a network layout where edges connect proteins to 1458 1399 results suggest that the HOA domain is an important med- an associated biological process (Fig. 4). The results show the 1459 1400 iator of RBM45 PPIs and that homo-oligomerization of RBM45 individual proteins that result in the identification of an 1460 1401 is required for many RBM45 PPIs. enriched biological process. For example, the identification 1461 1402 1462 of the “mRNA metabolic process” and “regulation of RNA 1403 1463 2.4. Gene ontology and pathway analysis splicing” terms results in large part from the many hnRNP 1404 1464 proteins in our list of RBM45-interacting proteins. Conversely, 1405 1465 To identify putative biological processes associated with the enrichment for “regulation of translation” results from 1406 1466 RBM45-interacting proteins, we performed enrichment ana- the presence of initiation and elongation factors (e.g., eIF 1407 1467 lysis in the Gene Ontology (GO) domain “Biological Process” proteins) in our list of RBM45-interacting proteins (Fig. 4). 1408 1468 (Fig. 3). The results of this analysis identified two predomi- Major canonical pathways associated with RBM45- 1409 1469 nant themes: (1) nuclear RNA processing and (2) cytoplasmic interacting proteins were identified using Ingenuity Pathway 1410 1470 fl Analysis (IPAs, QIAGEN Redwood City). Out of a total of 103 1411 RNA translation. RNA processing terms were chie y related 1471 “ ” “ pathways, 28 were significantly enriched (p-value lower than 1412 to splicing (e.g., regulation of RNA splicing , alternative 1472 ” 0.05). The top 5 pathways, ranked by significance and percent 1413 mRNA splicing ). Other nuclear RNA-associated terms 1473 “ ” “ ” “ ” “ 1414 included mRNA transport , regulation of mRNA stability , overlap are EIF2 Signaling , Regulation of eIF4 and p70S6K 1474 “ ” ” “ ” “ 1415 and nuclear export . Cytoplasmic translational themes were Signaling , mTOR Signaling , Telomere Extension by Telo- 1475 ” “ ” 1416 more diverse and included events directly to mRNA transla- merase , and RAN Signaling (Table 1 and Supplemental 1476 “ ” “ ” 1417 tion ( translation initiation , translation termination ), as Table 7). These results were consistent with associations 1477 1418 1478 1419 1479 1420 1480 1421 1481 1422 1482 1423 1483 1424 1484 1425 1485 1426 1486 1427 1487 1428 1488 1429 1489 1430 1490 1431 1491 1432 1492 1433 1493 1434 1494 1435 1495 1436 1496 1437 1497 1438 1498 1439 1499 1440 1500 1441 1501 1442 Fig. 3 – Enriched GO Biological Process terms. RBM45-interacting proteins were tested for GO Biological Process enrichment 1502 1443 using the right-sided hypergeometric test with Benjamini–Hochberg post-hoc p value correction. Terms with a p value of 0.001 1503 1444 or less were visualized in a network layout, where node size corresponds to term p value. The proportion of shared proteins 1504 1445 between terms was evaluated using the kappa statistic and nodes with a kappa score (κ) of at least 0.4 were connected with 1505 1446 edges on the graph, with edge width proportional to kappa score. Leading terms, those terms with the highest number of 1506 1447 proteins, are colored for emphasis. (For interpretation of the references to color in this figure legend, the reader is referred to 1507 1448 the web version of this article) . 1508

brain research ] ( ]]]]) ]]]– ]]] 13

1509 1569 1510 1570 1511 1571 1512 1572 1513 1573 1514 1574 1515 1575 1516 1576 1517 1577 1518 1578 1519 1579 1520 1580 1521 1581 1522 1582 1523 1583 1524 1584 1525 1585 1526 1586 1527 1587 1528 1588 1529 1589 1530 1590 1531 1591 1532 1592 1533 1593 1534 1594 1535 1595 1536 1596 1537 Fig. 4 – Leading terms with associated proteins. Leading terms from Fig. 3 were placed into a separate network and all 1597 1538 associated proteins from the list of RBM45-interacting proteins were visualized as nodes and connected to the appropriate 1598 1539 term. Where a protein is associated with multiple terms, multiple edges emanate from that protein and edges are color- 1599 1540 matched to their associated terms. (For interpretation of the references to color in this figure legend, the reader is referred to 1600 1541 the web version of this article). 1601 1542 1602 1543 1603 1544 found in the gene ontology analysis. Collectively, this view subcellular segregation, the ICQ is large, negative, and statis- 1604 1545 emphasizes the diverse array of biological functions served tically significant, while for random variations in intensity, 1605 1546 by RBM45-interacting proteins. the ICQ¼0, p40.05. 1606 1547 The results of this analysis are shown in Fig. 5B. We used 1607 1548 2.5. Co-localization analysis SMN as a negative control, as the staining for this protein is 1608 1549 predominantly cytoplasmic. As shown in Fig. 5B, by either 1609 1550 To assess the association of RBM45 and selected interacting measure of co-localization, the association between RBM45 1610 1551 proteins in cells, we used immunocytochemistry of our FLAG- and SMN is low, reflecting subcellular segregation, as antici- 1611 1552 RBM45 stable HEK293 cells together with digital deconvolu- pated. We then evaluated the extent of co-localization 1612 1553 tion and co-localization analysis. The results of this analysis between RBM45 and several RBM45-interacting proteins. 1613 1554 are shown in Fig. 5 and Supplemental Fig. 3. Because the FLAG-RBM45 staining was exclusively nuclear and we eval- 1614 1555 staining we observe for the majority of the proteins analyzed uated the co-localization of RBM45 with several nuclear 1615 1556 is predominantly nuclear (Supplemental Fig. 3), multiple hnRNP proteins. By both methods, the highest degree of co- 1616 1557 methods were used to provide a quantitative measure of localization was observed between RBM45 and hnRNP-A1 1617 1558 the extent of co-localization. We thus analyzed the co- (Fig. 5H). RBM45 also exhibited statistically significant co- 1618 1559 localization of FLAG-RBM45 and selected proteins using localization with hnRNP-A3, hnRNP-L, and Matrin 3, in 1619 1560 Manders coefficients (Bolte and Cordelieres, 2006) and the descending order of extent of co-localization (Fig. 5B). By 1620 1561 intensity correlation quotient (ICQ) (Li et al., 2004), together contrast, RBM45 co-localization with hnRNP-A2/B1 by either 1621 1562 with pixel intensity scatter plots. The ICQ evaluates the co- approach was lesser and did not reach statistical significance, 1622 1563 variation of pixel intensities for each protein and provides a despite a nuclear localization for both proteins (Fig. 5A). This 1623 1564 correlation-based metric (the ICQ, range 0.5 to 0.5) that finding highlights the utility of digital deconvolution and 1624 1565 reflects the degree to which protein staining intensities vary quantitative co-localization measures for assessing the true 1625 1566 in synchrony and associated statistical significance. If stain- extent of association between proteins by immunocytochem- 1626 1567 ing intensities vary in synchrony (co-localization), the ICQ is istry. We also observed a lack of co-localization between 1627 1568 large, positive, and statistically significant. For proteins with RBM45 and G3BP, the latter of which was predominantly 1628

14 brain research ] ( ]]]]) ]]]– ]]]

1629 1689 1630 1690 1631 1691 1632 1692 1633 1693 1634 1694 1635 1695 1636 1696 1637 1697 1638 1698 1639 1699 1640 1700 1641 1701 1642 1702 1643 1703 1644 1704 1645 1705 1646 1706 1647 1707 1648 1708 1649 1709 1650 1710 1651 1711 1652 1712 1653 1713 1654 1714 1655 1715 1656 1716 1657 1717 1658 1718 1659 1719 1660 1720 1661 1721 1662 1722 1663 1723 1664 1724 1665 Fig. 5 – Co-localization analysis. (A–G) The co-localization of RBM45 and the indicated proteins were evaluated using 1725 1666 immunocytochemistry together with image deconvolution and co-localization analysis. Representative images and pixel 1726 1667 intensity scatter plots are shown with cutouts at higher magnification to highlight detail. (H) Statistical analysis of protein co- 1727 1668 localization. M1¼RBM45 overlap with indicated protein. M2¼indicated protein overlap with RBM45. ICQ¼intensity 1728 1669 correlation quotient. p ICQ¼p value of ICQ. Manders coefficients (M1 and M2) measure the proportion of co-localizing proteins 1729 1670 in each channel of a two-channel image and are shown as mean7SEM. The intensity correlation quotient (ICQ) has a range of 1730 1671 0.5 (perfect segregation) to 0.5 (perfect co-localization), with random intensity variation resulting in a value 0. The 1731 1672 statistical significance of each ICQ value is shown at far right. SMN staining was used as a negative control. 1732 1673 1733 1674 1734 1675 1735 1676 cytoplasmic. The absence of statistically significant co- binding protein (RBP) in HEK293 cells. By employing two 1736 1677 localization between RBM45 and hnRNP-A2/B1 and G3BP complementary IP methods, regular IP and formaldehyde 1737 1678 may reflect the absence of required stimuli/signaling events crosslinking-IP, we detected 132 RBM45 PPIs with high con- 1738 1679 necessary for the interaction of these proteins. The associa- fidence. Our ability to identify numerous RBM45 PPIs with 1739 1680 tion of RBM45 and G3BP, for example, most likely occurs in high confidence was a result of our stringent IP–MS approach. 1740 1681 cytoplasmic stress granules that not observed under basal We identified 132 “true” interactors along with another 1741 1682 conditions (Li et al., 2015). 6 proteins matched to putative contaminants in the CRA- 1742 1683 Pome database (Mellacheruvu et al., 2013). Triplicate IPs were 1743 1684 analyzed by mass spectrometry. Identified proteins were 1744 1685 3. Discussion subjected to a manual thresholding approach (resulting in 1745 1686 132 hits) and a probabilistic approach (resulting in 131 hits) to 1746 1687 We used IP–MS to identify RBM45 PPIs and gain insight into remove non-specifically bound proteins and predict putative 1747 1688 the biological functions of this ALS/FTLD-associated RNA- PPIs. The resulting candidate proteins overlapped at 98.9%, 1748

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1749 highlighting the robustness of analytical method. RBPs were binding proteins share the regulation of specific RNA targets. 1809 1750 the most prominent protein family identified by our analy- We used our list of RBM45-interacting proteins to generate a 1810 1751 tical approach, both in overall number of proteins and list of putative RBM45 biological functions and associated 1811 1752 individual protein spectral counts. Taking the list of RBM45 pathways using Gene Ontology and pathway analysis. Two 1812 1753 PPIs, we next used enrichment and pathway analysis to link major themes emerged: nuclear RNA processing/splicing via 1813 1754 RBM45 PPIs to putative biological functions and pathways. hnRNPs and cytoplasmic translation via the eiF2 and eiF4 1814 1755 The results showed enrichment for nuclear RNA processing pathways (Figs. 3 and 4; Supplemental Table 7). The many 1815 1756 via hnRNPs and cytoplasmic translation functions via eiF2 splicing-associated proteins in our list (Fig. 4) suggest a role 1816 1757 and eiF4 pathways. Taken together, these results provide new for RBM45 in the regulation of splicing events. Dysregulation 1817 1758 insights into the PPIs, biological functions, and roles in ALS/ of RNA splicing is a well-characterized phenomenon in ALS/ 1818 1759 FTLD of RBM45. FTLD and can result from RBP cytoplasmic mis-localization, 1819 1760 These insights are necessary to further understand the aggregation, or both (Walsh et al., 2015). Loss of individual 1820 1761 role of RBM45 (and RBPs more generally) in ALS/FTLD. RBM45 RBP function due to these phenomena can have profound 1821 1762 is a component of ubiquitinated inclusions in neurons and effects on transcriptional regulation. For example, TDP-43 1822 1763 glial cells in ALS, FTLD, and AD patients (Collins et al., 2012). and FUS bind to more than 50% of the human transcriptome 1823 1764 The mechanisms mediating the protein's incorporation into and the loss of these proteins results in substantial global 1824 1765 inclusions are poorly understood, however. RBM45 is distinct alterations in transcription and splicing (Lagier-Tourenne 1825 1766 from other inclusion forming RBPs, such as TDP-43, FUS, et al., 2012; Polymenidou et al., 2011; Tollervey et al., 2011). 1826 1767 TAF15, and hnRNP-A1, in that it does not possess a prion-like We anticipate that future studies directly examining the role 1827 1768 domain (King et al., 2012). RBM45 does, however, contain a of RBM45 in the regulation of transcription and RNA splicing 1828 1769 homo-oligomerization (HOA) domain that mediates RBM45 will likewise reveal widespread RBM45 binding across the 1829 1770 self-association and association with other RBPs, including transcriptome and substantial influence on mRNA splicing 1830 1771 TDP-43 and FUS, suggesting a role for this domain in RBM45 decisions. 1831 1772 inclusion formation (Li et al., 2015). Consistent with this In further support of this notion, the identification of 1832 1773 notion, we identify numerous inclusion-forming RBPs that RBM45 PPIs with 19 members of the hnRNP family suggests 1833 1774 bind to RBM45 via our IP–MS approach, including hnRNP-A1, considerable functional overlap between RBM45 and this 1834 1775 hnRNP-A2/B1, TDP-43 and FUS (reviewed in (Peters et al., diverse class of proteins. Spectral count values for many of 1835 1776 2015). For several of these proteins, the HOA domain is these proteins were among the highest observed in our study 1836 1777 requisite for interaction (Fig. 2D). Thus, while the HOA (Table 1) and we accordingly predict considerable functional 1837 1778 domain is likely necessary for normal RBM45 functions, its overlap between RBM45 and the hnRNP family. hnRNPs 1838 1779 role in mediating RBM45 oligomerization and association participate in a variety of mRNA processing/maturation 1839 1780 with other RBPs suggests this domain also contributes to processes, including mRNA maturation, splicing, nuclear 1840 1781 the pathological aggregation of RBM45 and other RBPs in ALS/ export, and 30-end processing (Kim and Dreyfuss, 2001). 1841 1782 FTLD. The presence of prion-like domains in many RBM45 Abnormalities in the expression/function of hnRNPs are 1842 1783 interacting proteins and the lack of a prion domain in RBM45 associated with a number of human diseases, including ALS 1843 1784 also suggests that RBM45 aggregation may be driven by its by virtue of the recent demonstration that mutations in the 1844 1785 association with other aggregation-prone RBPs, as has been prion domains of hnRNP-A2/B1 and hnRNP-A1 cause familial 1845 1786 observed for RBPs such as PSF and NONO found in TDP-43/ forms of ALS (Kim et al., 2013). Our analysis of the co- 1846 1787 FUS positive aggregates (Dammer et al., 2012; Shelkovnikova localization of RBM45 and these proteins demonstrates that 1847 1788 et al., 2014). Matrin 3 is a nuclear matrix protein implicated in RBM45 co-localizes most highly with hnRNP-A1, followed by 1848 1789 binding and stabilizing mRNA and Matrin 3 mutations have hnRNP-A3 and hnRNP-L, with low, non-significant co-locali- 1849 1790 been linked to ALS (Johnson et al., 2014). While Matrin 3 has zation observed with hnRNP-A2/B1 (Fig. 5). 1850 1791 not been associated with cytoplasmic inclusions in ALS, The association of RBM45 with hnRNP-A1, together with 1851 1792 interactions between RBM45 and Matrin 3 within the nucleus the aggregation-prone prion-like domain of hnRNP-A1, may 1852 1793 may contribute to the regulation of mRNA stability and thus mediate both the function and aggregation of RBM45. 1853 1794 transport within the nucleus. The identification of numerous We observe a high degree of nuclear co-localization between 1854 1795 ALS-associated proteins within our RBM45 PPI list suggests these proteins (Fig. 5) and confirmed their physical, RNA- 1855 1796 that RBM45 can directly contribute to disease by virtue of its dependent interaction via co-immunoprecipitation (Fig. 2C). 1856 1797 association with these proteins. hnRNP-A1 serves many purposes in the nucleus, including 1857 1798 The aggregation of RBPs in ALS/FTLD confers toxicity both regulating the transcription of numerous genes (Jean-Philippe 1858 1799 by aggregation-induced toxic gain of function as well as et al., 2013). Transcriptional regulation by hnRNP-A1 is, in 1859 1800 aggregation-induced loss of normal RBP function. Thus, part, conferred by its ability to bind and relax G-quadruplex 1860 1801 understanding the normal functions of RBPs is critical to nucleic acid structures, including the fALS-linked c9ORF72 1861 1802 identifying molecular mechanisms of disease and potential GGGGCC hexanucleotide repeat expansion (Cooper-Knock 1862 1803 therapeutic targets. RBPs are typically multifunctional and et al., 2014; Fukuda et al., 2002). RBM45 may thus be seques- 1863 1804 act in both the nucleus and cytoplasm, influencing transcrip- tered to c9ORF72 repeat expansion G-quadruplex structures 1864 1805 tion, RNA splicing, RNA export, translation, and transport of in c9-linked fALS cases, causing a loss of normal RBM45 1865 1806 mRNAs (Dreyfuss et al., 2002). Interestingly, RBM45 associates functions. Indeed, we identify numerous c9ORF72 repeat 1866 1807 with many of the validated binding proteins via RNA- expansion binding proteins, including FUS, ELAVL1, hnRNP- 1867 1808 mediated interactions (Fig. 2), suggesting that RBM45 and its K, hnRNP-L, hnRNP-Q, and hnRNP-U, as RBM45 PPIs (Table 1) 1868

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1869 (Cooper-Knock et al., 2014; Mori et al., 2013). Despite its high with nucleocytoplasmic shuttling hnRNP's, such as hnRNP- 1929 1870 affinity for poly(G)/(C) RNA (Tamada et al., 2002), RBM45 A1, hnRNP-L, and hnRNP-K (Kim et al., 2000). 1930 1871 binding to c9ORF72 has not been shown, although discrepan- One limitation of the current approach is that our analyses 1931 1872 cies between experimental approaches and results suggest were performed exclusively in HEK293 cells. HEK293 cells 1932 1873 that additional c9-binding RBPs remain as yet unidentified have a unique gene expression profile, rapidly divide, and 1933 1874 (Cooper-Knock et al., 2014; Mori et al., 2013). have an unstable karyotype. Each of these properties could 1934 1875 We also found significant co-localization of RBM45 with influence the list of RBM45 PPIs detected in the present work. 1935 1876 hnRNP-L and hnRNP-A3 in the nucleus (Fig. 5). hnRNP-L is a Future studies are necessary to determine cell-type and 1936 1877 multifunctional protein that regulates transcript splicing (Hui phenotype-specific RBM45 PPIs and how these contribute to 1937 1878 et al., 2003b), stability (Hui et al., 2003a), and translation cellular physiology. One area of particular interest is the role 1938 1879 (Majumder et al., 2009). The protein affects splice site deci- of RBM45 in cell division and cell type specification. The 1939 1880 sions for a large number of transcripts and is capable of initial characterization of RBM45 demonstrated developmen- 1940 1881 inhibiting spliceosome assembly via coordinated action with tal regulation and neuronal enrichment of RBM45 expression, 1941 1882 hnRNP-A1 (Chiou et al., 2013; Hung et al., 2008). These results, suggesting that RBM45 and, by extension, RBM45 PPIs con- 1942 1883 together with their RNA-dependent physical interaction tribute to cell division and organismal development. Deli- 1943 1884 (Fig. 2C) and co-localization of RBM45 with these proteins neating which RBM45 PPIs occur in differentiated cell 1944 1885 (Fig. 5), provides further evidence of a role for RBM45 in populations, such as neurons, may likewise yield insight into 1945 1886 mRNA splicing decisions. hnRNP-A3 is involved in the RBM45 PPIs and cellular functions that lead to its incorpora- 1946 1887 nucleocytoplasmic trafficking of mRNA (Ma et al., 2002) and tion into inclusions in ALS/FTLD. While stress is known to 1947 1888 is involved in telomere maintenance and protection by virtue induce cytoplasmic stress granules to modulate translation, 1948 1889 of its direct binding to telomeres (Huang et al., 2010; Tanaka chronic stress has been proposed to induce the generation of 1949 1890 cytoplasmic inclusions from stress granules (Wolozin and 1950 et al., 2007). The protein is also a component of p62 positive/ 1891 Apicco, 2015). Further studies examining the RBM45 protein 1951 TDP-43 negative inclusions in c9ORF72-linked fALS motor 1892 complexes under stress conditions may identify biological 1952 neurons (Mori et al., 2013). hnRNP-A3 is a component mRNP 1893 pathways relevant to the induction of RBM45 aggregation and 1953 complexes that act to stabilize mRNA (Papadopoulou et al., 1894 inclusion formation. 1954 2012). The co-localization of RBM45 and hnRNP-A3 within 1895 Finally, we used multiple immunoprecipitation methods 1955 distinct nuclear foci (Fig. 5, Supplemental Fig. 3) suggests a 1896 coupled with mass spectrometry to increase the confidence 1956 possible role for RBM45 in this process as well. 1897 of our results. Two different tagged RBM45 constructs as well 1957 A variety of cytoplasmic RBP functions also contribute to 1898 as the presence or absence of a formaldehyde crosslinking 1958 cellular function and studies have repeatedly shown that loss 1899 method were used for immunoprecipitation. We used a 1959 of these functions negatively impact cellular viability. TDP- 1900 combination of cross-linking and regular IP to distinguish 1960 43, for example, associates with cytoplasmic stress granules 1901 weak and strong interactions, respectively. While commonly 1961 (Colombrita et al., 2009), regulates local mRNA translation 1902 used to identify PPIs, regular IP may also yield non- 1962 (Wang et al., 2008), and participates in RNA transport 1903 physiological protein associations resulting from artefactual, 1963 (Narayanan et al., 2013). Our results likewise suggest impor- 1904 non-specific binding after cell lysis (Mili and Steitz, 2004). 1964 tant cytoplasmic functions for RBM45 in both normal cellular 1905 Formaldehyde is a mild, cell-permeable and reversible cross- 1965 homeostasis and disease. We identified RNA transport as a 1906 linker with very short spacer length (2.3–2.7 Å) and cross- 1966 biological process putatively regulated by RBM45 (Fig. 3). The 1907 links only closely associated proteins (Klockenbusch and 1967 interaction of RBM45 with ELAVL1, a known RNA transport 1908 Kast, 2010). In vivo formaldehyde crosslinking-IP can help 1968 1909 protein (Kraushar et al., 2014), is consistent with a role for reduce interaction artifacts introduced after cell lysis and 1969 1910 RBM45 in the transport of mRNA and local translation (Fig. 4). help preserve transient and/or weak protein–protein interac- 1970 fi 1911 We also identi ed enrichment in numerous biological pro- tions and has been used for discovering novel protein–protein 1971 1912 cesses directly and indirectly related to cytoplasmic transla- interactions in many proteomics studies (Corgiat et al., 2014; 1972 1913 tion. A direct role for RBM45 in translation is predicted from Klockenbusch and Kast, 2010; Miernyk and Thelen, 2008; 1973 fi 1914 the identi cation of numerous elongation and initiation Sutherland et al., 2008; Zhang et al., 2009). Crosslinking-IP 1974 … 1915 factors (e.g., eiF4a, eiF5A, EEF2, [Fig. 4]) as RBM45 inter- also facilitates stringent immunoprecipitations via increased 1975 1916 actors. Twelve percent of the eiF2 signaling pathway respon- detergent concentration, sonication and extensive washes. 1976 1917 sible for charged tRNA delivery to the ribosome and start site Identification of a protein only in crosslinking-IP experiments 1977 1918 recognition is mapped by PPIs with RBM45, highlighting a suggests that the interaction with RBM45 is weak. However, 1978 1919 possible role of RBM45 in early translational events (Supple- one cannot predict the biologic significance of the interaction 1979 1920 mental Table 7). Indirect contributions to translation included with RBM45 based solely in whether the interaction is strong 1980 1921 the GO Biological Process “Protein Targeting to ER” (Fig. 3). ER or weak. Of the identified 132 proteins, 68 proteins were 1981 1922 stress occurs in ALS (Lautenschlaeger et al., 2012) and RNA- found solely in crosslinking-IP, while only 28 proteins were 1982 1923 binding proteins may directly associate with ER to modulate found exclusively in regular IP. It is possible that the protein- 1983 1924 its functions in certain cell/tissue types (Gautrey et al., 2005). binding sites in these 28 proteins were masked by the cross- 1984 1925 Despite these findings, immunocytochemical analysis shows linking reaction and thus not detected by crosslinking-IP. 1985 1926 an exclusively nuclear staining pattern for RBM45 in HEK293 Collectively, our results demonstrate that RBM45 associ- 1986 1927 cells (Supplemental Fig. 3). However we hypothesize that ates with a large and functionally diverse set of protein 1987 1928 RBM45 can mediate nuclear mRNA export via its association binding partners. Functions served by these proteins, 1988

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1989 particularly the hnRNPs, suggest plausible and previously 50 μg of either pre-crosslinked antibody or IgG. FLAG-IP was 2049 1990 unknown biological functions for RBM45. The identification performed using anti-FLAG M2 Affinity Gel (Sigma A2220), 2050 1991 of these functions and the association of RBM45 with numer- HA-IP was performed using anti-HA Agarose (Sigma A2095), 2051 1992 ous ALS-associated RBPs points to RBM45-mediated mechan- and IgG-IP control was performed using Mouse IgG-Agarose 2052 1993 isms of disease in ALS/FTLD and provides further insight into (Sigma A0919). IPs were performed at 4 1C for 2 h and the 2053 1994 the pathological aggregation of RBM45 occurring in neurode- beads were washed six times in IP buffer. The proteins were 2054 1995 generative disease. The association of RBM45 with the set of eluted with SDS sample buffer and heated at 95 1C for 5 min 2055 1996 proteins identified herein provides new directions for future for regular IP samples and heated for 20 min for formalde- 2056 1997 studies of RBM45's role in neuronal development, the regula- hyde crosslinking IP samples. The samples were then run on 2057 1998 tion of gene expression, and neurodegeneration. the Bolt 4–12% Bis-Tris Plus Gel (Life Technologies), and 2058 1999 stained using Bio-Safe Coomassie Stain (BioRad). 2059 2000 2060 2001 4. Experimental procedure 4.2.2. Protein digestion 2061 2002 Gel lanes in the molecular weight range between 10 kDa and 2062 2003 4.1. Cell culture and plasmid construction greater than 250 kDa were excised into individual fractions, 2063 2004 excluding the stained IgG-H (52 kDa) and IgG-L (25 kDa) 2064 ™ 2005 HEK293 (FreeStyle 293-F Cells, Invitrogen) cells were cul- bands. Bands fractions were then further reduced into cubes 2065 2006 tured in DMEM medium with 10% FBS and 1% Pen-Strep at of 1–2mm3, destained, washed, dried and further processed 2066 1 2007 37 C with 5% CO2. Transfection was performed using the using an established method (Shevchenko et al., 2006). 2067 2008 Lipofectamine 2000 (Life technologies) and stable cell lines Briefly, each fraction was reduced using 10 mM DTT (6 1C 2068 μ 2009 were selected in the presence of 500 g/ml G418 (Life Tech- for 30 min) and alkylated using 55 mM iodoacetamide (room 2069 2010 nologies) 48 h post-transfection. The RBM45 cDNA clone temperature for 30 min, in the dark), using multiple hydra- 2070 2011 plasmid, cGST-hRBM45 (HsCD00356971), was obtained from tion and dehydration cycles of the acrylamide gel. Fractions 2071 2012 the DNASU Plasmid Repository at Arizona State University, were then digested using 20 ng/mL of Trypsin Gold (Promega) 2072 fi 2013 Tempe. The cDNA was ampli ed by PCR using Phusion High- (37 1C, overnight). Finally, peptides were extracted, concen- 2073 2014 Fidelity DNA Polymerase (NEB) and sub-cloned into the trated to dryness under vacuum and stored at 20 1C until 2074 2015 pcDNA3 vector (Invitrogen). The 3xFLAG tag (DYKDHDG- LC–MS analysis. 2075 2016 DYKDHDIDYKDDDDK) or 2xHA tag (DYPYDVPDYAGGAAY- 2076 2017 PYDVPDYA) was appended to the N-terminus of specific 4.2.3. LC–MS analysis 2077 2018 proteins to generate the 3xFLAG-or 2xHA-tagged construct. Each fraction was reconstituted in 0.1% formic acid and 2078 2019 analyzed using online liquid chromatography on a 2079 2020 4.2. LC-MS/MS protein identification nanoAcquity-UPLC coupled to a Thermo LTQ Orbitrap Velos 2080 2021 mass-spectrometry. Samples were loaded onto a 100-mm 2081 2022 4.2.1. Immunoprecipitation diameter column (length 100 mm) packed with 3 mm Reprosil 2082 2023 Each immunoprecipitation (IP) was carried out in triplicate. Pur C18 AQ resin. Solvent A and B were 0.1% formic acid in 2083 2024 Stable cell lines expressing FLAG-RBM45, HA-RBM45, or water and acetonitrile, respectively. The gradient was 3% B to 2084 2025 pcDNA3 vector were grown on 10 cm plates till 90% confluent 40% B in 17 min followed by 40% B to 90% B in 0.5 min, then 2085 2026 and harvested. For regular IP, cells from one 10 cm plate were 90% B for 2 min and final re-equilibration for 10.5 min. The 2086 2027 lysed with 500 μl of 0.5% NP40 lysis buffer (50 mM HEPES pH flow rate was set to 500 nL/min The mass spectrometer was 2087 2028 7.6, 150 mM KCl, 2 mM EDTA, 0.5% NP40, 0.5 mM DTT and operated in positive ion mode using a spray voltage of 1.8 kV, 2088 2029 protease (Sigma P8340)/phosphatase (Calbiochem 524629)/ and a capillary temperature of 200 1C. Data were acquired in 2089 2030 RNase inhibitors (Ambion AM2694)) at 4 1C for 15 min. For top-15, data-dependent acquisition mode using a collision 2090 2031 formaldehyde crosslinking-IP, formaldehyde in-cell cross- voltage of 30 V. 2091 2032 linking was performed prior to IP as previously reported (Li 2092 2033 et al., 2015). Cells from one 10 cm plate were suspended in 4.3. Protein identification 2093 2034 1 ml PBS containing 0.1% formaldehyde and incubated at 2094 2035 room temperature for 7 min with gentle agitation. The sus- Mass spectra were extracted, deconvolved and deisotoped 2095 2036 pension was spun for 3 min at 1800g at room temperature using Proteome Discoverer 1.4.1.14 (Thermo Fisher Scientific, 2096 2037 and the supernatant was discarded. The pellet was washed Waltham, MA) and searched against a concatenated database 2097 2038 with 1 ml 1.25 M glycine in cold PBS twice to quench the (Homo sapiens, Mus musculus, UniprotKB/Swissprot) using 2098 2039 crosslinking reaction. The pellet was further washed in PBS, Mascot (Matrix Science, London, UK; version 1.4.1.14). Oxida- 2099 2040 lysed with 500 μl of 1% NP40 lysis buffer (50 mM HEPES pH 7.6, tion (Met), carbamidomethylation (Cys) were specified as 2100 2041 150 mM KCl, 2 mM EDTA, 1% NP-40, 0.5 mM DTT and pro- variable modifications. Peptides were allowed maximum 2101 2042 tease/phosphatase inhibitors) at 4 1C and sonicated in a water two trypsin missed cleavages with a mass tolerance of 2102 2043 bath sonicator (Misonix Sonicator 3000) at level 2 for 4 cycles 710 ppm, and a fragment ion mass tolerance of 70.8 Da. 2103 2044 (15 s on/30 s off). Search results were imported into Scaffold (Proteome Soft- 2104 2045 The lysates were first cleared by spinning at 16,000g at 41C ware Inc., Portland, OR), and identifications were confirmed 2105 2046 for 15 min to remove cell debris, pre-cleared using IgG– by X!Tandem (The GPM, v2010.12.01.1). Only proteins with 2106 2047 Agarose (Sigma A0919) for 1 h and further centrifugated. probabilities equal or higher than 99.0% were retained for 2107 2048 3 mg of total protein was used for immunoprecipitation with analysis (one or more peptide per protein contributing to a 2108

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2109 positive match). Computation of putative PPIs (manual and 4.5. Reciprocal immunoprecipitation 2169 2110 SAINTexpress) were based on exclusive spectrum counts, as 2170 2111 determined by Scaffold. Cells were cultured and processed as described previously. 2171 2112 In-cell RNase treatment was performed as described in Li 2172 2113 4.4. Bioinformatics, pathway analysis and gene ontology et al. (2015). 500 mg total protein and 2 mg antibody was first 2173 2114 analysis incubated at 4 1C for 1 h, and the entire mixture was added to 2174 2115 15 ml Protein A/G Agarose (Pierce) and rotated at 41 C for 3 h. 2175 2116 A combination of an unsupervised probabilistic approach The immunoprecipitates were washed 4 times and analyzed 2176 2117 (SAINTexpress, (Choi et al., 2012)) and a manual approach for immunoblot. The antibodies used for immunoprecipita- 2177 2118 was used to identify proteins potentially interacting with tions are as follows: mouse monoclonal hnRNP-L antibody 2178 2119 RBM45. For each protein–protein interaction, SAINTexpress (Novus Biological NB120-6106), rabbit monoclonal hnRNP-A1 2179 2120 predicted an individual probability based on spectral counts antibody (Cell Signaling 8443S), rabbit polyclonal Matrin-3 2180 2121 and reported average probabilities across all replicates (AvgP), antibody (Abcam ab70336), rabbit polyclonal RBM14 antibody 2181 2122 average fold-change, average spectral counts and a Bayesian (Proteintech 10196-1-AP). IgG-IP control was performed using 2182 2123 False Discovery Rate (BFDR) (Teo et al., 2014). Empty vector IPs rabbit IgG (Sigma I5006) and mouse IgG (Sigma I5381). 2183 2124 were used as experimental controls to provide a background 2184 2125 list of proteins binding non-specifically to the construct. The 4.6. Immunoblot 2185 2126 interactions provided by SAINTexpress were filtered for 2186 2127 protein fold change equal or greater than 2, for proteins Protein samples were mixed with 4 SDS loading buffer and 2187 2128 observed in at least 2 out of 3 replicates and with an AvgP denatured by heating (95 1C for 5 min for regular IP samples 2188 2129 equal or greater than 0.7, as recommended (Choi et al., 2012). and 95 1C for 20 min for crosslinking IP samples), resolved on 2189 2130 For manual elucidation of candidate PPIs, only proteins the Bolt 4–12% Bis-Tris Plus Gel (Life Technologies), and 2190 2131 observed in at least 2 out of 3 replicates were retained in transferred to Immobilon-FL PVDF membrane (Millipore). 2191 2132 RBM45 IPs. Fold-change was calculated as the sum of exclu- The membranes were blocked with Odyssey Blocking Buffer 2192 2133 sive spectral counts across RBM45 replicates divided by the (LiCOR) for 1 h. The antibodies were diluted in Odyssey 2193 2134 sum of the exclusive spectral counts of that protein in the Blocking Buffer with 0.1% Tween-20. Primary antibody incu- 2194 2135 vector control replicates. Any protein with a fold-change bation was performed at room temperature for 1 h or 4 1C 2195 2136 smaller than 2 was filtered out. overnight. The IRDye-conjugated secondary antibody (LiCOR) 2196 2137 PPIs with the highest degree of confidence, e.g. valid incubation was performed at room temperature for 1 h. The 2197 2138 across the unsupervised and manual approaches were then membranes were scanned using the Odyssey CLx Infrared 2198 2139 analyzed using Ingenuity Pathway Analysis software (IPAs, Imaging System (LiCOR). The primary antibodies used for 2199 2140 QIAGEN Redwood City). The default IPA parameters were immunoblot are as follows: mouse monoclonal FLAG M2 2200 2141 utilized along with Uniprot identifiers for mapping proteins antibody (Sigma F3165, 1:5000), rabbit monoclonal RBM45 C- 2201 2142 within IPA. The reference set for analysis was the Ingenuity terminal antibody (custom-made, 1:3000), rabbit monoclonal 2202 2143 Knowledge Base. Direct and indirect relationships were hnRNP-A1 antibody (Cell Signaling 8443S, 1:3000), mouse 2203 2144 included but only from proteins that were experimentally monoclonal hnRNP-L antibody (Novus Biological NB120- 2204 2145 observed. IPA mapped 127 out of 131 proteins to known 6106, 1: 10,000), mouse monoclonal hnRNP-A2B1 antibody 2205 2146 pathways. P-value and percent overlap were used to rank (Santa Cruz sc-32316, 1:3000), rabbit monoclonal Matrin-3 2206 2147 potentially significant pathways. antibody (Abcam ab151714, 1:10,000), rabbit polyclonal 2207 2148 To identify biological processes associated with the list of RBM14 antibody (Proteintech 10196-1-AP, 1:5000), rabbit poly- 2208 2149 RBM45 interacting proteins, we performed enrichment ana- clonal TDP-43 antibody (Proteintech 10782-2-AP, 1:3000), rab- 2209 2150 lysis in the Gene Ontology (GO) Biological Process domain bit monoclonal GAPDH antibody (Cell Signaling 2118S, 2210 2151 using Cytoscape (Shannon et al., 2003) together with the 1:5000). The appropriate secondary antibodies conjugated 2211 2152 ClueGo plugin (Bindea et al., 2009). We performed enrichment with LiCOR IRDye 800CW or IRDye 680RD antibodies made 2212 2153 analysis using the right-sided hypergeometric test with in goat (1: 15,000) were used for immunoblot experiments. 2213 2154 Benjamini–Hochberg post-hoc correction. GO terms were 2214 2155 considered significant at the po0.001 level and the resultant 4.7. Immunocytochemistry 2215 2156 significant terms were visualized in a network layout where 2216 2157 GO Biological Process terms were visualized as color-coded For immunocytochemistry, HEK293 cells were grown on 2217 2158 circular nodes, with node size corresponding to enrichment p number 1.5 glass coverslips. Cells were washed with 1X PBS 2218 2159 value. The overlap of proteins associated with any two and fixed in 4% paraformaldehyde for 10 min. After fixation 2219 2160 Biological Process terms was evaluated using the kappa and further washing, cells were permeabilized by immersion 2220 2161 statistic and nodes were connected where the κ value was in 1X PBS containing 0.1% Triton X-100 for 15 min. After 2221 2162 Z0.4 using edges, with edge thickness corresponding to further washing, cells were blocked by incubation in SuperB- 2222 2163 kappa score. We then took leading terms, those GO Biological lock (Scytek) for 1 h. Subsequently, primary antibody solu- 2223 2164 Process terms with the highest number of associated pro- tions were applied and allowed to incubate for 2 h. Following 2224 2165 teins, and visualized these in a network layout where Biolo- primary antibody incubations, coverslips were washed four 2225 2166 gical Process terms were connected by edges to their times in a 1:10 mixture of SuperBlock:1X PBS. Secondary 2226 2167 associated proteins. All final figures were assembled using antibodies were applied following these washes, allowed to 2227 2168 Adobe Illustrator CS5 (Adobe Systems; San Jose, CA, USA). incubate for 1 h, and washed four times as above. Cell nuclei 2228

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2229 were visualized by staining with a 300 nM DAPI solution for 2289 fi 2230 10 min followed by washing with 1X PBS. Coverslips were Competing nancial interests 2290 2231 mounted on glass slides using 2,20-thiodiethanol (TDE) 2291 2232 according to the method of Staudt et al. (2007). In brief, RB is founder of Iron Horse Diagnostics, Inc., a biotechnology 2292 2233 coverslips were immersed in a series of increasing concen- company focused on developing diagnostic and prognostic 2293 2234 trations of TDE (10%, 25%, 50%, and 97%). The final TDE tests for neurologic diseases. 2294 2235 solution has a refractive index of 1.518 to match that of the 2295 2236 immersion oil used in imaging the slides. 2296 2237 The primary antibodies used for immunofluorescence Acknowledgments 2297 2238 were as follows: rabbit monoclonal RBM45 C-terminal anti- 2298 This work was supported by National Institutes of Health Q5 2239 body (custom-made, 1:250), rabbit monoclonal hnRNP-A1 2299 2240 antibody (Cell Signaling 8443S, 1:800), mouse monoclonal Grants R01NS061867, R56NS061867, and R01NS068179 to RB, 2300 2241 hnRNP-A2B1 antibody (Santa Cruz sc-32316, 1:250), rabbit NIH Grant F31NS080614 to MC, an award from the Achieve- 2301 2242 polyclonal hnRNP-A3 antibody (Sigma AV41195, 1:200), ment Rewards for College Scientists Foundation, Inc. Pitts- 2302 2243 mouse monoclonal hnRNP-L antibody (Novus Biological burgh Chapter to MC, and the ALS Association Milton Q6 2303 2244 NB120-6106, 1:1000), rabbit monoclonal Matrin-3 antibody Safenowitz Post-Doctoral Fellowship for ALS Research to YL. 2304 2245 (Abcam ab151714, 1:500), mouse monoclonal G3BP antibody 2305 2246 (BD Transduction Laboratories, 1:250), mouse monoclonal 2306 Appendix A. Supplementary material 2247 SMN antibody (Sigma S2944, 1:400), and mouse monoclonal 2307 2248 FLAG M2 antibody (Sigma F3165, 1:1000). The secondary 2308 Supplementary data associated with this article can be found 2249 antibodies used for immunofluorescence were goat-anti-Cy2 2309 in the online version at http://dx.doi.org/10.1016/j.brainres. 2250 (rabbit) and goat-anti-Cy5 (Mouse) (Millipore, 1:1000 for both). 2310 2016.02.047. 2251 2311 2252 4.8. Microscopy, digital deconvolution, and co-localization 2312 2253 2313 analysis references 2254 2314 2255 An Observer Z1 microscope (Zeiss) was used for all image 2315 2256 acquisitions using a 63 (1.4 NA) objective and LED light 2316 Bakkar, N., et al., 2015. RBM45 modulates the antioxidant 2257 source. Images were acquired as three-dimensional stacks 2317 response in amyotrophic lateral sclerosis through interactions 2258 m 2318 with a Z sampling interval of 0.240 m. Images were shading with KEAP1. Mol. Cell. Biol. 35, 2385–2399. 2259 corrected and background subtracted. Following acquisition, Bindea, G., et al., 2009. ClueGO: a cytoscape plug-in to decipher 2319 2260 images were deconvolved using Huygens Essential deconvo- functionally grouped gene ontology and pathway annotation 2320 2261 lution software (SVI). Deconvolution and chromatic shift networks. Bioinformatics 25, 1091–1093. 2321 2262 correction were performed using a measured PSF obtained Bolte, S., Cordelieres, F.P., 2006. A guided tour into subcellular 2322 colocalization analysis in light microscopy. J. Microsc. 224, 2263 by volume imaging of 200 mm fluorescent beads (Life Tech- 2323 213–232. 2264 nologies) together with the Huygens Essential PSF Distiller 2324 Chiou, N.T., Shankarling, G., Lynch, K.W., 2013. hnRNP L and 2265 2325 application. Deconvolution was performed using the soft- hnRNP A1 induce extended U1 snRNA interactions with an 2266 ware's classic maximum likelihood estimation algorithm. exon to repress spliceosome assembly. Mol. Cell 49, 972–982. 2326 2267 Deconvolved images were used to analyze the co- Choi, H., et al., 2012. SAINT-MS1: protein–protein interaction 2327 2268 localization of RBM45 and selected RBM45 interacting pro- scoring using label-free intensity data in affinity purification- 2328 2269 teins identified by IP–MS. Co-localization analysis was per- mass spectrometry experiments. J. Proteome Res. 11, 2329 2619–2624. 2270 formed using ImageJ (Schneider et al., 2012) in conjunction 2330 Collins, M., et al., 2012. The RNA-binding motif 45 (RBM45) protein 2271 2331 with the JaCoP plugin (Bolte and Cordelieres, 2006). Images accumulates in inclusion bodies in amyotrophic lateral 2272 2332 were automatically thresholded for analysis using the sclerosis (ALS) and frontotemporal lobar degeneration with 2273 method of Costes et al. (2004) and the M1 and M2 overlap TDP-43 inclusions (FTLD-TDP) patients. Acta Neuropathol. 2333 2274 coefficients (Bolte and Cordelieres, 2006) and intensity corre- 124, 717–732. 2334 2275 lation quotient (ICQ) (Li et al., 2004) were calculated. Statis- Colombrita, C., et al., 2009. TDP-43 is recruited to stress granules 2335 in conditions of oxidative insult. J. Neurochem. 111, 2276 tical significance of the ICQ was evaluated using the normal 2336 1051–1061. 2277 approximation of the sign test as in (Li et al., 2004). 2337 Cooper-Knock, J., et al., 2014. Sequestration of multiple RNA 2278 2338 recognition motif-containing proteins by C9orf72 repeat 2279 expansions. Brain 137, 2040–2051. 2339 2280 Author contributions Corgiat, B.A., Nordman, J.C., Kabbani, N., 2014. Chemical cross- 2340 2281 linkers enhance detection of receptor interactomes. Front. 2341 2282 YL and RB designed the project. YL and PP designed the IP–MS Pharmacol. 4, 171. 2342 2283 experiments. YL performed the validation IP-WB experi- Costes, S.V., et al., 2004. Automatic and quantitative measure- 2343 2284 ments. KG, PP and YL performed the mass spectrometry data ment of protein-protein colocalization in live cells. Biophys. J. 2344 86, 3993–4003. 2285 analysis. MC performed the microscopy experiments and 2345 Dammer, E.B., et al., 2012. Coaggregation of RNA-binding proteins 2286 constructed the GO networks. JA, KT and RG helped with in a model of TDP-43 proteinopathy with selective RGG motif 2346 2287 the IP–MS experiments. TT performed the LC–MS analysis. YL, methylation and a role for RRM1 ubiquitination. PLoS One 7, 2347 2288 MC, PP and RB wrote the manuscript. e38658. 2348

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