Direct interaction between hnRNP-M and CDC5L/PLRG1 proteins affects alternative splice site choice David Llères, Marco Denegri, Marco Biggiogera, Paul Ajuh, Angus Lamond

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David Llères, Marco Denegri, Marco Biggiogera, Paul Ajuh, Angus Lamond. Direct interaction be- tween hnRNP-M and CDC5L/PLRG1 proteins affects alternative splice site choice. EMBO Reports, EMBO Press, 2010, 11 (6), pp.445 - 451. ￿10.1038/embor.2010.64￿. ￿hal-03027049￿

HAL Id: hal-03027049 https://hal.archives-ouvertes.fr/hal-03027049 Submitted on 26 Nov 2020

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. scientificscientificreport report Direct interaction between hnRNP-M and CDC5L/ PLRG1 proteins affects alternative splice site choice David Lle`res1*, Marco Denegri1*w,MarcoBiggiogera2,PaulAjuh1z & Angus I. Lamond1+ 1Wellcome Trust Centre for Gene Regulation & Expression, College of Life Sciences, University of Dundee, Dundee, UK, and 2LaboratoriodiBiologiaCellulareandCentrodiStudioperl’IstochimicadelCNR,DipartimentodiBiologiaAnimale, Universita’ di Pavia, Pavia, Italy

Heterogeneous nuclear ribonucleoprotein-M (hnRNP-M) is an and affect the fate of heterogeneous nuclear RNAs by influencing their abundant nuclear protein that binds to pre-mRNA and is a structure and/or by facilitating or hindering the interaction of their component of the spliceosome complex. A direct interaction was sequences with other pre-mRNA processing factors (Martinez- detected in vivo between hnRNP-M and the human spliceosome Contreras et al, 2007). hnRNP-M is an abundant component of proteins cell division cycle 5-like (CDC5L) and pleiotropic human hnRNP complexes that can influence pre-mRNA splicing by regulator 1 (PLRG1) that was inhibited during the heat-shock regulating its own pre-mRNA splicing (Hase et al, 2006) or by affecting stress response. A central region in hnRNP-M is required for the regulation of alternative splicing of fibroblast growth factor receptor interaction with CDC5L/PLRG1. hnRNP-M affects both 50 and 30 2 (Hovhannisyan & Carstens, 2007). An antibody against hnRNP-M alternative splice site choices, and an hnRNP-M mutant lacking inhibited splicing in vitro, and heat-shock-induced inhibition of the CDC5L/PLRG1 interaction domain is unable to modulate splicing was associated with a loss of hnRNP-M from spliceosomal alternative splicing of an adeno-E1A mini-gene substrate. complexes (Gattoni et al, 1996). Proteomic analyses of in vitro purified Keywords: alternative pre-mRNA splicing; FLIM–FRET; CDC5L; spliceosomes detected hnRNP-M in the pre-spliceosomal H-complex hnRNP-M; spliceosome and throughout the spliceosome assembly (Rappsilber et al, 2002; EMBO reports advance online publication 14 May 2010; doi:10.1038/embor.2010.64 Wahl et al, 2009). The interactions of hnRNP-M with other components of the spliceosome machinery remains unclear. INTRODUCTION In this study, we combine a large-scale proteomic analysis with The spliceosome comprises small nuclear ribonucleoprotein biochemical approaches and live-cell imaging, including Fo¨rster (snRNP) subunits, and additional protein splicing factors (Wahl resonance energy transfer–fluorescence lifetime imaging micro- et al, 2009), including the conserved cell division cycle 5-like scopy (FLIM–FRET) and fluorescence recovery after photobleaching (CDC5L) complex (called the nineteen complex NTC in yeast), (FRAP), to analyse interactions between hnRNP-M and the pre-mRNA which is essential for spliceosome assembly and catalysis. The splicing machinery. We show that hnRNP-M interacts directly human CDC5L and human pleiotropic regulator 1 (PLRG1) in vivo with CDC5L and PLRG1, and that hnRNP-M can modulate proteins are ‘core’ components of this complex and are directly both 50 and 30 alternative splicing choices. associated with and required for the first catalytic step of pre-mRNA splicing (Ajuh et al, 2000; Makarova et al, 2004). RESULTS AND DISCUSSION Pre-mRNAs associate co-transcriptionally with heterogeneous A comparative proteomic analysis of CDC5L and SKIP-containing nuclear ribonucleoproteins (hnRNPs; Dreyfuss et al, 1993). complexes isolated from HeLa nuclear extracts was performed by Many hnRNP proteins modulate alternative splicing of pre-mRNAs immuno-affinity purification (IP) using CDC5L and SKIP anti- bodies, respectively (supplementary Fig S1A–E and supplementary Tables S2–S5 online). Mass spectrometric analysis showed that 1 Wellcome Trust Centre for Gene Regulation & Expression, College of Life Sciences, both complexes contained previously characterized CDC5L and University of Dundee, MSI/WTB/JBC Complex, Dow Street, Dundee DD1 5EH, UK 2Laboratorio di Biologia Cellulare and Centro di Studio per l’Istochimica del CNR, SKIP interaction partners (Makarov et al, 2002) and additional Dipartimento di Biologia Animale, Universita’ di Pavia, Piazza Botta 10, Pavia 27100, Italy proteins involved in RNA processing (supplementary Table S1A w Present address: Molecular Cardiology IRCCS Fondazione Salvatore Maugeri, online), especially members of the hnRNP family. The hnRNP-M Via Salvatore Maugeri 4, Pavia 27100, Italy zPresent address: Dundee Cell Products Ltd, James Lindsay Place, Dundee protein was abundant in both cases with 37 hnRNP-M peptides Technopole, Dundee DD1 5JJ, UK detected, that is, 47.1% of the final sequence coverage *These authors contributed equally to this work (supplementary Fig S1F–H online). +Corresponding author. Tel: þ 44 (0) 1382 345473; Fax: þ 44 (0) 1382 348072; E-mail: [email protected] hnRNP-M–CDC5L complex interactions Received 13 March 2010; revised 2 April 2010; accepted 6 April 2010; Antibodies directed against either CDC5L or PLRG1 proteins were published online 14 May 2010 used to immunoprecipitate proteins from whole-cell extracts. The

&2010 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports 1 hnRNP-M interacts directly with the CDC5L complex scientificreport D. Lle`res et al

A

kDa Input IP: anti-hnRNP-MNegative ctrl kDa Input IP: anti-PLRG1IP: anti-CDC5LNegative ctrl kDa Input IP: anti-hnRNP-MNegative ctrl 64 CDC5L 191 97 PLRG1 97 64 51 hnRNP-M 5 67 64 WB: anti-CDC5L 39

1 2 34 8 9 10 WB: anti-hnRNP-M WB: anti-PLRG1

1.0 B DAPIhnRNP-M CDC5L Merge C

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Fluorescence recovery Fluorescence hnRNP-M–EGFP_CDC5L siRNA

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97 0.6 CDC5L hnRNP-M–EGFP (37°C) 64 recovery Fluorescence hnRNP-M–EGFP (42°C) 1 2 3 4 5 6 7 8 WB: anti-CDC5L WB: anti-CDC5L 0.4 0 5 10 15 20 25 30 Time after photobleaching (s)

Fig 1 | hnRNP-M interacts with components of the CDC5L complex. (A) Co-IP from total HeLa cell extracts using either anti-PLRG1 (lane 2) or anti-CDC5L (lane 3) and probed with anti-hnRNP-M (D2IF7). Co-IP with anti-hnRNP-M and blotted with anti-CDC5L (lane 6). Co-IP with anti- hnRNP-M and blotted with anti-PLRG1 (lane 9). (B) Co-immunofluorescence labelling was performed using anti-hnRNP-M with either anti-CDC5L (upper row) or anti-PLRG1 (lower row). Nuclei were stained with DAPI. Scale bar, 10 mm. (C) Mobility of hnRNP-M–GFP in control cells (scrambled short interfering RNA; black squares) and CDC5L-knockdown HeLa cells (CDC5L short interfering RNA; white squares) was monitored by FRAP. The relative fluorescence recovery kinetics are represented. Each data point shows the average±s.d. of more than 10 cells. (D) Heat shock. Co-IP using anti-hnRNP-M either at 37 1C (left panel) or on heat shock for 1 h at 42 1C (right panel) and probed with anti-CDC5L. hnRNP-M is associated with CDC5L under physiological conditions (lane 3) but not after heat shock (lane 7). (E) Mobility of hnRNP-M–GFP at 37 1C (black squares) or on heat shock (white squares) was investigated by FRAP. The fluorescence recovery kinetics are shown. Each data point shows the average±s.d. of more than 10 cells. CDC5L, cell division cycle 5-like; DAPI, 40,6-diamidino-2-phenylindole; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; hnRNP, heterogeneous nuclear ribonucleoprotein; IP, immuno-affinity purification; PLRG1, pleiotropic regulator 1.

isolated proteins were analysed by western blotting using an treatment of extracts with both RNase-A and DNase-I, suggesting hnRNP-M antibody (Fig 1A). These results show specific co-IP that association of hnRNP-M with these complexes might be specific. of hnRNP-M, using both antibodies (Fig 1A, lanes 2 and 3). We infer that a subset of the pool of hnRNP-M proteins might be in Reciprocal co-IP, using an hnRNP-M antibody, followed by complex with CDC5L–PLRG1 because less than 10% of the protein detection with either CDC5L (Fig 1A, lane 6) or PLRG1 antibody is recovered in the IP, relative to the input (Fig 1A, lanes 1–3). (Fig 1A, lane 9) confirmed specific association of hnRNP-M In vivo distribution of hnRNP-M was compared with both with the CDC5L–PLRG1 complex. The IP protocols included CDC5L and PLRG1 proteins by immunostaining experiments

2 EMBO reports &2010 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION hnRNP-M interacts directly with the CDC5L complex D. Lle`res et al scientificreport

(Fig 1B, top and bottom rows, respectively). Both CDC5L and In vivo hnRNP-M–CDC5L–PLRG1 interactions PLRG1 antibodies showed a speckled nuclear staining pattern as We investigated if hnRNP-M and CDC5L–PLRG1 proteins interact described previously (Ajuh et al, 2001). The hnRNP-M antibody directly in vivo, by using FLIM–FRET. hnRNP-M–ECFP (enhanced instead showed a diffuse localization throughout the nucleoplasm, cyan fluorescent protein) was transiently coexpressed in HeLa as seen previously (Carmo-Fonseca et al, 1991). Partial colocal- cells together with either enhanced yellow fluorescent protein ization between hnRNP-M and both CDC5L and PLRG1 is (EYFP)-CDC5L or EYFP–PLRG1 proteins (Fig 2). As a positive observed in the diffuse area of the nucleoplasm, with little or no control, FLIM–FRET assays were performed on HeLa cells colocalization in the splicing speckles (Fig 1B, ‘Merge’ arrow- coexpressing ECFP–PLRG1 and EYFP–CDC5L, which interact heads). The nuclear localization of hnRNP-M was compared with directly (supplementary Fig S3 online). protein markers for various nuclear compartments (e.g. SC-35, Sm In contrast with cells expressing hnRNP-M–ECFP alone (Fig 2A), proteins and coilin; supplementary Fig S2A online) indicating that coexpression of either EYFP–CDC5L (Fig 2B) or EYFP–PLRG1 (Fig 2C) its association with CDC5L–PLRG1 is likely to occur within the decreased the mean fluorescence lifetime of the donor ECFP, diffuse nuclear compartment. indicating a FRET interaction (Fig 2D). The diffuse nuclear compartment contains nascent transcripts As the FLIM–FRET assay measures the FRET efficiency for that can be detected by transmission electron microscopy as each pixel, inspection of the FRET efficiency distribution perichromatin fibrils (Biggiogera & Fakan, 2008). Subnuclear throughout the nucleus shows that the highest FRET values are distribution of both hnRNP-M and CDC5L proteins was analysed predominantly in the diffuse nuclear domain with lower values in by immunoelectron microscopy by using CDC5L and hnRNP-M the speckles (Fig 2B,C, arrows indicate lower FRET). Further antibodies in thin sections of HeLa cell nuclei (supplementary analysis of the fraction of the hnRNP-M–ECFP molecules showing Fig S2B online). a FRET interaction (Fig 2B,C, ‘FRET population %’ panels) Immunolabelling of CDC5L was observed on the peri- indicated that less than 30% of the total hnRNP-M protein pool chromatin fibrils, stained using the regressive EDTA technique was involved in this interaction. (supplementary Fig S2B online, i, arrowheads), and also at the As the CDC5L subcomplex is implicated in the activation of the periphery of the interchromatin granule clusters (supplementary second catalytic step of pre-mRNA splicing (Bessonov et al, 2008), Fig S2B online, i, asterisk). Double labelling performed using the direct interactions between hnRNP-M and the CDC5L sub- hnRNP-M and CDC5L antibodies (black and white arrowheads, complex detected by FLIM–FRET might correspond either to active respectively) showed both proteins localized in tight proximity sites of pre-mRNA splicing or to functional complexes that will be to a single RNA fibril, stained with either terbium citrate involved in splicing (House & Lynch, 2008). In combination with (supplementary Fig S2B online, ii) or by using the regressive the electron microscopic and heat-shock data, this suggests that EDTA technique (supplementary Fig S2B online, iii, iv). These the interactions between hnRNP-M and both CDC5L and PLRG1 data show the colocalization of hnRNP-M and CDC5L proteins are either co-transcriptional or require nascent transcription. on individual RNA fibrils. The mobility of green fluorescent protein (GFP)-tagged hnRNP-M wild type in control cells Identification of interaction domain (scrambled short interfering RNA) and CDC5L-knockdown HeLa The interaction between hnRNP-M and CDC5L/PLRG1 proteins cells (CDC5L short interfering RNA) was monitored by FRAP. was confirmed in vitro using a binding assay with in vitro CDC5L knockdown caused an increase in the mobility of translated hnRNP-M and Escherichia coli-expressed glutathione-S- hnRNP-M (Fig 1C), suggesting it is regulated by interaction with transferase (GST)–CDC5L and GST–PLRG1 (supplementary Fig S4B,C the CDC5L protein. online). No direct interaction was observed between GST–SPF30 The combination of fluorescence and electron microscopic and in vitro translated hnRNP-M (supplementary Fig S4B,C online, data support the proteomic and co-IP data, indicating that a subset lane 2), consistent with previous data (Ajuh et al, 2001). of hnRNP-M associates in vivo with complexes containing the To map the region of hnRNP-M that binds directly to CDC5L CDC5L–PLRG1 proteins. and PLRG1 proteins, deletion mutants of hnRNP-M were made (supplementary Fig S4A online) and the size and level of Heat shock disrupts hnRNP-M interactions expression were evaluated for each mutant (supplementary We tested whether hnRNP-M association with CDC5L and PLRG1 Fig S5A online). Full-length hnRNP-M and mutants 2 and 3 were would be disrupted after heat shock, which inhibits transcription pulled down by both GST–CDC5L (supplementary Fig S4B of most pre-mRNAs and blocks splicing (Bond, 1988; Biamonti, online, lanes 3, 5 and 6) and GST–PLRG1 (supplementary 2004). IPs were performed from both control (i.e. 37 1C) and heat- Fig S4C online, lanes 4, 6 and 7), respectively. By contrast, little shocked (1 h at 42 1C) HeLa cell extracts using an hnRNP-M or none of mutants 1, 4, 5 and 6 were pulled down with either antibody. Isolated proteins were analysed with CDC5L antibody GST–CDC5L (supplementary Fig S4B online, lanes 4 and 7; (Fig 1D). The CDC5L protein was specifically detected together supplementary Fig S4D online, lanes 2 and 3) or with GST–PLRG1 with hnRNP-M at 37 1C, but not at 42 1C (Fig 1D, lanes 3 and 7). (supplementary Fig S4C online, lanes 5 and 8; supplementary Fig The levels of hnRNP-M show little or no change on heat S4D online, lanes 7 and 8). IP of cell extracts expressing YFP- shock (supplementary Fig S2C online). Loss of binding between mutant 1 and mutant 3 using an antibody against CDC5L showed hnRNP-M and the CDC5L complex after heat shock stress was that mutant 3, but not mutant 1, could be co-immunoprecipitated addressed by FRAP experiments, showing increased mobility of (supplementary Fig S5B online, lanes 6 and 7). Together, these hnRNP-M–EGFP after heat shock (Fig 1E). This suggests that the results indicate that the central region of hnRNP-M, spanning association of hnRNP-M with CDC5L is dependent on nascent amino acids 248–344, is required for binding to both CDC5L pre-mRNA production. and PLRG1.

&2010 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports 3 hnRNP-M interacts directly with the CDC5L complex scientificreport D. Lle`res et al

A Fluorescence FRET protein (EGFP), expressed in HeLa cells and analysed by FRAP Intensity Intensity lifetime efficiency (%) (Fig 3A,B). These data show a difference in their rate of recovery hnRNP M-ECFP from photobleaching, with mutant 6(248344aa) showing more than 10-fold higher rate of fluorescence recovery as compared with wild- type hnRNP-M. Furthermore, mutant 6(248344aa) shows a mobile fraction corresponding to essentially all the expressed protein, as

1.7  (NS) 2.3 0 5 10 25 compared with about 85% of the wild-type hnRNP-M protein. The direct binding of hnRNP-M mutant 6(248344aa) to both B FRET population (%) CDC5L and PLRG1 in vivo was analysed using the FLIM–FRET hnRNP M-ECFP EYFP-CDC5L assay (Fig 3C). hnRNP-M mutant 6(248344aa)–ECFP was transiently expressed in HeLa cells alone (Fig 3C, upper panels) or together with either EYFP–CDC5L (middle panels) or EYFP–PLRG1 (lower panels). Coexpression of the tagged CDC5L or PLRG1 proteins did not decrease the mean fluorescence lifetime of mutant 1.7  (NS) 2.3 0 5 10 25 10 20 30 50 6(248344aa)–ECFP, indicating no FRET and therefore no protein– protein interaction. In agreement, no difference in the mobility of C hnRNP M-ECFP EYFP-PLRG1 the mutant 6(248344aa)–EGFP was observed by FRAP after heat shock (supplementary Fig S6 online). Together, the data are consistent with hnRNP-M wild-type but notmutant6(248344aa) protein, interacting directly with the CDC5L and PLRG1 proteins in vivo and thereby reducing its mobility. The 1.7  (NS) 2.3 0 5 10 25 10 20 30 50 FRAP data indicate roughly 10–20% of the tagged hnRNP-M engaged in the splicing machinery in vivo, consistent with the binding fraction D 2.2 of less than 30% estimated by FLIM–FRET. )

NS 2.1 hnRNP-M is involved in alternative splice site choice 2.0 We investigated whether the direct interactions of hnRNP-M with 1.9 CDC5L and PLRG1 were important for pre-mRNA splicing activity.

1.8 As hnRNP-M was reported to promote exon skipping and exon inclusion (Hovhannisyan & Carstens, 2007), we analysed the ability 1.7 of wild-type hnRNP-M to affect alternative splice site choice on two Fluorescence lifetime ( Fluorescence 1.6 separate pre-mRNA substrates (Fig 4). By using expression of calcitonin/dhfr pre-mRNA from pDC20 reporter (Bai et al, 1999), the effect of increasing in vivo levels of hnRNP-M by transient coexpression in HeLa cells was analysed (Fig 4A) and compared with hnRNP-M–ECFPhnRNP-M–ECFP/hnRNP-M–ECFP/ overexpression of the ASF/SF2 protein. Overexpression of exogenous EYFP–CDC5LEYFP–PLRG1 hnRNP-M activated the distal 30 splice site, increasing the level of the Fig 2 | hnRNP-M interacts directly with CDC5L and PLRG1 in vivo.HeLa 418-bp band (Fig 4A, lane 4). By contrast, overexpression of ASF/SF2 cells were transfected with hnRNP-M–ECFP (A) alone or in combination with protein promoted preferential use of the proximal 30 splice site, either (B) EYFP-CDC5L or (C) EYFP-PLRG1 and analysed using the FLIM– increasing the ratio of the 196/418-bp bands (Fig 4A, compare lane 3 FRET technique. The fluorescence lifetime t (NS) was detected using the with lanes 1 and 4). These data show that increased expression of TCSPC method, and its spatial distribution was analysed using the SPCimage hnRNP-M has the opposite effect on alternative 30 splice site choice software. Fluorescence lifetimes are depicted using a continuous pseudo- to ASF/SF2 (Fig 4A, graph). colour scale representing time values ranging from 1.7 to 2.3 NS.TheFRET A more complex effect on alternative splice site choice is efficiency percentage is calculated and depicted using discrete colours provided by the E1A mini-gene reporter, in which five distinct RNA representing FRET values ranging from 0 to 25%. The FRET species (%) was isoforms can be generated by alternative splicing (Fig 4B). As quantified for both hnRNP-M–CDC5L and hnRNP-M–PLRG1 interactions. previously observed (Caceres et al,1994),overexpressionofASF/SF2 Scale bars, 10 mm. (D) Statistical analysis from three independent experiments protein strongly activated the 13S 50 splice site, enhancing the levels in at least 15 different cells for each condition is presented. CDC5L, cell of the 13S RNA isoform and reducing the levels of 12S and 10S division cycle 5-like; ECFP, enhanced cyan fluorescent protein; EYFP, RNAs (Fig 4C, lane 3). By contrast, overexpression of hnRNP-M enhanced yellow fluorescent protein; FLIM–FRET, Fo¨rster resonance activated the most distal 50 splice site, increasing the levels of the 9S energy transfer–fluorescence lifetime imaging microscopy; hnRNP, RNA isoform and decreasing the levels of the 13S isoform, with little heterogeneous nuclear ribonucleoprotein; PLRG1, pleiotropic regulator 1; or no change in the 12S isoform (Fig 4C, lanes 5–8, and Fig 4D). The TCSPC, time-correlated single photon counting. effect of hnRNP-M was concentration-dependent. This indicates that hnRNP-M facilitates preferential use of the distal over the proximal 50 To test whether this binding region affects the properties of the splice site in E1A pre-mRNA splicing. Our data are consistent with hnRNP-M protein in vivo, both wild-type hnRNP-M and hnRNP- previous evidence that hnRNP-M can regulate splicing in metazoans. M mutant 6(248344aa) protein (lacking the central CDC5L/PLRG1- Furthermore, studies have shown that Hrp59, the homologue of binding region) were tagged with enhanced green fluorescent hnRNP-M in Drosophila melanogaster, can regulate the splicing of its

4 EMBO reports &2010 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION hnRNP-M interacts directly with the CDC5L complex D. Lle`res et al scientificreport

A Pre 0.000 s 1.336 s 2.670 s 4.004 s 6.672 s B 1.0

0.8 hnRNP-M–EGFP 2.642 s ) Pre 0.000 s 0.663 s 1.323 s 1.982 s

(248–344aa 0.6 Fluorescence recovery

–EGFP hnRNP-M–EGFP

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0.4 051015 20 25 30 Time after photobleaching (s)

Mean fluorescence Mutant 6 –ECFP FRET (%) C (248–344aa) lifetime

1.7 (NS) 2.3 0 5 10 25 Mutant 6(248–344aa)–ECFP EYFP–CDC5L

 Mutant 6(248–344aa)–ECFP EYFP–PLRG1 1.7 (NS) 2.3 0 5 10 25

1.7 (NS) 2.3 0 5 10 25

Fig 3 | The nuclear dynamic of hnRNP-M–EGFP and hnRNP-M mutant 6(248344aa)–EGFP as observed by FRAP. (A) A time-lapse series showing nucleoplasmic hnRNP-M–EGFP (top panels) and mutant 6–EGFP (bottom panels) FRAP. The photobleached area is outlined with yellow circles. (B) The kinetics of the relative fluorescence recovery of hnRNP-M–EGFP (black squares) and mutant 6–EGFP (white squares) are represented. The mobile fraction (%) and the half-life time recovery were calculated, respectively: hnRNP-M–EGFP, 84.4±5.3%, 6.40±0.04 s; mutant 6(248344 aa)–EGFP, 100.0±3.2%, 0.47±0.02 s. Each data point shows the average±s.d. of more than 10 cells. Scale bars, 10 mm. (C) HeLa cells transfected with hnRNP-M mutant 6(248344 aa)–ECFP alone (top row) or with either EYFP–CDC5L (middle row) or EYFP–PLRG1 (bottom row) were analysed by FLIM–FRET. Scale bars, 10 mm. CDC5L, cell division cycle 5-like; FRAP, fluorescence recovery after photobleaching; hnRNP, heterogeneous nuclear ribonucleoprotein; PLRG1, pleiotropic regulator 1. own transcript as well as that of another transcript by acting as a Finally, we investigated whether the function of hnRNP-M in negative regulator of splicing (Hase et al, 2006). alternative splicing was affected by the central region mapped as Overexpression of hnRNP-M caused an enhancement in the essential for binding to the CDC5L/PLRG1 proteins. Overexpression levels of the 10S RNA isoform (Fig 4C,D, compare lanes 5–8 with of hnRNP-M mutant 6(248344aa) was compared with full-length lane 4), indicating stimulation of the distal 30 splice site. Consistently, hnRNP-M using the E1A mini-gene assay (Fig 4C, lanes 8 and 9). we observed by using FRET a direct in vivo interaction between The mutant 6(248344aa) protein caused minimal change to the RNA hnRNP-M and both subunits of the splicing factor U2AF, which binds isoform pattern, whereas overexpression of wild-type hnRNP-M to the polypyrimidine tract adjacent to the 30 intron–exon junction enhanced the level of the 10S and reduced that of the 13S isoforms (supplementary Fig S7A,B online). This interaction was stronger with (Fig 4D). No direct interactions between mutant 6(248344aa)–EGFP the U2AF65 subunit, which could be co-immunoprecipitated using and both mCherry-tagged U2AF65 and U2AF35 were detected by anti-GFP antibodies (supplementary Fig S7C online). FLIM–FRET in vivo (supplementary Fig S7D online). We conclude

&2010 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports 5 hnRNP-M interacts directly with the CDC5L complex scientificreport D. Lle`res et al

A Distal 1.2

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1 234 hnRNP-M–ECFP

9S hnRNP-M–ECFP BC SF/SF2 12S μg 13S μg μg μg μg mutantIsoforms 6–ECFP Cos-1 CFP-N1GFP-A ECFP 0.5 1 2 5 5 E1A exon 1 ′ ′ ′ Unspliced 5′ 5 5 3 13S 5′ 3′ 5′ 3′ 12S EIA exon 2 11S 11S 10S PCR products 5′ splice site selection 13S 10S 12S 9S PCR products 3′ splice site selection 11S 9S 10S 12 3 456789

100 D 13S 12S 80 11S 10S 9S 60

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0 E1A CFP-N1 ASF/SF2 0.5 μg 1.0 μg 2.0 μg 5.0 μg mutant 6

CFP-N1–hnRNP-M

Fig 4 | hnRNP-M is involved in alternative splice site choice through its CDC5L/PLRG1-binding region. (A) Representation of pDC20 mini-gene containing the alternative 30 splice sites. RT–PCR analysis of HeLa cells co-transfected with pDC20 and either the ECFP-N1 vector (lane 2) or hnRNP- M–ECFP (lane 4). Overexpression of hnRNP-M activates the distal 30 splice site choice of the chimeric calcitonin/dhfr pre-mRNA. The relative distal- to-proximal 30 splice site ratio was quantified (right panel). (B) Representation of E1A reporter mini-gene containing the alternative 50 splice sites. The arrows indicate the positions of the exon primers used for RT–PCR. (C) Analysis of cells co-transfected with E1A and ECFP-N1 vector (lanes 2

and 4) or a gradient of hnRNP-M–ECFP (lanes 5–8) or the mutant 6(248344aa)–ECFP (lane 9). (D) The relative amounts of the various isoforms were quantified. CDC5L, cell division cycle 5-like; ECFP, enhanced cerculean fluorescent protein; GFP, green fluorescent protein; hnRNP, heterogeneous nuclear ribonucleoprotein; PLRG1, pleiotropic regulator 1; RT–PCR, reverse transcription–PCR.

that loss of the CDC5L/PLRG1 interaction domain in hnRNP-M It is established from studies on ASF/SF2 and spliceosome proteins, correlates with a loss of ability to modulate alternative splice site that splicing factors can contribute to both constitutive and alternative selection in this assay. splicing mechanisms and show pre-mRNA substrate-specific effects

6 EMBO reports &2010 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION hnRNP-M interacts directly with the CDC5L complex D. Lle`res et al scientificreport

(Cazalla et al, 2005). We propose that hnRNP-M provides another Ajuh P, Sleeman J, Chusainow J, Lamond AI (2001) A direct interaction between example of this phenomenon and our data suggest that interactions the carboxyl-terminal region of CDC5L and the WD40 domain of PLRG1 is essential for pre-mRNA splicing. JBiolChem276: 42370–42381 with the core spliceosome factors in the CDC5L complex might Bai Y, Lee D, Yu T, Chasin LA (1999) Control of 30 splice site choice in vivo by influence the mechanism of splice site selection. In this regard, ASF/SF2 and hnRNP A1. Nucleic Acids Res 27: 1126–1134 detection of additional interactions between hnRNP-M and U2AF65 Bessonov S, Anokhina M, Will CL, Urlaub H, Luhrmann R (2008) Isolation of by FRET and IP assays suggests that the effect of hnRNP-M on 30 an active step I spliceosome and composition of its RNP core. Nature 0 452: 846–850 splice-site selection could result from titration of the 3 splice-site- Biamonti G (2004) Nuclear stress bodies: a heterochromatin affair? Nat Rev binding factor U2AF. A similar titration mechanism has been Mol Cell Biol 5: 493–498 proposed for SRp53, which interacts with the U2AF65-related protein Biggiogera M, Fakan S (2008) Visualization of nuclear organization by HCC1 and can activate weak 30 splice sites in addition to its role at 50 ultrastructural cytochemistry. Methods Cell Biol 88: 431–449 splice-sites (Cazalla et al, 2005). All three U2AF-type proteins, that is, Bond U (1988) Heat shock but not other stress inducers leads to the disruption of a sub-set of snRNPs and inhibition of in vitro splicing in HeLa cells. U2AF65, PUF60 and HCC1, interact with SRp54, which has been EMBO J 7: 3509–3518 0 implicated in early 3 splice site recognition (Zhang & Wu, 1996). Caceres JF, Stamm S, Helfman DM, Krainer AR (1994) Regulation of These factors, together with the U2AF35 subunit and SR proteins, alternative splicing in vivo by overexpression of antagonistic splicing determine the 30 splice site ‘exon definition complex’ and bend the factors. Science 265: 1706–1709 Carmo-Fonseca M, Pepperkok R, Sproat BS, Ansorge W, Swanson MS, pre-mRNA (Matlin et al, 2005). Lamond AI (1991) In vivo detection of snRNP-rich organelles in the In summary, by using many experimental approaches, we nuclei of mammalian cells. EMBO J 10: 1863–1873 show a direct involvement of hnRNP-M in the spliceosome Cazalla D, Newton K, Caceres JF (2005) A novel SR-related protein is required machinery through its interaction with the CDC5L/PLRG1 for the second step of pre-mRNA splicing. Mol Cell Biol 25: 2969–2980 spliceosomal subcomplex. The data show that hnRNP-M can Chiodi I, Biggiogera M, Denegri M, Corioni M, Weighardt F, Cobianchi F, Riva S, Biamonti G (2000) Structure and dynamics of hnRNP-labelled influence both constitutive and alternative splicing, and provide nuclear bodies induced by stress treatments. J Cell Sci 113: 4043–4053 new insight into the mechanism whereby hnRNP-M can modulate Chusainow J, Ajuh PM, Trinkle-Mulcahy L, Sleeman JE, Ellenberg J, both 50 and 30 splice site choice. Lamond AI (2005) FRET analyses of the U2AF complex localize the U2AF35/U2AF65 interaction in vivo and reveal a novel self-interaction METHODS of U2AF35. RNA 11: 1201–1214 Antibodies and plasmids. The following antibodies were used: Datar KV, Dreyfuss G, Swanson MS (1993) The human hnRNP M proteins: PLRG1 and CDC5L (Ajuh et al, 2001); PLRG1 (Everest Biotech, identification of a methionine/arginine-rich repeat motif in ribonucleoproteins. Nucleic Acids Res 21: 439–446 Oxford, UK); hnRNP-M 2A6 (sc-20001) and 1D8 (Datar et al, 1993); Dreyfuss G, Matunis MJ, Pinol-Roma S, Burd CG (1993) hnRNP proteins and hnRNP D2IF7 (Gattoni et al, 1996); GFP (Roche Diagnostics, the biogenesis of mRNA. Annu Rev Biochem 62: 289–321 Indianapolis, IN, USA); SC35 (Sigma, St Louis, MO, USA); Sm Ellis JD, Lleres D, Denegri M, Lamond AI, Caceres JF (2008) Spatial mapping (Abcam, Cambridge, UK); E1A reporter plasmid (Caceres et al, of splicing factor complexes involved in exon and intron definition. J Cell 1994); pDC20 reporter (Wang et al,2002);andEYFP–CDC5L,EYFP– Biol 181: 921–934 Gattoni R, Mahe D, Mahl P, Fischer N, Mattei MG, Stevenin J, Fuchs JP (1996) PLRG1, EYFP–U2AF35 and EYFP–U2AF65 (Chusainow et al, 2005). The human hnRNP-M proteins: structure and relation with early heat Protein binding assays. We performed the assays as described by shock-induced splicing arrest and chromosome mapping. Nucleic Acids Ajuh et al (2001) using 0.2 nmol of the appropriate recombinant Res 24: 2535–2542 protein and 8–10 ml of the in vitro translation reaction. Hase ME, Yalamanchili P, Visa N (2006) The Drosophila heterogeneous nuclear ribonucleoprotein M protein, HRP59, regulates alternative Alternative splicing assays. We performed alternative splicing splicing and controls the production of its own mRNA. J Biol Chem 281: assays using E1A and pDC20 mini-gene reporters as described by 39135–39141 Caceres et al (1994) and Wang et al (2002). House AE, Lynch KW (2008) Regulation of alternative splicing: more than just Heat shock. Heat-shock treatments were performed as described the ABCs. J Biol Chem 283: 1217–1221 by Chiodi et al (2000). Hovhannisyan RH, Carstens RP (2007) Heterogeneous ribonucleoprotein m is a splicing regulatory protein that can enhance or silence splicing of Fluorescence lifetime imaging microscopy. We acquired and alternatively spliced exons. J Biol Chem 282: 36265–36274 analysed FLIM–FRET measurements as described by Lleres et al Lleres D, Swift S, Lamond AI (2007) Detecting protein–protein interactions (2007) and Ellis et al (2008). in vivo with FRET using multiphoton fluorescence lifetime imaging Supplementary information is available at EMBO reports online microscopy (FLIM). Curr Protoc Cytom 12: 10 Makarov EM, Makarova OV, Urlaub H, Gentzel M, Will CL, Wilm M, (http://www.emboreports.org). Luhrmann R (2002) Small nuclear ribonucleoprotein remodeling during ACKNOWLEDGEMENTS catalytic activation of the spliceosome. Science 298: 2205–2208 We thank Drs M.S. Swanson, J.P. Fuchs, J.F. Caceres, P. Ouyang, Makarova OV, Makarov EM, Urlaub H, Will CL, Gentzel M, Wilm M, G. Biamonti and J. Tazi for reagents; Dr Bartosz Pilch from Prof Matthias Luhrmann R (2004) A subset of human 35S U5 proteins, including Prp19, function prior to catalytic step 1 of splicing. EMBO J 23: 2381–2391 Mann’s lab for performing mass spectrometry analysis; the Lamond lab Martinez-Contreras R, Cloutier P, Shkreta L, Fisette JF, Revil T, Chabot B (2007) members for helpful suggestions; and Dr S. Swift of the CLS Light hnRNP proteins and splicing control. Adv Exp Med Biol 623: 123–147 Microscopy Facility for advice and technical assistance. This work was Matlin AJ, Clark F, Smith CW (2005) Understanding alternative splicing: supported by the Wellcome Trust and the European Alternative Splicing towards a cellular code. Nat Rev Mol Cell Biol 6: 386–398 Network (Eurasnet). A.I.L. is a Wellcome Trust Principal Research Fellow. Rappsilber J, Ryder U, Lamond AI, Mann M (2002) Large-scale proteomic CONFLICT OF INTEREST analysis of the human spliceosome. Genome Res 12: 1231–1245 The authors declare that they have no conflict of interest. Wahl MC, Will CL, Luhrmann R (2009) The spliceosome: design principles of a dynamic RNP machine. Cell 136: 701–718 REFERENCES Wang P, Lou PJ, Leu S, Ouyang P (2002) Modulation of alternative pre-mRNA Ajuh P, Kuster B, Panov K, Zomerdijk JC, Mann M, Lamond AI (2000) splicing in vivo by pinin. Biochem Biophys Res Commun 294: 448–455 Functional analysis of the human CDC5L complex and identification Zhang WJ, Wu JY (1996) Functional properties of p54, a novel SR protein active of its components by mass spectrometry. EMBO J 19: 6569–6581 in constitutive and alternative splicing. Mol Cell Biol 16: 5400–5408

&2010 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports 7 Supplementary information

Supplementary Materials and methods

Expression of recombinant proteins in Escherichia coli

Expression of recombinant proteins in E. coli was done as previously described (Ajuh &

Lamond, 2003). CDC5L, PLRG1 cloned into the pGEX-6P3 vector were used to transform E.coli strain BL21(D3) (Stratagene). Overnight cultures were grown form a single colony, then diluted 1:10 in fresh LB medium with ampicillin (100 µg/ml) and

grown at 25-30˚C to an OD600 of 0.7-1.0 before induction with 0.04 mM IPTG. Three hours post-induction, cells were pelleted and resuspended in 10 ml of phosphate- bufffered saline (PBS) containing 0.5% Triton X-100 and protease inhibitor cocktail

(Boehringer). Cell lysis was achieved by sonication. The cell debris was removed by centrifugation at 10000 g for 15 min at 4˚C. Pre-swollen glutathione-Sepharose beads pre-equilibrated in PBS were added to the supernatant (1 mg/l culture). The beads were incubated with the crude protein extract for 2h at 4˚C with rocking. Beads were collected and washed three times in PBS. Proteins were eluted from the beads by incubating in 25 mM glutathione in 50 mM Tris-HCl, pH 8.0. The proteins were dialysed into either PBS or a buffer containing 20 mM HEPES, pH 8.0, 20% glycerol, 100 mM KCl, 0.2 mM

EDTA and 1 mM dithiothreitol and stored -80˚C. Protein concentrations were determined

either by measuring the OD280 of the purified protein or by using the Coomassie Plus

Protein Assay reagent (Perbio Science, UK) according to the manufacturer’s instructions.

In vitro Transcription and Translation

The in vitro transcription and translation experiments were done with the T7 RNA polymerase transcription/translation systems using rabbit reticulocyte lysate (TNT

Systems, Promega) and L-[35S]Methionine (Amersham Pharmacia Biotech, AG1094) to produce 35S-labeled proteins according to the manufacturer’s recommendations. Aliquots of these reactions were used for protein-protein interaction assays as described below.

In vitro Mutagenesis of the hnRNP M cDNAs

Deletion mutants were generated with specific primers designed to interrupt the full- length protein in different regions by PCR strategy. The cDNAs were sequenced to confirm the presence of the appropriate region amplified. Regions of the cDNAs involved in binding and overlapping regions were subcloned into the vectors pET-30a (Novagen) for use protein-protein interaction assays or in pEYFP-N1 (CLONTECH) vectors for expression in mammalian cells.

Co-immunoprecipitation and immunoblotting

Cells were lysed with RIPA buffer, 50mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40,

0.5% deoxycholate and protease inhibitors in presence of Rnase/Dnase I. Lysates were precleared with nonspecific immunoglobulin G-Sepharose (Amersham) and then immunoprecipitated with anti-CDC5L or anti-hnRNP M (2A6 Antibody) with 50 µl of a

50% slurry of protein G-Sepharose for 1 h at 4°C. The beads were washed twice with

RIPA buffer, and once with washing buffer (50mM Tris-HCl, pH 7.5, 0.25 M NaCl,

0.1% NP-40, 0.05% deoxycholate and protease inhibitor cocktail (Boehringer).

Immunoprecipitated proteins were resuspended in SDS PAGE loading buffer (Invitrogen) and heated at 70°C for 10 min. Protein samples usually 10-25 μl was loaded on a 4-12% gradient gel (Invitrogen). The separated proteins were transferred onto Hybond-C extra membrane (Amersham) according to the manufacturer’s instructions. The membranes carrying the transferred proteins were blocked with 5% non-fat milk powder in PBS/0.1%

Tween-20. The membranes were incubated with primary antibody for 1hr at room temperature or over night at 4˚C, washed and incubated with the appropriate secondary antibody. Protein bands were detected using the ECL plus kit (Amersham) according to the manufacturer’s instructions. For the input control were loaded 5-10 % of the total amount of the sample.

Fluorescence Recovery after Photobleaching (FRAP)

HeLa cells were cultured and transiently transfected in glass-bottomed dishes (WILCO;

Intracel). Before imaging, growth medium was replaced with phenol-red free CO2- independent medium (Invitrogen). Hela cells transiently expressing EGFP-fusion proteins were imaged using a 60x oil immersion lens (NA 1.4) and photobleached by using the

“photokinetic experiment” function of the DeltaVision Spectris microscope. Briefly, a small region inside nucleus was photobleached with a 488 nm laser (100% laser power for the duration of 0.05s), and time-lapse sequences of single optical sections for imaging

GFP fluorescence were collected with an exposure time of 0.1s for each image. The fluorescence intensities in the bleached and non-bleached area before and after laser photobleaching were quantified using SoftWoRX software. The calculated recovery half times and the mobile fraction of the proteins were carried out with Excel and Prism softwares.

Immunoelectron microscopy

For electron microscope immunocytochemistry, HeLa cells were trypsinized, fixed with

4% paraformaldehyde in DMEM at 4˚C for 2 h, rinsed in phosphate buffer (pH 7.2) and then embedded in 2% low gelling agarose. The specimens were placed into 0.5 M NH4Cl solution in buffer for 30 min at 4˚C to block free aldehyde groups, dehydrated in ethanol at room temperature and embedded in LR White resin. Ultrathin sections on formvar- carbon-coated nickel grids were incubated on a drop of normal goat serum diluted 1:100 in PBS, for 3 minutes. For the double labelling, a polyclonal CDC5L (1:100) and a monoclonal 1D8 anti-hnRNP M (1:5; Upstate) antibodies were used and incubate in PBS-

0.05% Tween 0.1% BSA at 4°C for 17 hrs. After rinsing with PBS/Tween and PBS, the grids were incubated with NGS. The grids were incubated with goat anti-Rabbit IgG

(Jackson Immunoresearch Laboratories) coupled with goat anti-rabbit 12 nm colloidal gold and goat anti-mouse (6 nm) diluted 1:20 in PBS. All the incubations were carried out for 30 min at room temperature. As a control, some grids were floated on the incubation mixture without the primary antibody, treated as above and incubated with the appropriate secondary antibody. All the grids were rinsed with PBS and distilled water, and finally stained with the EDTA regressive technique for RNP-containing nuclear components detection (Bernhard, 1969), or with terbium citrate to reveal specifically

RNA (Biggiogera & Fakan, 2008). Stained specimens were observed with a Zeiss EM900 electron microscope equipped with a 30 µm objective aperture and operating at 80 kV cDNA Cloning and Sequencing

The hnRNP M cDNA was cloned from a HeLa cDNA library (Clontech) by PCR. The amplification product corresponds to the registered Swiss Prot.P52272-2. The hnRNP M primers contained Bgl II and Sal I sites added to the 5’ ends of the 3’ end of the primers, respectively. Purified PCR product from hnRNP M and relative mutants cDNAs were digested with the appropriate enzymes and cloned using standard methods into the compatible sites of the vectors pGEX-4T1 (Amersham Pharmacia Biotech), pET-30a (Novagen), pECFP-N1, pEYFP-N1 (CLONTECH). The plasmids DNA were sequenced

(University of Dundee, sequencing service). pGEX-4T1 clones were used for expression of recombinant protein in Escherichia coli, whereas the pECFP-C1 and pEYFP-N1 vectors were used for expression in mammalian cells. pET-30a clones were used for in vitro transcription/translation experiments.

Supplementary Figure Legends

Figure S1. Purification of CDC5L complexes in HeLa nuclear extract and Graphical presentation of LC MS/MS analysis of the peptide INEILSNALK from hnRNP-M

(Q9BWL9).

(A) CDC5L complexes were purified by affinity chromatography using anti-CDC5L and anti-SKIP antibodies as described in the materials and methods section. Peak fractions from the antibody columns were analysed by SDS PAGE and the protein bands revealed using Coomassie blue (Invitrogen Ltd) staining according to the manufacturer’s instructions. Lanes 1 and 2 contained control eluates from a non-specific total IgG column. Lanes 3 and 4 contained peak fractions from the anti-CDC5L column and lanes

5 and 6 contained peak fractions from the anti-SKIP antibody column.

(B and C) Western blotting of fractions from the anti-CDC5L and anti-SKIP antibody columns using anti-SKIP and anti-CDC5L antibodies respectively. Lane 1 is the positive control containing HeLa nuclear extract. Lanes 2 and 3 represent the negative control i.e. peak fractions from the control rabbit IgG column. Lanes 4-8 contained fractions from the anti-CDC5L and anti-SKIP antibody columns.

(D and E) Western blot analysis of peak fractions from the anti-CDC5L antibody column and the anti-SKIP antibody column respectively using anti-PLRG1 antibodies. Lane 1 in each panel is the positive control containing HeLa nuclear extract and Lane 2 is the IgG control. Lanes 3 and 4 contained peak fractions from the anti-CDC5L and anti-SKIP antibody columns respectively. The arrows on the right of each panel indicate the PLRG1 and antibody bands respectively. Note that there was some leaching of IgG off the antibody columns during elution using urea. (F) Total ion intensity at any point of liquid chromatography with the mark at the time where the particular peptide was observed and sequenced. (G) Mass spectra of several peptides obtained at the marked time in the first panels. (Insets) The zoom on the selected peptide indicated with the m/z ratio tag. (H)

Tandem MS spectra of selected peptide. Parent ions, if they were viewable, were marked with m/z ratio tag. The C-terminus containing ion series (y ions), used to identify the peptide, were marked with dots. 37 peptides from hnRNP-M protein were identified, giving the final sequence coverage of 47,1%.

Figure S2. (A) Nuclear distribution of hnRNP M proteins with markers of the nuclear speckles compartment like SC35 (top and middle row) and Sm proteins (bottom row).

(Top row) Transiently transfected cells expressing EGFP-hnRNP M were immunostained with an anti-SC35 antibody. (Middle row) Transiently transfected cells expressing EGFP-

SC35 were immunostained with an anti-hnRNP M (D2IF7 antibody). (Bottom row).

Transiently transfected cells expressing EGFP-hnRNP M were immunostained with a monoclonal antibody anti-Sm proteins (Y12 antibody). Bar, 10μm.

(B) Electron microscope immunocytochemistry. Low magnification of a cell nucleus after RNP staining. (i) The labelling for CDC5L is present on the perichromatin fibrils

(arrowheads) and at the periphery of the interchromatin granule clusters (asterisk). Some labelling is present at the nucleolus (No). Cy, cytoplasm, Nu, nucleus. (ii) Single RNA fibril after terbium staining and double labelling for CDC5L (white arrowheads, 12 nm) and hnRNP M (black arrowheads, 6 nm). Both antibodies are associated with RNA. (iii- iv) represent two examples of perichromatin fibrils double labelled for CDC5L and hnRNP M, and stained for RNP with the EDTA technique. (C) Heat shock. Co-IP using anti-hnRNP M (2A6) either at 37˚C (left panel) or upon heat shock 1h at 42˚C (right panel) and probed with anti-hnRNP M (1D8).

Figure S3. hCDC5L directly interacts with PLRG1 in vivo.

HeLa cells transiently transfected with ECFP-PLRG1 alone (panel A) or cotransfected with ECFP-PLRG1 and EYFP-CDC5L fusion proteins (panel B) were analyzed using the multiphoton FLIM-FRET technique. The mean fluorescence lifetime τ (ns) is detected and the spatial distribution of the mean lifetime values at each pixel is shown.

Fluorescence lifetimes are presented in continuous pseudo-colour scale representing time values ranging from 1.7 to 2.3 ns. A significant decrease of the τ (ns) of ECFP-PLRG1 in the presence of EYFP-CDC5L is observed in comparison with the donor ECFP-PLRG1 alone. Then, the percentage of FRET efficiency is calculated and its spatial distribution

(FRET Efficiency %) is depicted using discrete colours scale representing FRET efficiency values ranging from 0 to 25%. Bars, 10 μm.

Figure S4. Identification of the hnRNP M domain required for CDC5L/PLRG1 binding.

(A) Linear diagram representing domains of hnRNP M and relative truncated mutants used. On the right side, the interaction results are summarized. (B) GST pull down using

GST-CDC5L purified protein with either hnRNP M full length (lane 3), or hnRNP M mutants 1-4 (lanes 4-7) [35S]-Methionine labelled. (C) GST pull down using GST-

PLRG1 purified protein with either hnRNP M full length (lane 4), or hnRNP M mutants

1-4 (lanes 5-8) [35S]-Methionine labelled. (D) Pull down results using either GST-

CDC5L or GST-PLRG1 proteins in the presence of either hnRNP M full lenght [35S]-

Methionine labelled (lanes 1&6, respectively), or mutant 5 (lanes 2 & 7), or mutant 6

(lanes 3 & 8).

Figure S5. In vitro mapping of the minimal domain of protein-protein interaction between hnRNP M and CDC5L complex. (A) Molecular size and level of expression of hnRNP M full length and truncated hnRNP M mutants 1-6 [35S]-Methionine labelled used in this study (lanes 1-7). (B) Immunoprecipitation experiments using a polyclonal antibody anti-CDC5L from HeLa cells transiently transfected with either full-length hnRNP M-YFP (lane 5), or hnRNP M mutant 1-YFP (lane 6), or hnRNP M mutant 3-

YFP (lane 7). The Western blot is probed with a monoclonal antibody anti-GFP. The related inputs are shown (lanes 1-4). The full-length hnRNP M protein (lane 5) and hnRNP M- YFP mutant 3 (lane 7) are interacting with hCDC5L as indicated by the black arrowhead. Note that the asterisk is indicating a nonspecific band present in all the lanes.

Figure S6. Nuclear dynamic of hnRNP M mutant 6(248-344aa)-EGFP before and after

Heat shock stress. The kinetics of the relative fluorescence recovery of hnRNP M mutant 6(248-344aa)-EGFP in physiological conditions (black squares) and after Heat shock (white squares) are represented.

Figure S7. In vivo interaction between hnRNP M and both subunits of splicing factor U2AF by FLIM-FRET.

(A) HeLa cells transiently transfected with hnRNP M-ECFP alone or cotransfected with either hnRNP M-ECFP and EYFP-U2AF65 fusion proteins, or hnRNP M-ECFP and

EYFP-U2AF35 fusion proteins were analyzed using the multiphoton FLIM-FRET technique. The mean fluorescence lifetime τ (ns) is detected and the spatial distribution of the mean lifetime values at each pixel is shown. Fluorescence lifetimes are presented in continuous pseudo-colour scale representing time values ranging from 1.7 to 2.3 ns. Bars,

10 μm. (B) The statistical analysis is presented as histogram bars. (C) Total cells extracts from untransfected HeLa cells or transiently transfected HeLa cells with either 1ug

EYFP-U2AF35, or EYFP-U2AF65, or EYFP-C1 DNA plasmid were co- immunoprecipitated using antibody against GFP and probed with an antibody anti- hnRNP M (D2IF7 antibody). (D) HeLa cells transiently transfected with hnRNP M- mutant 6-EGFP alone or cotransfected with either hnRNP M mutant 6-EGFP and mCherry-U2AF65 fusion proteins, or hnRNP M mutant 6-EGFP and mCherry-U2AF35 fusion proteins were analyzed by FLIM-FRET. The mean fluorescence lifetime τ (ns) is detected and its the spatial distribution is shown. Fluorescence lifetimes are presented in continuous pseudo-colour scale representing time values ranging from 1.7 to 2.4 ns. Bars,

10 μm.

Ajuh P, Lamond AI (2003) Identification of peptide inhibitors of pre-mRNA splicing derived from the essential interaction domains of CDC5L and PLRG1. Nucleic Acids Res 31: 6104-6116

Bernhard W (1969) A new staining procedure for electron microscopical cytology. J Ultrastruct Res 27: 250-265

Table I (A)

Accession Number Protein Name Comments SWISS‐PROT:Q09161 80 kDa nuclear cap binding protein (1) SWISS‐PROT:Q07955 ASF‐1/SF2 (2) Contains SR domain SWISS‐PROT:Q9Y3B4 branch site protein p14 (2) SWISS‐PROT:P11142 CCAP1 (2) Also called HSP73 or HS7C SWISS‐PROT:Q9UI29 CCAP2 Also called HSPC148 or AD‐002 TREMBL:Q15394 CCAP4 (2) TREMBL:Q92616 CCAP6 (2) SWISS‐PROT:Q9UPN6 CCAP7 (RNA binding motif protein 16) Contains SR domain SWISS‐PROT:O60289 CCAP8 TREMBL: Q99974 CDC5L (2) SWISS‐PROT:Q9P2I0 CPSF 100 kDa subunit (2) functions in in pre‐mRNA 3'‐end formation SWISS‐PROT:Q10570 CPSF 160 (1) SWISS‐PROT:O95639 CPSF 30 kDa subunit (1) SWISS‐PROT:Q9UKF6 CPSF 73 kDa subunit (2) TREMBL:Q12996 CSTF3 77 kDa subunit (2) Involved in mRNA polyadenylation SWISS‐PROT:Q99848 EBNA1 binding protein 2 (1) Involved in processing of the 27S pre‐ rRNA SWISS‐PROT:Q15717 ELAV‐like protein 1 (2) SWISS‐PROT:O75400 Formin binding protein 3 (Fas‐ligand Interacts with phosphorylated CTD of associated factor 1) (1) POLR2A, SF1, SRPK1 SWISS‐PROT:P35637 FUS/TLS (2) RNA‐binding protein Associates with PTB, SRm160, and SR proteins SWISS‐PROT:Q13344 FUS‐like protein SWISS‐PROT:Q8WXD5 Gem‐associated protein 6 SWISS‐PROT:P57678 Gemin 4 (2) Component of the of the core SMN complex TREMBL:O02916 HLARK (2) RNA binding SWISS‐PROT:Q99729 HnRNP A/B (1) SWISS‐PROT:Q13151 HnRNP A0 (1) SWISS‐PROT:P09651 HnRNP A1 (1) SWISS‐PROT:P22626 HnRNP A2/B1 (2) SWISS‐PROT:P51991 HnRNP A3 (1) SWISS‐PROT:P07910 HnRNP C1/C2 (2) SWISS‐PROT:Q14103 HnRNP D0 (1) SWISS‐PROT:P52597 HnRNP F (2) SWISS‐PROT:P38159 HnRNP G SWISS‐PROT:P38159 HnRNP G (1) SWISS‐PROT:P31943 HnRNP H (2) SWISS‐PROT:Q07244 HnRNP K (2) SWISS‐PROT:P31943 HnRNP L (2) TREMBL:Q9BWL9 HnRNP M (2) TREMBL:Q96LC1 HnRNP Q (1) SWISS‐PROT:O43390 HnRNP R (1) SWISS‐PROT:Q00839 HnRNP U (2) SWISS‐PROT:Q15365 HnRNP‐E1 (2) TREMBL:Q16630 HPBRII‐7 protein (1) RNA binding TREMBL:Q9UMS4 hPRP19 (2) Has 1‐U box and WD40 motifs, may be involved in protein ubiquitination TREMBL:Q8WVV9 Hypothetical protein (2) RNA binding and mRNA processing activity inferred from electronic annotation TREMBL:Q9NUY0 Hypothetical protein FLJ11063 (1) Contains SR domain. Closely related to yeast protein Luc7 TREMBL:O75921 Lodestar RNA Pol II termination factor, also present in the spliceosome complex TREMBL:Q96S32 LUC7L (2) Contains SR motifs, yeast Luc7p is part of the U1 snRNP SWISS‐PROT:Q86U44 N6‐adenosine‐methyltransferase 70 kDa methylates adenosine residues of subunit (1) some mRNAs, may function in the efficiency of mRNA splicing, transport or translation

TREMBL:Q9NU36 Novel protein similar to small nuclear ribonucleoprotein polypeptide A' (SNRPA1) (1) SWISS‐PROT:Q12906 Nuclear factor associated with dsRNA (NFAR) Interacts with FUS and SMN, (1) SWISS‐PROT:Q13573 Nuclear protein SKIP (2) SWISS‐PROT:Q14978 Nucleolar 130 kDa protein (Nopp140) (1) Has intrinsic GTPase and ATPase activities SWISS‐PROT:Q15233 P54nrb, NONO (2) Has 2 RRM motifs SWISS‐PROT:O43660 PLRG1 (2) TREMBL:Q86U42 Poly(A) binding protein II (PABII) (1) Nuclear import may involve the nucleocytoplasmic transport receptor transportin and RAN‐GTP TREMBL:Q6PKG5 Poly(RC)‐binding protein 2, isoform b (2) SWISS‐PROT:Q01780 Polymyositis/scleroderma autoantigen 2 (2) Component of the exosome multienzyme ribonuclease complex, has a 3'‐5' exonuclease activity SWISS‐PROT:P26599 Polypyrimidine tract‐binding protein 1 (PTB) (2) SWISS‐PROT:P08129 PP1‐alpha 1 catalytic subunit (2) Protein is also involved in some of the other functional groups SWISS‐PROT:P30154 PP2A, 65 kDa regulatory subunit A, beta Protein is also involved in some of the isoform (1) other functional groups SWISS‐PROT:Q92989 Pre‐mRNA cleavage complex II protein Clp1 Interacts with cleavage factor I and (1) CPSF SWISS‐PROT:O94913 Pre‐mRNA cleavage complex II protein Pcf11 (1) TREMBL:O43809 Pre‐mRNA cleavage factor I 25kDa TREMBL:Q8N684 Pre‐mRNA cleavage factor I, 59 kDa (2) SWISS‐PROT:Q08170 Also called splicing factor, Pre‐mRNA splicing factor SRP75 arginine/serine‐rich 4 SWISS‐PROT:Q92733 Proline rich protein PRCC SWISS‐PROT:Q13523 PRP4 kinase TREMBL:Q9Y658 Prp5‐like DEAD‐box protein (1) DEAD (Asp‐Glu‐Ala‐Asp) box polypeptide 46 TREMBL:O14547 hPRP8 (2) SWISS‐PROT:P23246 PSF (PTB‐associated splicing factor) (2) May bind DNA TREMBL:Q8NFB1 PTBP2 Contains 4 RRM motifs interacts with SAP145 and with SWISS‐PROT:Q9Y580 RBM7 SRp20 TREMBL:Q9BTE4 RNA binding motif protein 5 (1) Putative tumor suppressor LUCA15 TREMBL:Q86YB3 ROD1 Contains 4 RRMs SWISS‐PROT:Q15459 SAP 114 (2) SWISS‐PROT:Q15393 SAP 130 (2) SWISS‐PROT:Q13435 SAP 145 (2) SWISS‐PROT:O75533 SAP 155 (2) SWISS‐PROT:Q15427 SAP 49 (1) SWISS‐PROT:Q12874 SAP 61 (2) SWISS‐PROT:Q15428 SAP 62 TREMBL:Q96BK1 SF3b DEAD‐box protein (2) SWISS‐PROT:P13641 SmD1 SWISS‐PROT:P43331 SmD2 SWISS‐PROT:P43331 SmD3 (2) SWISS‐PROT:P08578 SmE SWISS‐PROT:Q16637 SMN (2) TREMBL:O75934 SPF27 (2) SWISS‐PROT:Q96I25 SPF45 (2) SWISS‐PROT:Q16629 Splicing factor 9G8 Contains SR domain SWISS‐PROT:Q05519 Splicing factor arginine/serine‐rich 11 (1) SWISS‐PROT:Q14498 Splicing factor HCC1 (2) Contains SR domain SWISS‐PROT:Q01130 Splicing factor SC35 (1) Contains SR domain TREMBL:Q8IYB3 SRM160 (Ser/Arg‐related nuclear matrix Contains SR domain protein Srrm1) (2) SWISS‐PROT:P23152 SRP20 Pre‐mRNA splicing factor (1) Contains SR domain SWISS‐PROT:Q13243 SRP40 (1) Contains SR domain SWISS‐PROT:Q13247 SRP55 (1) Contains SR domain TREMBL:Q8WU30 SR‐related CTD associated factor 6 (2) Contains SR domain,

SWISS‐PROT:Q8WXA9 SRrp86 Contains SR domain SWISS‐PROT:Q13148 TAR DNA‐binding protein‐43 (2) Regulates CFTR splicing, promotes, binds RNA TREMBL:Q8WWH5 TruB pseudouridine synthase‐like protein 1 pseudouridylate synthase, may be (2) involved in tRNA processing TREMBL:Q16083 U1 snRNP 1 SNRP homolog protein (1) SWISS‐PROT:P08621 U1 snRNP 70 kDa (2) SWISS‐PROT:P09012 U1A SWISS‐PROT:P08579 U2 snRNP B"" (1) SWISS‐PROT:P09661 U2A SWISS‐PROT:Q01081 U2AF 35 kDa (2) SWISS‐PROT:P26368 U2AF 65 kDa (2) TREMBL:O15042 U2‐associated SR140 (2) SWISS‐PROT:O43395 U4/U6 hPrp3 2 TREMBL:O43290 U4/U6.U5 110 kDa (SART‐1) (2) SWISS‐PROT:Q96RK9 U4/U6.U5 65 kDa TREMBL:O43188 U5 100 kDa (1) SWISS‐PROT:O94906 U5 102 kDa (2) SWISS‐PROT:O75643 U5 200 kDa (2) TREMBL:O95320 U5 40 kDa (2) SWISS‐PROT:Q15029 U5116 kDa (2) SWISS‐PROT:Q9H0D6 Xrn2p/Rat1p, 5'‐3' exoribonuclease 2 (2) functions in RNA metabolism, RNA synthesis and RNA trafficking

Table I continued (B) Accession number Protein Name Comment SWISS‐PROT:O15160 DNA‐directed RNA polymerase I 40 kDa polypeptide (RPA39) (2) SWISS‐PROT:O60563 Cyclin T1 (1) Also called positive transcription elongation factor B (P‐TEFB), facilitates the transition from abortive to production elongation by phosphorylating the CTD of RNAP II SWISS‐PROT:O96019 BAF53A (2) SWISS‐PROT:P05423 DNA‐directed RNA polymerase III 53 kDa subunit RPC4 SWISS‐PROT:P16989 DNA‐binding protein A (CSDA) (1) SWISS‐PROT:P19388 DNA‐directed RNA polymerase II 23 kDa polypeptide (1) SWISS‐PROT:P24468 COUP transcription factor 2 (1) SWISS‐PROT:P24928 DNA‐directed RNA polymerase II largest subunit (1) SWISS‐PROT:P40763 Acute‐phase response factor (STAT3) SWISS‐PROT:P48382 DNA‐binding protein RFX5 transcription activation from class II MHC promoters SWISS‐PROT:Q14839 Chromodomain helicase‐DNA‐ Member of the SNF2/RAD54 helicase binding protein 4 (1) family SWISS‐PROT:Q96GM5 BAF60A (2) SWISS‐PROT:Q9BTC0 Death associated transcription factor 1 (2) Activity‐dependent neuroprotector SWISS‐PROT:Q9H2P0 Probable transcription factor (2) SWISS‐PROT:Q9NPF5 DNMAP1 (2) Has a transcription repression role SWISS‐PROT:Q9NVU0 DNA‐directed RNA polymerases III 80 kDa polypeptide TREMBL:O43539 BAF57 (2) DNA‐binding subunit of the mammalian SWI/SNF‐like chromatin‐remodelling BAF complexes TREMBL:O75175 CCR4‐NOT transcription complex subunit 3 TREMBL:Q14673 Bcl‐2‐associated transcription factor Death‐promoting transcriptional 1 (2) repressor TREMBL:Q15044 CoREST protein (2) Possible nuclear receptor and silencing mediator for retinoid and thyroid

human receptors (SMRT) TREMBL:Q8IXK4 BRAF‐HDAC complex protein BHC110 (2) TREMBL:Q96E12 BAF170 subunit (2) Binds chromatin and contains 1 MYB‐like domain, involved in regulation of transcription from Pol II promoter TREMBL:Q9H9A5 CCR4‐NOT transcription complex, Global regulator of RNA polymerase subunit 10 II transcription TREMBL:Q9Y5B9 Chromatin‐specific transcription elongation factor FACT 140 kDa subunit SWISS‐PROT:P22670 Enhancer factor C Has RNA polymerase II transcription factor activity, enhancer binding TREMBL:Q15723 ETS transcription factor SWISS‐PROT:Q9H9B1 Euchromatic histone represses transcriptional by methyltransferase 1 recruiting HP1 proteins to methylated histones SWISS‐PROT:Q96AE4 FUSE binding protein 1 (1) DNA helicase V, acts both as activator and repressor of transcription. TREMBL:Q9NQD0 GAS41 protein oncogenic transcription factor glioma amplified sequence 41/NuMA binding protein 1 SWISS‐PROT:P78347 General transcription factor II‐I (2) SWISS‐PROT:P51531 Global transcription activator Has helicase activity, cooperates SNF2L2 (SNF2‐alpha) (1) with nuclear hormone receptors to promote transcriptional activation SWISS‐PROT:Q13227 GPS2 subunit of the N‐CoR‐HDAC3 complex, modulates p53 transactivation SWISS‐PROT:Q9NRL2 hACF1 (Bromodomain adjacent to with CHRAC1, POLE3 and zinc finger domain protein 1A) (2) ISWI/SNF2H proteins, it forms the ISWI chromatin remodelling complex SWISS‐PROT:P54198 HIRA protein (2) SWISS‐PROT:Q13547 Histone deacetylase 1 (2) important role in transcriptional regulation, cell cycle progression and development SWISS‐PROT:Q92769 Histone deacetylase 2 (2) TREMBL:O43719 HIV‐1 transcriptional elongation factor TAT cofactor TAT‐SF1 (1) TREMBL:O60264 HSNF2H (2) DEAD box helicas SWISS‐PROT:Q96JB3 Hypermethylated in cancer 2 protein (Hic‐2 or Hic‐3) TREMBL:Q8N5H6 Hypothetical protein KIAA1197 (2) SWISS‐PROT:P43243 Matrin 3 (2) May play a role in transcription, has 2 RRMs TREMBL:O60535 Methyl‐CpG binding protein 2 (1) Component of the MeCP1 complex that contains HDAC1 and HDAC2, binds DNMT1, MIZF, XAB1, SIN3A, p66alpha and p66beta TREMBL:O95983 Methyl‐CpG binding protein MBD3 component of the NuRD and the (1) MeCP1 complex, binds HDAC1, MTA2, DNMT1, p66alpha and p66beta SWISS‐PROT:Q9UBU8 MORF4L1 (Transcription factor‐like Functions in growth regulation and protein MRG15) (1) replicative senescence; upregulation of MORF4L1 may be needed for cell cycle progression SWISS‐PROT:Q96T58 Msx2‐interacting protein May repress transcription by recruiting of large complexes containing histone deacetylase, interacts with steroid receptor RNA coactivator SRA SWISS‐PROT:O94776 MTA1 L1 (2) May act as repressor and activator SWISS‐PROT:Q9BTC8 MTA3 SWISS‐PROT:O14686 Myeloid/lymphoid or mixed‐lineage leukemia protein 2 (1) TREMBL:Q96RE7 NAC1 (2) SWISS‐PROT:O75376 N‐COR1 (2) represses transcription by interaction with the human N CoR/mSin3/HDAC1 complex

SWISS‐PROT:Q9Y618 N‐CoR2 (SMRT) (1) SWISS‐PROT:Q8IXH7 Negative elongation factor C/D (1) SWISS‐PROT:Q8WX92 NELF‐B, Cofactor of BRCA1 (2) TREMBL:Q13051 NF1 TREMBL:Q12905 NF45 (2) ENSEMBL:ENSP00000337001 NF‐KB2 (p49/p100) SWISS‐PROT:P08651 Nuclear factor 1 C‐type (1) Capable of activating transcription and replication

TREMBL:Q96CB8 nuclear factor SBBI22 (1) DNA binding protein SWISS‐PROT:Q9BZK7 Nuclear receptor co‐ recruits the ubiquitin/19S repressor/HDAC3 complex subunit proteasome complex to nuclear TBLR1 (2) receptor‐regulated transcription units SWISS‐PROT:P19338 Nucleolin (1) Involved in pre‐RNA transcription SWISS‐PROT:Q01085 Nucleolysin TIAR (2) Contains 3 RRM motifs SWISS‐PROT:P10588 Orphan nuclear receptor EAR‐2 (1) nuclear steroid/thyroid hormone receptor, DNA binding SWISS‐PROT:P49116 Orphan nuclear receptor TR4 (1) TREMBL:Q9BVS6 p60 (1) Involved in positive regulation of transcription from Pol II promoter TREMBL:Q8N4P9 PAX transcription activation domain interacting protein 1 (1) TREMBL:Q8IW31 PCAF associated factor 65 beta SWISS‐PROT:Q9Y4A5 PCAF‐associated factor 350/400 kDa Steroid receptor estrogen TREMBL:O15450 PELP1 (1) coactivator SWISS‐PROT:Q13610 Periodic tryptophan protein 1 homolog (1) TREMBL:Q9Y4X7 Pogo transposable element with ZNF domain (1) TREMBL:Q9H314 Polybromo‐1 (1) SWISS‐PROT:Q06587 Polycomb complex protein RING1 (1) SWISS‐PROT:O75676 Ribosomal protein S6 kinase alpha 4 mediates the growth‐factor and (2) stress induced activation of the transcription factor CREB SWISS‐PROT:Q01844 RNA‐binding protein EWS SWISS‐PROT:Q9Y265 RuvB‐like 1 (2) DNA helicase, regulation of transcription from Pol II promoter SWISS‐PROT:Q9Y230 RuvB‐like 2 (2) DNA helicase, regulation of transcription from Pol II promoter, DNA repair SWISS‐PROT:Q96ST3 Sin3a TREMBL:Q15355 SMAP (1) SWISS‐PROT:Q9H4L7 SMARCAD1 (2) SWI/SNF‐related, matrix associated, actin‐dependent regulator of chromatin subfamily A containing DEAD/H box 1 SWISS‐PROT:Q12824 SMARCB1 (2) SWI/SNF related, matrix associated, actin dependent regulator of chromatin subfamily B member 1 TREMBL:Q9Y6P7 SNAP190 Involved in transcription from Pol II and Pol III promoters TREMBL:Q8IWH2 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily c, member 1 (2) TREMBL:Q96PK5 SYT interacting protein SIP (1) Regulator of transcription from Pol II promoter, may be involved in histone deacetylation SWISS‐PROT:Q92804 TATA‐binding protein associated factor 2N (TAF(II)68) (1)

SWISS‐PROT:O14981 TBP‐associated factor 172 (2) Member of the SNF2/RAD54 helicase family TREMBL:Q9H4P2 TFIIIC 63 TREMBL:Q12789 TFIIIC box B‐binding subunit (1) Functions in RNA Pol III‐mediated transcription, essential for transcription of 5S rRNA and other stable nuclear and cytoplasmic RNAs

TREMBL:Q16632 TFIIIC2 subunit (2) Needed for RNA polymerase III‐ mediated transcription SWISS‐PROT:Q96FV9 THOC1 (1) Involved in RNA Pol II transcription elongation, links proteins involved in RNA export and transcription SWISS‐PROT:Q8NI27 THOC2 (1) Component of the THO/TREX complex containing THOC1, THOC2, THOC3, THOC4 and NFX1/UAP56

SWISS‐PROT:P41212 Transcription factor ETV6 (2) TREMBL:Q9Y5Q9 Transcription factor IIIC102 (1) Functions in RNA polymerase III‐ mediated transcription, component of the DNA‐binding TFIIIC2 subcomplex that directly binds tRNA and virus‐associated RNA promoters TREMBL:Q9UKN8 Transcription factor IIIC90 (1) Required for RNA Pol III transcritption of small nuclear and cytoplasmic RNAs, including 5S RNA & tRNA SWISS‐PROT:Q15014 Transcription factor‐like protein Involved in growth regulation and MRGX (MORF4L2) (1) replicative senescence SWISS‐PROT:Q16514 Transcription initiation factor TFIID subunit 12 (1) SWISS‐PROT:Q16594 Transcription initiation factor TFIID subunit 9 (1) SWISS‐PROT:Q13263 Transcription intermediary factor 1‐ complex with a KRAB‐domain beta (2) transcription factor and increases the efficiency of KRAB‐mediated repression by recruiting SETDB1 to histone H3 TREMBL:Q8WXI9 Transcription repressor p66 (1) SWISS‐PROT:Q00577 Transcriptional activator protein transcription activator that binds the PUR‐alpha (1) purine‐rich single strand of the PUR element located upstream of the MYC gene; also involved in DNA replication initiation Transcriptional repressor p66 alpha TREMBL:Q96F28 Putative transcriptional repressor (2) SWISS‐PROT:P25490 Transcriptional repressor protein Function as an activator or a YY1 (1) repressor is determined by the presence of other proteins SWISS‐PROT:Q9NPJ6 TRAP p36 subunit, Vitamin D3 component of the large multiprotein receptor‐interacting protein complex complexes ARC/DRIP and PC2 36 kDa (2) SWISS‐PROT:Q9Y2W1 TRAP150 (2) Coactivator of transcription SWISS‐PROT:Q93074 TRAP230 Component of the large multiprotein complexes TRAP and ARC/DRIP SWISS‐PROT:Q99816 Tumor susceptibility gene 101 Contains one UEV domain required protein (2) for interaction the with ubiquitin TREMBL:Q9Y652 Vitamin D receptor‐interacting protein (DRIP92) (1) SWISS‐PROT:Q14320 XAP‐5 protein (1) SWISS‐PROT:Q15906 YL‐1 protein, Transcription factor‐ negative regulator of transcription like 1 (2) from Pol II promoter SWISS‐PROT:P52739 Zinc finger protein 131 transcription factor SWISS‐PROT:Q9UQR1 Zinc finger protein 148 (2) Component of RNA Pol II core complex, represses transcription of some genes including gastrin, stromelysin and enolase SWISS‐PROT:Q12899 Zinc finger protein 173 (1) SWISS‐PROT:Q9Y2X9 Zinc finger protein 281 (2) negative regulator of transcription from Pol II promoter TREMBL:Q8N3I1 Zinc finger protein 289, ID1 Binds DNA, putative transcription regulated (2) factor SWISS‐PROT:P17030 Zinc finger protein KOX19 (1) krueppel C2H2‐type zinc‐finger protein family

SWISS‐PROT:Q92785 Zinc‐finger protein ubi‐d4 (1) May be involved in the induction of apoptosis by extracellular signals

Table I continued (C)

Accession number Protein Name Comments TREMBL:Q9BY44 CDA02 (2) Translation initiation factor activity SWISS‐PROT:P05198 eIF‐2‐alpha (1) SWISS‐PROT:P20042 eIF‐2‐beta (1) SWISS‐PROT:P41091 eIF‐2‐gamma (2) SWISS‐PROT:Q13347 eIF‐3 beta (2) SWISS‐PROT:O75821 eIF‐3 delta (2) Binds to the 18S rRNA SWISS‐PROT:P55884 eIF‐3 eta promotes the binding of methionyl‐ tRNAi and mRNA SWISS‐PROT:O15372 eIF‐3 gamma (1) SWISS‐PROT:P60228 eIF3e REFSEQ_NP:NP_004944 eIF‐4‐gamma 1 isoform 4 (1) SWISS‐PROT:P78344 eIF‐4‐gamma 2 (2) Role in the switch from cap‐ dependent to IRES‐mediated translation during mitosis SWISS‐PROT:P04720 Elongation factor (2) Promotes the GTP‐dependent binding of aminoacyl‐tRNA to the A‐site of ribosomes during protein synthesis TREMBL:Q9UPW3 ERFS (HBS1) (2) SWISS‐PROT:P04765 Eukaryotic initiation factor 4A‐I RNA cap binding (DDX2A) (2) SWISS‐PROT:Q1424 Eukaryotic initiation factor 4A‐II Involved in regulation of (DDX2B) (2) translational initiation SWISS‐PROT:P46055 Eukaryotic peptide chain release factor subunit 1 (2) SWISS‐PROT:Q9Y262 Eukaryotic translation initiation factor 3 subunit 6 interacting protein (1) SWISS‐PROT:P04792 HSPB1 (1) TREMBL:Q9Y6E2 HSPC028 (1) May be involved in translation initiation TREMBL:Q14281 Hypothetical protein KIAA0005 (2) May be involved in translation initiation TREMBL:Q15394 Hypothetical protein KIAA0005 (2) May have translation initiation factor activity (inferred from electronic annotation) TREMBL:Q9BX72 IFP38 (1) Translation initiation factor activity TREMBL:O15386 Mov34 homolog (2) Human eukaryotic initiation factor 3 subunit, possible roles in RNA binding macromolecular assembly SWISS‐PROT:P06748 Nucleolar phosphoprotein B23 Function in the assembly and/or transport of ribosome SWISS‐PROT:P11940 Poly(A)‐binding protein 1 (PABP 1) (1) SWISS‐PROT:Q14671 Pumilio 1 Regulates translation and mRNA stability, binding to 3' UTRs of mRNA targets SWISS‐PROT:Q8TB72 Pumilio 2 (1) TREMBL: Q9HAU5 Regulator of nonsense transcripts 2, RNA surveillance protein UPF2 (2) SWISS‐PROT:P30050 ribosomal protein L12 (1) SWISS‐PROT:P26373 ribosomal protein L13 (2) SWISS‐PROT:P14118 ribosomal protein L19 (1) SWISS‐PROT:P35268 ribosomal protein L22 (2) SWISS‐PROT:P29316 ribosomal protein L23a (1) SWISS‐PROT:P83731 ribosomal protein L24 SWISS‐PROT:P39023 ribosomal protein L3 (1) SWISS‐PROT:P12947 ribosomal protein L31 (2) SWISS‐PROT:Q9Y3U8 ribosomal protein L36 (1) SWISS‐PROT:P36578 ribosomal protein L4 (1) SWISS‐PROT:P11518 ribosomal protein L7a (1) SWISS‐PROT:P25120 ribosomal protein L8 (1) SWISS‐PROT:P05388 ribosomal protein P0 (2) SWISS‐PROT:Q02546 ribosomal protein S13

SWISS‐PROT:P06366 ribosomal protein S14 (1) REFSEQ_XP:XP_086097 ribosomal protein S16 (1) SWISS‐PROT:P25232 ribosomal protein S18 (1) SWISS‐PROT:P39019 ribosomal protein S19 (1) SWISS‐PROT:P25111 ribosomal protein S25 (1) SWISS‐PROT:P23396 ribosomal protein S3 (1) REFSEQ_XP:XP_039702 ribosomal protein S3a (2) REFSEQ_XP:XP_292099 ribosomal protein S6 (1) SWISS‐PROT:Q9UBS0 ribosomal protein S6 kinase (2) SWISS‐PROT:Q8TDN6 Ribosome biogenesis protein Brix (1) Involved in ribosome large subunit biogenesis SWISS‐PROT:P57772 Selenocysteine‐specific elongation factor needed for the incorporation of (1) selenocysteine into proteins TREMBL:O14897 TNF‐alpha stimulated ABC protein (2) Nucleic acid and ATP binding ENSEMBL:ENSP00000323063 translation initiation factor 2, subunit 3 gamma, 52kDa REFSEQ_XP:XP_290345 translation initiation factor 3, subunit 5 epsilon, 47kDa SWISS‐PROT:Q9UI10 Translation initiation factor eIF‐2B delta SWISS‐PROT:O60841 Translation initiation factor IF‐2 (2) TREMBL:Q92616 Translational activator GCN1 (2) SWISS‐PROT:P49411 Tu translation elongation factor (1)

Table II

Accession number Protein Name Comment SWISS‐PROT:Q9UKV3 Acinus (1) Has ATPase activity SWISS‐PROT:O43823 A‐kinase anchor protein 8 Involved in mitosis and signal transduction SWISS‐PROT:Q9H1A4 Anaphase promoting complex subunit 1 (2) SWISS‐PROT:Q9UJX6 Anaphase promoting complex subunit 2 SWISS‐PROT:Q13042 Anaphase promoting complex subunit 6 APC is a cell cycle‐regulated ubiquitin (APC6) (2) ligase that controls progression through mitosis SWISS‐PROT:Q9UJX3 Anaphase promoting complex subunit 7 SWISS‐PROT:Q9UJX2 Anaphase promoting complex subunit 8 Cell cycle‐regulated E3 ubiquitin ligase (2) that controls progression through mitosis and the G1 SWISS‐PROT:P30260 CDC27 (2) Interacts with RB TREMBL:Q86TR8 CDC42‐binding protein kinase beta (2) TREMBL:Q86TR8 CDC42BPB protein (2) Possible small GTPase regulatory/interacting protein activity SWISS‐PROT:P50750 CDK9 (2) Has kinase activity toward CTD of RNA Pol II SWISS‐PROT:P06493 Cell division control protein 2 homolog, Component of the kinase complex that p34, CDK1 (1) phosphorylates the repetitive CTD of RNA pol II. SWISS‐PROT:Q9NYV4 Cell division cycle 2‐related protein kinase 7 (1) SWISS‐PROT:Q00526 Cell division protein kinase 3 (1) SWISS‐PROT:O95239 Chromosome‐associated kinesin KIF4A Needed for mitotic chromosomal (2) positioning and bipolar spindle stabilization SWISS‐PROT:O95347 Chromosome‐associated protein E (hCAP‐E) (2)

SWISS‐PROT:Q14008 CH‐TOG protein (2) Functions in organizing spindle poles TREMBL:Q9BQT5 CLIP‐associating protein CLASP1 Required to maintain dynamic normal behaviour of microtubules at kinetochores SWISS‐PROT:Q8WVM7 Cohesin subunit SA‐1 (1) Required for the cohesion of sister chromatids after DNA replication, interacts directly with RAD21 in cohesin complex

SWISS‐PROT:Q15021 Condensin subunit 1 (2) Complex required for conversion of interphase chromatin into mitotic‐like condense chromosomes SWISS‐PROT:Q15003 Condensin subunit 2 (2) SWISS‐PROT:Q9BPX3 Condensin subunit 3 (1) SWISS‐PROT:Q14185 Dedicator of cytokinesis protein 1 (1) SWISS‐PROT:Q92608 DOCK2 (1) SWISS‐PROT:Q9H7D0 DOCK5 Needed for cytokinesis SWISS‐PROT:Q8NF50 DOCK8 Needed for cytokinesis SWISS‐PROT:O60216 Double‐strand‐break repair protein Component of the cohesin complex, rad21 homolog (1) involved in chromosome cohesion during cell cycle, in DNA repair, and in apoptosis

SWISS‐PROT:O60610 DRF1 (2) Required for cytokinesis, stress fiber formation, and transcriptional activation of the serum response factor SWISS‐PROT:Q9NSV4 DRF3 (1) SWISS‐PROT:Q9Y295 DRG1 (2) Member of the the GTP1/OBG family SWISS‐PROT:Q13561 Dynactin 2 (2) Plays a role in prometaphase chromosome alignment and spindle organization during mitosis SWISS‐PROT:P50570 Dynamin 2 (2) TREMBL:Q8IUH1 FEEL‐1 protein May be involved in negative regulation of angiogenesis SWISS‐PROT:P15170 G1 to S phase transition protein 1 homolog (1) SWISS‐PROT:P14635 G2/mitotic‐specific cyclin B1 (2) Control of the cell cycle at the G2/M transition TREMBL:Q7Z5K2 HWAPL (1) Associated with cervical carcinogenesis and tumor progression TREMBL:Q7Z638 hypothetical protein FLJ20403 isoform DNA binding, related to AKAP95 1 (1) TREMBL:Q9Y6Y3 IDN3‐B protein (1) Possible involvement in sister chromatid cohesion SWISS‐PROT:Q99661 Kinesin‐like protein KIF2C (2) Binds centromeric, has microtubule motor activity TREMBL:Q96Q78 Kinetochore protein Nuf2 (2) LAP4 protein (Scribble homolog TREMBL:Q14160 Tumor suppressor activity protein) SWISS‐PROT:O43684 Mitotic checkpoint protein BUB3 (2) SWISS‐PROT:O43683 Mitotic checkpoint serine/ threonine‐ protein kinase BUB1 (2) SWISS‐PROT:O60566 Mitotic checkpoint serine/ threonine‐ protein kinase BUB1 beta (2) Mitotic control protein dis3 homolog TREMBL:Q7Z48 (2) TREMBL:Q9UHD8 MLL septin‐like fusion protein MSF‐A GTP binding (2) SWISS‐PROT:Q9UBU8 MORF4L1 (Transcription factor‐like Functions in growth regulation and protein MRG15) (1) replicative senescence; upregulation of MORF4L1 may be needed for cell cycle progression Neurofibromatosis‐related protein NF‐ SWISS‐PROT:P21359 Negative regulator of cell proliferation 1 TREMBL:Q7Z2E6 Nipped‐B‐like protein (1) May be involved in sister chromatid cohesion SWISS‐PROT:P35579 NMMHC‐A (2) Cellular myosin, may play a role in cytokinesis, cell shape, and specialized functions such as secretion and TREMBL:Q8N330 Nuclear interacting partner of ALK TREMBL:Q9UNL7 NUMA protein (2) Involved in the segregation of proteins at mitosis SWISS‐PROT:Q9ULW0 p100 Associated with the spindle pole and with the mitotic spindle SWISS‐PROT:Q92888 p115‐RhoGEF (2) SWISS‐PROT:Q12834 p55CDC Needed for full ubiquitin ligase activity of the cyclosome TREMBL:Q9BRX2 Pelota protein TREMBL:Q7Z7C9 Pericentrin B SWISS‐PROT:P48059 PINCH protein (2) Focal adhesion protein part of the complex ILK‐PINCH PP2A, subunit A, PR65‐alpha isoform Possible roles in all the other functional SWISS‐PROT:P30153 (1) groups (splicing)

SWISS‐PROT:Q8WUM4 Programmed cell death 6‐interacting protein (1) TREMBL:Q14680 Protein kinase PK38 SWISS‐PROT:O15297 Protein phosphatase 2C delta isoform Involved in growth inhibitory pathways (WIP1) activated in response to DNA damage in a p53‐dependent manner. TREMBL:O14777 Retinoblastoma‐associated protein HEC (2) SWISS‐PROT:Q15291 Retinoblastoma‐binding protein 5 (2) TREMBL:Q07666 Sam68 (2) Involved in cell cycle progression, signal transduction transcription, RNA splicing and export, translation SWISS‐PROT:Q16181 Septin 7 (1) Homologous to CDC10 in Saccharomyces cerevisiae SWISS‐PROT:O14965 Serine/threonine kinase 6 (1) Also called Aurora‐A or Breast‐tumor‐ amplified kinase SWISS‐PROT:Q96GD4 Serine/threonine protein kinase 12 Also called aurora B, regulator for the onset of cytokinesis during mitosis SWISS‐PROT:Q96GD4 Serine/threonine protein kinase 12 (Aurora‐B) (1) SWISS‐PROT:Q99986 Serine/threonine protein kinase VRK1 (1) SWISS‐PROT:O14757 Serine/threonine‐protein kinase Chk1 Negative regulator of cell division, DNA (2) damage response, signal transduction resulting in cell cycle arrest SWISS‐PROT:Q13153 Serine/threonine‐protein kinase PAK 1 Enzyme activity is inhibited in cells (1) undergoing apoptosis Serine/threonine‐protein kinase PLK SWISS‐PROT:P53350 (2) SWISS‐PROT:P29353 SHC transforming protein TREMBL:Q9BVQ7 Spermatogenesis associated 5‐like 1 (1) May have nucleotide binding activity SWISS‐PROT:Q8N3U4 stromal antigen 2 Cohesin subunit SA‐2 Interacts directly with RAD21 SWISS‐PROT:Q14683 Structural maintenance of chromosome Also involved in DNA repair 1like 1 protein (2) SWISS‐PROT:Q9UQE7 Structural maintenance of chromosome 3 protein (2) SWISS‐PROT:Q9NTJ3 Structural maintenance of Also called Chromosome‐associated chromosomes 4‐like 1 protein (2) polypeptide C, hCAP‐C or XCAP‐C homolog Suppressor of G2 allele of SKP1 SWISS‐PROT:Q9Y2Z0 Component of ubiquitin ligase complex homolog SWISS‐PROT:P23919 Thymidylate kinase SWISS‐PROT:P33981 TTK protein kinase (1) Can phosphorylate proteins on serine, threonine, and tyrosine Component of the centrosome and TREMBL:Q13312 TXBP181 (2) spindle SWISS‐PROT:P41240 Tyrosine‐protein kinase CSK (1) Phosphorylates a tyrosine on the SRC kinase SWISS‐PROT:Q9UGP9 WD‐repeat protein 5 (1) Involved in osteoblast differentiation TREMBL:Q9BZZ5 XAGL protein (2) A putative antiapoptotic factor, interacts with FGF‐2

Table III

Accession number Protein Name Comment SWISS‐PROT:P18085 ADP‐ribosylation factor 4 (ARF 4) (1) GTP‐binding protein, an ADP‐ ribosyltransferase, involved in protein trafficking; may modulate vesicle budding SWISS‐PROT:P26437 ADP‐ribosylation factor 5 (ARF 5) (1) SWISS‐PROT:P42024 Alpha‐centractin (1) Involved in vesicle‐mediated transport SWISS‐PROT:Q9Y6D5 Brefeldin A‐inhibited GEP 2 (1) Facilitates the activation of ARF1/ARF5/ARF6 through replacement of GDP with GTP.

SWISS‐PROT:Q14204 CCAP5 (Dynein heavy chain) (2) Has ATPase activity SWISS‐PROT:P53621 Coatomer alpha subunit (2) SWISS‐PROT:P53618 Coatomer beta subunit (2) SWISS‐PROT:P48444 Coatomer delta subunit (2) SWISS‐PROT:Q9Y678 Coatomer gamma subunit (2) SWISS‐PROT:Q9UBF2 Coatomer gamma‐2 subunit (2) SWISS‐PROT:P35606 Coatomer protein complex, subunit beta 2 (beta prime) (2) SWISS‐PROT:O75131 Copine III (1) SWISS‐PROT:Q14203 Dynactin 1 (1) SWISS‐PROT:P50570 Dynamin 2 SWISS‐PROT:Q14677 Epsin 4 SWISS‐PROT:Q9UPT5 Exocyst complex component Exo70 (2) SWISS‐PROT:O00471 Exocyst complex component Sec10 (2) SWISS‐PROT:Q9NV70 Exocyst complex component Sec3 (1) SWISS‐PROT:Q96KP1 Exocyst complex component Sec5 (2) SWISS‐PROT:O60645 Exocyst complex component Sec6 (1) SWISS‐PROT:Q96A65 Exocyst complex component Sec8 (2) TREMBL:Q96HN3 Exportin 5 Mediates nuclear export of double‐stranded RNA binding proteins & translation factors TREMBL:Q96CP8 Exportin 6 (1) Nuclear export receptor that is specific for profilin.actin complexes SWISS‐PROT:P14625 GRP94 (2) Functions in the processing and transport of secreted proteins SWISS‐PROT:O75146 HIP1R (2) May link the endocytic machinery to the actin cytoskeleton ENSEMBL:ENSP00000288063 homolog of yeast mRNA transport regulator 3 (1) TREMBL:Q96GS8 Hypothetical protein ABCF3 (2) Contains ATP‐binding cassette (ABC) transporter activity SWISS‐PROT:Q16629 Importin 4 (2) TREMBL:O95373 Importin 7/RanBP7 SWISS‐PROT:Q96P70 Importin 9 (2) SWISS‐PROT:P52294 Importin alpha‐1 subunit (1) SWISS‐PROT:P52292 Importin alpha‐2 subunit (2) SWISS‐PROT:Q14974 Importin beta‐1 subunit (2) SWISS‐PROT:Q92973 Importin beta‐2 subunit (Transportin) (2) SWISS‐PROT:P55060 Importin‐alpha re‐exporter (2) SWISS‐PROT:O00410 Karyopherin (Importin) beta 3 (2) SWISS‐PROT:P41214 Ligatin SWISS‐PROT:P78406 mRNA‐associated protein mrnp 41 (1) Involved in nuclear export of poly(A)+ RNA SWISS‐PROT:Q14764 MVP (2) May be involved in nucleo‐ cytoplasmic transport Implicated in nuclear protein SWISS‐PROT:P12270 Nucleoprotein TPR import SWISS‐PROT:Q13492 Phosphatidylinositol‐binding clathrin assembly protein (2) SWISS‐PROT:P46060 Ran GTPase‐activating protein 1 (2) SWISS‐PROT:P49792 Ran‐binding protein 2 (2) SWISS‐PROT:Q9UN86 Ras‐GTPase‐activating protein binding May be involved in mRNA protein 2 (1) transport SWISS‐PROT:Q96EE3 Sec13‐like protein (2)

SWISS‐PROT:Q15436 Sec23A SWISS‐PROT:Q15437 Sec23B (1) SWISS‐PROT:P53992 Sec24C TREMBL:Q9NQW1 Sec31B‐1 (1) TREMBL:O95149 SNURPORTIN1 (1) SWISS‐PROT:Q9UMY4 Sorting nexin 12 SWISS‐PROT:O60749 Sorting nexin 2 SWISS‐PROT:O60763 Transcytosis associated protein SWISS‐PROT:Q96AX1 Vacuolar protein sorting 33A Involved in vesicle‐mediated protein trafficking SWISS‐PROT:Q9H267 Vacuolar protein sorting 33B (2) Involved in vesicle‐mediated protein trafficking SWISS‐PROT:P46459 Vesicle‐fusing ATPase (2) Functions as a fusion protein required for the delivery of cargo proteins to all compartments of the Golgi stack independent of vesicle origin

Table IV (A) Accession Number Name Comments TREMBL:Q86XH3 ALC1 (2) SWISS‐PROT:P12956 ATP‐dependent DNA helicase II, 70 kDa Involved in chromosome subunit (1) translocation SWISS‐PROT:Q92499 ATP‐dependent helicase DDX1 (1) Has a role in spliceosome assembly SWISS‐PROT:Q14562 ATP‐dependent helicase DHX8 (1) Functions in nuclear export of spliced mRNA by releasing the RNA from the spliceosome SWISS‐PROT:Q08211 ATP‐dependent RNA helicase A (1) SWISS‐PROT:Q13838 ATP‐dependent RNA helicase p47 (2) SWISS‐PROT:P26196 ATP‐dependent RNA helicase p54 SWISS‐PROT:P54132 Bloom's syndrome protein (1) Involved in DNA repair and replication SWISS‐PROT:Q12873 Chromodomain helicase‐DNA‐binding Putative Transcription regulator, protein 3 (1) component of the nucleosome remodelling and histone deacetylase (NuRD) repressive complex SWISS‐PROT:Q14839 Chromodomain helicase‐DNA‐binding Transcription regulator, protein 4 (1) component of the nucleosome remodelling and histone deacetylase (NuRD) repressor complex. SWISS‐PROT:Q9P2D1 Chromodomain‐helicase‐DNA‐binding Member of the SNF2/RAD54 protein 7 (1) helicase family SWISS‐PROT:Q92841 DDX17 (RNA‐dependent helicase p72) (2) DEAD box polypeptide 17 isoform p82 SWISS‐PROT:Q9UMR2 DDX19 (2) Involved in mRNA export from the nucleus SWISS‐PROT:Q14240 DDX2B Involved in cap recognition and it is an ATP‐dependent single stranded DNA‐binding protein SWISS‐PROT:Q9UJV9 DDX41 (2) Needed during post‐ transcriptional gene expression TREMBL:Q9H0S4 DDX47, Hypothetical protein Putative DEAD/H helicase SWISS‐PROT: P38919 DDX48 (2) SWISS‐PROT:P17844| DDX5 (RNA‐dependent helicase p68) (2) TREMBL:Q9Y658 DEAD (Asp‐Glu‐Ala‐Asp) box polypeptide 46 Prp5‐like DEAD‐box protein (1) ENSEMBL:ENSP00000244357 DEAD/H (Asp‐Glu‐Ala‐Asp/His) box polypeptide 3 (2) ENSEMBL:ENSP00000295561 DEAH (Asp‐Glu‐Ala‐His) box polypeptide 15 (1) SWISS‐PROT:P04765 Eukaryotic initiation factor 4A‐I RNA cap binding, functions in translation TREMBL:Q9H6S0 FLJ21940 protein (1)

SWISS‐PROT:Q96AE4 FUSE binding protein 1 (1) DNA helicase V, acts both as activator and repressor of transcription.

SWISS‐PROT:P51531 Global transcription activator SNF2L2 Cooperates with nuclear hormone (SNF2alpha) (1) receptors to promote transcriptional activation SWISS‐PROT:Q9HCK8 HELSNF1 (2) Possible role in the regulation of transcription TREMBL:O60264 HSNF2H (2) involved in regulation of transcription from Pol II promoter TREMBL:Q9NUU7 Hypothetical protein FLJ11126 (2) TREMBL:Q8NCI1 hypothetical protein FLJ20105 TREMBL:Q9BVP6 nuclear RNA helicase, DECD variant of DEAD box family (2) SWISS‐PROT:Q9NR30 Nucleolar RNA helicase II (DDX21) (1) Proliferation‐associated SNF2‐like protein TREMBL:Q8N6P4 (2) SWISS‐PROT:P42285 Protein KIAA0052 SWISS‐PROT:P42285 Protein KIAA0052 TREMBL:Q9NVY6 R27090_2 (2) SWISS‐PROT:P46100 RAD54L (1) Involved in DNA damage repair, may be a global transcriptional regulator SWISS‐PROT:Q92900 Regulator of nonsense transcripts 1 (2) SWISS‐PROT:Q9Y265 RuvB‐like 1 (2) DNA helicase, regulation of transcription from Pol II promoter SWISS‐PROT:Q9Y230 RuvB‐like 2 (2) DNA helicase, regulation of transcription from Pol II promoter, DNA repair TREMBL:Q9NVP9 SETX (SEN1 homolog) (2) Defects in SETX may cause amyotrophic lateral sclerosis 4 (ALS4) TREMBL:Q96BK1 SF3b DEAD‐box protein (2) TREMBL:Q96BK1 SF3b DEAD‐box protein (2) TREMBL:Q9HBD4 SMARCA4 (BRG1) (2) Contains bromodomain, functions in chromatin remodelling/replication SWISS‐PROT:Q9H4L7 SMARCAD1 (2) SWI/SNF‐related, matrix associated, actin‐dependent regulator of chromatin subfamily A containing DEAD/H box 1 TREMBL:O43630 SUV3 (suppressor of var1, 3‐like 1) (1) Binds RNA TREMBL: Q86YA5 SWI/SNF‐related matrix‐associated actin‐ Also called SMARCA3 protein, ATP dependent regulator of chromatin, subfamily and DNA binding. Component of a, member 3 (2) ubiquitin ligase complex SWISS‐PROT:O14981 TBP‐associated factor 172 (2) Regulates transcription in association with TBP TREMBL:Q9UL03 tumor suppressor protein DICE1 DEAD/H box polypeptide TREMBL:Q8WV26 Werner helicase interacting protein

Table IV continued (B)

Accession number Protein Name Comment TREMBL:Q9UMS4 hPRP19 (2) Has 1‐U box and WD40 motifs, may be involved in protein ubiquitination SWISS‐PROT:O60216 Double‐strand‐break repair protein Component of the cohesin complex, rad21 homolog (1) involved in chromosome cohesion during cell cycle, in DNA repair, and in apoptosis SWISS‐PROT:P07992 DNA excision repair protein ERCC‐1 SWISS‐PROT:P09874 poly(ADP‐ribosyl)transferase SWISS‐PROT:P28340 DNA polymerase delta catalytic subunit Has DNA synthesis (polymerase) and (2) an exonucleolytic activities SWISS‐PROT:P35249 Activator 1 37 kDa subunit, RFCp37 (2) Also involved in the elongation of multiprimed DNA templates SWISS‐PROT:P35250 Activator 1 40 kDa subunit, RFCp40 (2) Required in elongation of primed DNA templates by DNA Pol delta and epsilon SWISS‐PROT:P39748 FEN1 (2) Genome stabilization factor that prevents flaps from causing structures that lead to duplications and deletions SWISS‐PROT:P40692 DNA mismatch repair protein Mlh1 (1) SWISS‐PROT:P40937 Activator 1 36 kDa subunit, RFCp36 (2) Also involved in elongation of primed DNA templates by DNA polymerase delta and epsilon SWISS‐PROT:P40938 Activator 1 38 kDa subunit, RFCp38 (2) Also functions in complex with above proteins SWISS‐PROT:P43246 DNA mismatch repair protein MSH2 (2) DNA mismatch repair SWISS‐PROT:P49959 Double‐strand break repair protein MRE11A (2) SWISS‐PROT:P52701 DNA mismatch repair protein MSH6 (2) SWISS‐PROT:P54132 Bloom's syndrome protein (1) part of the BRCA1‐associated genome surveillance complex (BASC), DNA helicase, Rec Q‐like SWISS‐PROT:P78527 DNA‐dependent protein kinase catalytic required for double‐strand break subunit (2) repair and V(D)J recombination, also involved in modulation of transcription SWISS‐PROT:Q07864 DNA polymerase epsilon, catalytic Involved in DNA repair and in subunit A (1) chromosomal DNA replication SWISS‐PROT:Q16531 DNA damage binding protein 1 (DDB1) Functions in the repair of UV‐damaged DNA TREMBL:O60934 Nibrin (2) Functions as part of a complex which also includes rad50 and mre11

SWISS‐PROT:Q9NS91 RAD18 (2) Post‐replication repair functions in gap‐filling of a daughter strand on replication of damaged DNA TREMBL:Q9BXK4 Rad3 related protein (1) TREMBL:Q92878 RAD50 (2) 3'‐5' exonuclease, single‐stranded DNA specific endodeoxyribonuclease SWISS‐PROT:P46100 RAD54L (1) May be a global transcriptional regulator, has DNA helicase activity SWISS‐PROT:Q13315 Serine‐protein kinase ATM (1) Also involved in signal transduction, cell cycle control, vesicle and/or protein transport SWISS‐PROT:Q93068 Ubiquitin‐like protein SMT3C precursor Ubiquitin conjugating enzyme, SWISS‐PROT:Q9UKK3 Vault poly(ADP‐ribose) polymerase (1) Part to the PARP family May be involved in postreplication SWISS‐PROT:Q9C0J8 WD‐repeat protein WDC146 (2) repair

Table IV continued (C)

Accession number Protein Name Comments SWISS‐PROT:Q9Y5N6 Origin recognition complex subunit 6 (2) SWISS‐PROT:Q9UBD5 Origin recognition complex subunit 3 (2) SWISS‐PROT:Q16576 RBBP‐7 (2) SWISS‐PROT:Q13416 Origin recognition complex subunit 2 (2) SWISS‐PROT:Q09028 CAF‐1 subunit C Mediates chromatin assembly in DNA replication and DNA repair SWISS‐PROT:Q07864 DNA polymerase epsilon, catalytic subunit A Also involved in DNA repair and (1) in chromosomal DNA replication SWISS‐PROT:Q00577 Transcriptional activator protein PUR‐alpha Transcription activator that (1) binds the purine‐rich single strand of the PUR element located upstream of the MYC gene; also involved in DNA replication initiation SWISS‐PROT:P40938 Activator 1 38 kDa subunit (2) May also be involved in cellular DNA damage responses by interacting with hRAD17 SWISS‐PROT:P40937 Activator 1 36 kDa subunit (2) May also be involved in cellular DNA damage responses by interacting with hRAD17 SWISS‐PROT:P35250 Activator 1 40 kDa subunit (2) Functions in cellular DNA damage responses by interacting with hRAD17 SWISS‐PROT:P35249 Activator 1 37 kDa subunit (2) Involved in cellular DNA damage responses by interacting with hRAD17 SWISS‐PROT:P33992 MCM5 DNA replication licensing SWISS‐PROT:P28340 DNA polymerase delta catalytic subunit (2) Has DNA synthesis (polymerase) and an exonucleolytic activities SWISS‐PROT:P27694 Replication protein A 70 kDa DNA‐binding subunit (2) SWISS‐PROT:P11388 DNA topoisomerase II (TOP2A) (2) SWISS‐PROT:P11387 DNA topoisomerase I 1 SWISS‐PROT:O43929 Origin recognition complex subunit 4 (2) SWISS‐PROT:O43913 Origin recognition complex subunit 5 (2)

Table IV continued (D)

Accession Number Protein Name Comments SWISS‐PROT:O00231 26S proteasome regulatory subunit S9 (2) Present in PA700 complex SWISS‐PROT:O00232 26S proteasome non‐ATPase regulatory Present in PA700 complex subunit 12 (2) SWISS‐PROT:O43242 26S proteasome regulatory subunit S3 (2) Also called proteasome subunit p58 SWISS‐PROT:P17980 26S proteasome regulatory subunit 6A (2) TAT‐binding protein 1, Interacts HIV tat transactivator SWISS‐PROT:P35998 proteasome 26S ATPase subunit 2 (2) SWISS‐PROT:P43686 26S proteasome regulatory subunit 6B (2) SWISS‐PROT:P47210 26S proteasome regulatory subunit 8 (2) Interacts with thyroid hormone receptor and with retinoid X receptor (RXR) SWISS‐PROT:Q03527 26S protease regulatory subunit 4 (2) Interacts with SCA7, AAA ATPase family

SWISS‐PROT:Q13200 26S proteasome regulatory subunit S2 (2) Interacts with intracellular domain of TNFR type 1 SWISS‐PROT:Q13616 Cullin homolog 1 (2) Component of the SCF E3 ubiquitin ligase complex, which mediates the ubiquitination of proteins involved in cell cycle progression, SWISS‐PROT:Q92524 26S protease regulatory subunit S10B (2) SWISS‐PROT:Q99460 26S proteasome non‐ATPase regulatory 26S proteasome subunit p112 subunit 1 (2) SWISS‐PROT:Q9UNM6 26S proteasome regulatory subunit S11 (2) Also called p40.5, component of PA700 complex TREMBL:O15386 Mov34 homolog TREMBL:Q15324 Alpha‐spectrin Has E2/E3 enzymatic activities Contains ubiquitin‐protein ligase TREMBL:Q8WWI1 LIM domain only 7 activity TREMBL:Q96HS8 26S proteasome‐associated UCH37 binding Interacts with UCHL5 protein 1 TREMBL:Q99496 DinG protein (Ring finger protein BAP‐1) Binds to the RING finger domain of (1) BRCA1 TREMBL:Q9BUI0 E3 ubiquitin protein ligase URE‐B1 2 Has one HECT domain TREMBL:Q9BV89 CGI 21 (USP39 protein) (1) TREMBL:Q9GZZ9 Hypothetical protein DKFZp434C2415 (2) Ubiquitin‐activating enzyme E1‐domain containing 1, isoform 1 TREMBL:Q9H0G2 Hypothetical protein DKFZp434G0222 (1) TREMBL:Q9UMS4 hPRP19 (2) Splicing factor SWISS‐PROT:Q9Y2Z0 Suppressor of G2 allele of SKP1 homolog (2) Involved in regulation of cell cycle TREMBL: Q86YA5 SWI/SNF‐related matrix‐associated actin‐ Also called SMARCA3 protein, ATP and dependent regulator of chromatin, DNA binding. Has ATP‐dependent subfamily a, member 3 (2) helicase activity SWISS‐PROT:Q14669 TRIP12 (1) Part of PA700, an ATP‐dependent multisubunit protein that activates the proteolytic activities of the 20S proteasome SWISS‐PROT:Q14694 Ubiquitin carboxyl‐terminal hydrolase 10 SWISS‐PROT:Q9UPU5 Ubiquitin carboxyl‐terminal hydrolase 24 SWISS‐PROT:Q9Y5K5 Ubiquitin carboxyl‐terminal hydrolase Component of the 19S regulatory isozyme L5 (2) complex of the 26S proteasome TREMBL:Q9NUJ6 Ubiquitin specific protease 31 (1) SWISS‐PROT:O76069 Ubiquitin‐conjugating enzyme E2 M (2) SWISS‐PROT:Q16781 Ubiquitin‐conjugating enzyme E2 N (2) SWISS‐PROT:Q16763 Ubiquitin‐conjugating enzyme E2‐24 kDa ENSP00000309527 Ubiquitin‐conjugating enzyme E2‐24 kDa TREMBL:Q9BVX5 Ubiquitin‐conjugating enzyme E2Q (1) SWISS‐PROT:P11441 Ubiquitin‐like protein GDX (2) SWISS‐PROT:Q93068 Ubiquitin‐like protein SMT3C precursor Ubiquitin conjugating enzyme, may be involved in DNA repair

Table V Accession Number Protein Name Comments SWISS‐PROT:P19474 52 kDa Ro protein Belongs to a large family of related RING/Bbox/coiled‐coil (RBCC) tripartite motif proteins SWISS‐PROT:P54619 5'‐AMP‐activated protein kinase, gamma1 subunit (1) SWISS‐PROT:Q01813 6‐phosphofructokinase, type C (2) SWISS‐PROT:P11021 78 kDa glucose‐regulated protein precursor (2) TREMBL:O15441 Aac11 (1) May be anti‐apoptotic factor TREMBL:Q9NVP0 Actin binding protein anillin (2) SWISS‐PROT:P02570 Actin, cytoplasmic 1 (2) SWISS‐PROT:O15142 Actin‐like protein 2 (2) SWISS‐PROT:Q10567 Adapter‐related protein complex 1 beta 1 subunit (2) SWISS‐PROT:O00203 Adapter‐related protein complex 3 beta 1 subunit (1) TREMBL:Q8WXA6 AF15q14 isoform 2 TREMBL:Q13727 AHNAK related protein (2) May be involved in neuronal cell differentiation TREMBL:Q8N1C0 Alpha2(E)‐catenin (2) SWISS‐PROT:Q9NVD7 Alpha‐parvin (2) Localized to focal adhesions TREMBL:Q15324 Alpha‐spectrin (2) TREMBL:Q96GR3 Ang2 Involved in growth of cancer cells SWISS‐PROT:P50995 Annexin A11 ENSEMBL:ENSP00000277916 Annexin VII isoform 2 (2) TREMBL:O73453 APM‐1 protein Zinc finger protein with growth inhibitory activity SWISS‐PROT:Q96P48 ARAP1 (1) TREMBL:Q8N2Y0 ARH protein (1) Putative LDL receptor adaptor protein SWISS‐PROT:Q9Y2J2 Band 4.1‐like protein 3 TREMBL:O43770 BCL7C protein (1) TREMBL:O43209 Beta tubulin (2) Has GTPase activity TREMBL:Q9H4B7 Beta tubulin 1 class VI (1) Has GTPase activity SWISS‐PROT:Q9HBI1 Beta‐parvin (2) Focal adhesion protein, involved in regulation of cell adhesion and cytoskeleton organization TREMBL:Q8IY86 BH3 interacting domain death agonist Regulation of apoptosis isoform 1 ENSEMBL:ENSP00000339071 bromodomain containing 7 (1) TREMBL:Q9UH59 Bromodomain protein CELTIX1 SWISS‐PROT:P25440 Bromodomain‐containing protein 2 SWISS‐PROT:O60885 Bromodomain‐containing protein 4 TREMBL:Q9BZQ1 C1orf25 Contains zinc finger and RNA binding motifs TREMBL:Q8NI28 C7orf13 SWISS‐PROT:P52907 CapZ alpha‐1 (2) SWISS‐PROT:Q9BWT7 Caspase recruitment domain protein 10 Activates NF‐kappa‐B via BCL10 and IKK SWISS‐PROT:O60716 Catenin delta‐1 TREMBL:Q9UBP9 CED‐6 (2) Involved in apoptosis and signal transduction SWISS‐PROT:P42024 Centrosome‐associated actin homolog TREMBL:Q9Y3E3 CGI‐145 protein TREMBL:O95766 CGI‐43 protein TREMBL:Q9UNS2 COP9 complex subunit 3 (1) TREMBL:O15387 COP9 signalosome subunit 6 (1) D‐3‐phosphoglycerate dehydrogenase SWISS‐PROT:O43175 (2) SWISS‐PROT:P15924 Desmoplakin (2) TREMBL:Q96QI3 DJ501N12.5.1 (Novel protein) (1) TREMBL:Q96QD5 DJ85M6.4 (2) Possibly involved in intracellular signaling cascade TREMBL:Q9H830 DKFZP434D193 protein (2) TREMBL:Q9P2A8 DKFZP586J0619 protein (2) TREMBL:Q8IUH3 DRB1 (2) Contains RRM motifs Dual specificity mitogen‐activated SWISS‐PROT:P46734 protein

kinase kinase 3, MAPKK 3 (1) SWISS‐PROT:O14733 Dual specificity mitogen‐activated Functions in signal transduction protein kinase kinase 7 (2) TREMBL:Q8TBT7 Dynamin 1‐like (2) GTP binding, GTPase activity TREMBL:Q9Y6G9 Dynein light chain GTPase activity, mediates signal transduction SWISS‐PROT:O43237 Dynein light intermediate chain 2 (2) SWISS‐PROT:Q9H8V3 ECT2 protein Interacts specifically to RhoA, RhoC and Rac proteins SWISS‐PROT:Q9H4M9 EH‐domain containing protein 1 (2) SWISS‐PROT:Q9H223 EH‐domain containing protein 4 (2) TREMBL:Q9UFB8 ENAH (2) Involved in microfilament assembly and cell motility TREMBL:Q9NR12 ENIGMA protein (2) Contains 1 PDZ/DHR and 3 LIM zinc‐ binding domains SWISS‐PROT:O94842 Epidermal Langerhans cell protein LCP1 (1) SWISS‐PROT:P58107 Epiplakin (2) TREMBL:Q96K06 EPS8R2 (2) TREMBL:Q9BQ06 FANCD2 SWISS‐PROT:Q9UNN5 FAS‐associated factor 1 Potentiates FAS‐induced apoptosis TREMBL:Q96IT0 FASN protein (2) FH1/FH2 domains‐containing protein SWISS‐PROT:Q9Y613 (2) TREMBL:Q8TEN5 FLJ00158 protein TREMBL:Q8TEF1 FLJ00246 protein TREMBL:Q8NC46 FLJ21919 protein (2) TREMBL:Q9Y2M2 Fls485 SWISS‐PROT:Q9UGJ1 Gamma tubulin SWISS‐PROT:Q9BSJ2 GCP‐2 (2) Gamma‐tubulin complex component SWISS‐PROT:Q96CW5 GCP‐3 (2) Gamma‐tubulin complex component SWISS‐PROT:Q92974 GEF‐H1 (2) Guanyl‐nucleotide exchange factor SWISS‐PROT:O00178 glyceraldehyde‐3‐phosphate dehydrogenase SWISS‐PROT:P04406 glyceraldehyde‐3‐phosphate dehydrogenase SWISS‐PROT:P49915 GMP synthase [glutamine‐hydrolyzing] SWISS‐PROT:Q14789 Golgi autoantigen, golgin subfamily B member 1 TREMBL:Q86XS8 Goliath protein SWISS‐PROT:P16520 Guanine nucleotide‐binding protein G(I)/G(S)/G(T) beta subunit 3 (1) SWISS‐PROT:P04895 Guanine nucleotide‐binding protein G Heterotrimeric G‐protein GTPase (S), alpha subunit activity SWISS‐PROT:O95837 Guanine nucleotide‐binding protein, alpha‐14 subunit (1) TREMBL:Q8TD90 HCA3 protein SWISS‐PROT:O75616 HERA‐A GTP and RNA binding TREMBL:Q7Z6R0 HIVEP1 TREMBL:O15324 HPV16 E1 binding protein (2) Putative ATPase SWISS‐PROT:P31689 HSDJ (2) Functions in protein folding SWISS‐PROT:O43301 HSP 12A (2) SWISS‐PROT:P04792 HSP 27 kDa protein (2) SWISS‐PROT:P17066 HSP 70 kDa protein 6 SWISS‐PROT:Q12931 HSP 75 kDa, precursor (2) SWISS‐PROT:P07900 HSP1 90kDa protein alpha (2) SWISS‐PROT:P10809 HSP60 (2) Chaperonin with possible function in macromolecular assembly SWISS‐PROT:P08238 HSP90B (2) TREMBL:Q9NWV8 HSPC142 protein (1) TREMBL:Q9H0E9 Hypothetical protein TREMBL:Q9NWB6 Hypothetical protein Contains SR domain TREMBL:Q96AG0 Hypothetical protein (1) Nucleic acid binding, putative nuclease TREMBL:Q8N0T9 Hypothetical protein (2) TREMBL:Q96B70 Hypothetical protein (LENG9) Nucleic acid binding TREMBL:Q9H077 Hypothetical protein DKFZp586K0717 (1) TREMBL:Q7Z314 Hypothetical protein DKFZp686P06215

TREMBL:Q9NVI1 Hypothetical protein FLJ10719 (2) TREMBL:Q9NSK1 Hypothetical protein FLJ12889 (2) Similar to leucine‐rich repeat proteins TREMBL:Q9NXK9 Hypothetical protein FLJ20187 (2) TREMBL:Q9NXD3 Hypothetical protein FLJ20311 ENSEMBL:ENSP00000329896 Hypothetical protein FLJ20506 TREMBL:Q9H6S7 Hypothetical protein FLJ21924 TREMBL:Q8N274 Hypothetical protein FLJ33834 (2) TREMBL:Q8NB16 hypothetical protein FLJ34389 (1) May be a protein‐tyrosine kinase as inferred from electronic annotation TREMBL:Q8N8M9 Hypothetical protein FLJ39190 TREMBL:Q8NBY1 Hypothetical protein FLJ90669 (1) ATP binding, has protein‐tyrosine kinase and transferase activities TREMBL:Q9Y3I0 Hypothetical protein HSPC117 (2) TREMBL:O60306 Hypothetical protein KIAA0560 (1) TREMBL:Q9Y4B5 Hypothetical protein KIAA0802 TREMBL:O94940 Hypothetical protein KIAA0859 (1) May have S‐adenosylmethionine‐ dependent methyltransferase activity TREMBL:Q8N1G4 Hypothetical protein KIAA1185 (1) Contains Leucine‐rich repeats (LRRs) TREMBL:Q9ULF3 Hypothetical protein KIAA1267 TREMBL:Q9ULE1 Hypothetical protein KIAA1279 (1) TREMBL:Q96EF4 Hypothetical protein KIAA1360 (2) ATP binding/protein kinase activity inferred from electronic annotation TREMBL:Q9HCD6 Hypothetical protein KIAA1636 Contains 11 ANK and 3 TPR protein‐ protein interaction motifs TREMBL:Q96JP2 Hypothetical protein KIAA1783 (2) TREMBL:Q96JN1 Hypothetical protein KIAA1794 (2) TREMBL:Q96EK9 Hypothetical protein MGC20419 (1) ENSEMBL:ENSP00000326603 hypothetical protein XP_371105 (1) TREMBL:Q9Y6Y3 IDN3‐B protein SWISS‐PROT:O95163 IkappaB kinase complex‐associated protein TREMBL:Q13572 Inositol 1,3,4‐trisphosphate 5/6‐kinase SWISS‐PROT:Q13418 Integrin‐linked protein kinase 1 (1) Focal adhesion protein component of the complex ILK‐PINCH TREMBL:Q9UG39 KIAA0650 protein (2) TREMBL:Q9Y2L0 KIAA1007 protein (2) TREMBL:Q86VH1 KIF27A (1) TREMBL:Q9C0F5 kinesin family member 21A (2) Contains WD repeats SWISS‐PROT:P52732 Kinesin‐like protein KIF11 (2) SWISS‐PROT:P02545 Lamin A/C (2) TREMBL:Q9BTT6 LANO adaptor protein SWISS‐PROT:Q9UQ13 Leucine‐rich repeat protein SHOC‐2 (1) SWISS‐PROT:Q13136 Liprin‐alpha 1, Protein tyrosine Regulates the disassembly of focal phosphatase receptor type f adhesions polypeptide‐interacting protein alpha 1 TREMBL:Q96IA3 LOC90268 protein (1) SWISS‐PROT:P40121 Macrophage capping protein (2) SWISS‐PROT:Q14168 MAGUK p55 subfamily member 2 SWISS‐PROT:P45985 MAP kinase 4 Involved in Signal transduction SWISS‐PROT:P36507 MAPKK 2 (1) SWISS‐PROT:Q96TA1 Meg 3 (2) SWISS‐PROT:Q9UNF1 Melanoma‐associated antigen D2 (1) TREMBL:Q14244 Microtubule associated protein SWISS‐PROT:Q96PK2 Microtubule‐actin crosslinking factor 1, isoform 4 SWISS‐PROT:Q9NU22 Midasin (2) ATPase and chaperone TREMBL:Q86VM3 MYB‐binding protein 1A SWISS‐PROT:Q09666 Neuroblast differentiation associated protein AHNAK (2) SWISS‐PROT:O60443 Nonsyndromic hearing impairment protein 5 (2) TREMBL:Q96PC7 NOPAR2 TREMBL:Q8NF92 NP220 (2) Has Zn finger and RRM SWISS‐PROT:P57740 Nuclear pore complex protein Nup107 SWISS‐PROT:P49790 Nuclear pore complex protein Nup153 (1) SWISS‐PROT:O75694 Nuclear pore complex protein Nup155

(2) SWISS‐PROT:Q8N1F7 Nuclear pore complex protein Nup93 TREMBL:Q9UHZ9 Nuclear protein SDK3 SWISS‐PROT:Q9BZE4 Nucleolar GTP‐binding protein (1) SWISS‐PROT:Q8NFH4 Nucleoporin Nup37 (1) TREMBL:Q96T89 OSA1 nuclear protein (2) Interacts with Brahma chromatin‐ remodeling complex. TREMBL:O95747 OSR1 (2) Has protein serine/threonine kinase activity TREMBL:Q99570 p150 (1) ATP binding, protein kinase activity, protein amino acid phosphorylation TREMBL:Q9BTU2 p30 DBC protein (2) TREMBL:Q9H245 Parafibromin (2) Tumor‐suppressor gene TREMBL:Q7Z5G2 Phosphatidylinositol 3‐kinase‐related protein kinase (1) SWISS‐PROT:P08237 Phosphofructokinase (2) SWISS‐PROT:P48059 PINCH protein (2) Focal adhesion protein part of the complex ILK‐PINCH, functions in regulation of cell survival, cell proliferation and cell differentiation TREMBL:Q99738 Pinin (2) SWISS‐PROT:Q9Y446 Plakophilin 3 (1) Belongs to the beta‐catenin family SWISS‐PROT:Q15149 Plectin (2) REFSEQ_NP:NP_958784 plectin 1, intermediate filament binding protein 500kDa isoform 8 (2) SWISS‐PROT:P30153 PP2A alpha (2) Coordinates the assembly of the catalytic subunit and a variable regulatory B subunit TREMBL:Q8IZZ7 Proline‐rich protein 73 (1) SWISS‐PROT:Q9H939 Proline‐serine‐threonine phosphatase‐ interacting protein 2 SWISS‐PROT:Q02297 Pro‐NRG1 SWISS‐PROT:P42655 Protein kinase C inhibitor protein‐1 Multifunctional regulator of the cell KCIP‐1 signaling processes mediated by both kinases. SWISS‐PROT:Q16513 Protein kinase C‐like 2 (2) TREMBL:Q9BQI8 Putative 40‐2‐3 protein (1) SWISS‐PROT:Q9NTK5 Putative GTP‐binding protein PTD004 (1) SWISS‐PROT:Q96T37 Putative RNA‐binding protein 15 (1) TREMBL:Q9NVL3 Putative RNA‐binding protein Luc7‐like Contains SR motif domain 2 (1) TREMBL:Q9NYH0 Putative serine‐rich protein (2) Binds double stranded RNA TREMBL:Q9Y4B3 R29828_1 (1) TREMBL:Q9H0H5 Rac GTPase activating protein 1 (2) SWISS‐PROT:P46940 Ras GTPase‐activating‐like protein GTPase inhibitor/activator, signal IQGAP1 (2) transduction TREMBL:Q8IY67 RAVER1 TREMBL:Q7Z6H7 RCD‐8 Contains WD40 motifs SWISS‐PROT:Q13123 Red protein (1) Chromatin binding SWISS‐PROT:P28702 Retinoic acid receptor RXR‐beta TREMBL:O75116 Rho‐associated protein kinase 2 (2) Promotes formation of focal adhesion complexes SWISS‐PROT:Q9BXT8 RING finger protein 17 TREMBL:Q96NV9 RIO kinase 1 isoform 1 (1) TREMBL:Q96DC1 RME8 protein (1) DnaJ homolog subfamily C member 13 TREMBL:O94803 RNA binding protein (1) SWISS‐PROT:Q9UKM9 RNA‐binding protein Raly (2) Contains RRM motif TREMBL:Q99628 Ro ribonucleoprotein‐binding protein 1 (1) TREMBL:Q96HM2 Sarcoma antigen NY‐SAR‐95 (1) TREMBL:Q96RG0 Ser/Thr protein kinase PAR‐1Balpha SWISS‐PROT:Q9Y6E0 Serine/threonine protein kinase 24 Functions in signal transduction SWISS‐PROT:O96013 Serine/threonine‐protein kinase PAK 4 (2) TREMBL:Q96IR9 Shroom‐related protein (2) TREMBL:Q9Y266 SIG‐92 (2)

SWISS‐PROT:P09132 Signal recognition particle 19 kDa protein (1) SWISS‐PROT:P13624 Signal recognition particle 54 kDa protein (2) SWISS‐PROT:Q9UHB9 signal recognition particle 68 kDa (2) SWISS‐PROT:O76094 signal recognition particle 72 kDa (2) TREMBL:Q9H3U8 SMAP‐1b (2) TREMBL:Q8TDL7 SPATA5 protein (2) ATP/nucleotide binding SWISS‐PROT:Q01082 Spectrin beta chain (2) SWISS‐PROT:Q14247 Src substrate cortactin (1) may function in cellular growth regulation and transformation through tyrosine phosphorylation in transformed cells TREMBL:Q9H2K7 STE20‐like kinase (1) SWISS‐PROT:Q9H4L4 SUMO‐1 specific protease 3 (SENP3) Releases UBL1, SMT3H1 and SMT3H2 monomers from sumoylated substrates TREMBL:Q8TAQ2 SWI/SNF related SWISS‐PROT:Q92797 Symplekin (2) telomeric poly(ADP‐ribose) SWISS‐PROT:Q9C0C2 TAB182 polymerase SWISS‐PROT:Q9Y490 Tallin 1 (2) Focal adhesion component SWISS‐PROT:Q9Y4G6 Tallin 2 Targets PIP5K1C to focal adhesion plaques and strongly activates its kinase activity SWISS‐PROT:P17987 T‐complex protein 1, alpha subunit (2) Chaperone, involved in the folding of proteins upon ATP hydrolysis SWISS‐PROT:P78371 T‐complex protein 1, beta subunit (2) SWISS‐PROT:P50991 T‐complex protein 1, delta subunit (2) T‐complex protein 1, epsilon subunit SWISS‐PROT:P48643 (2) SWISS‐PROT:Q99832 T‐complex protein 1, eta subunit (2) T‐complex protein 1, gamma subunit SWISS‐PROT:P49368 (2) SWISS‐PROT:P50990 T‐complex protein 1, theta subunit (2) SWISS‐PROT:P40227 T‐complex protein 1, zeta subunit (2) SWISS‐PROT:Q9NVV9 THAP domain protein 1 SWISS‐PROT:P42166 Thymopoietin (2) Involved in the structural organization of the nucleus and in the post‐mitotic nuclear assembly TREMBL:Q8N532 TUBA6 protein (1) ENSEMBL:ENSP00000318197 Tubulin alpha‐3/alpha‐7 (1) SWISS‐PROT:P05215 Tubulin alpha‐4 chain (2) SWISS‐PROT:Q9BQE3 Tubulin alpha‐6 chain SWISS‐PROT:P05217 Tubulin beta‐2 chain (2) SWISS‐PROT:Q13509 Tubulin beta‐4 chain (1) SWISS‐PROT:P05218 Tubulin beta‐5 chain (2) SWISS‐PROT:P23258 Tubulin gamma‐1 chain (2) REFSEQ_NP:NP_006073 tubulin, alpha, ubiquitous (2) SWISS‐PROT:Q03169 Tumor necrosis factor, alpha‐induced protein 2 SWISS‐PROT:Q12792 twinfilin isoform 1, Protein tyrosine kinase 9 (2) TREMBL:Q13079 Ubinuclein SWISS‐PROT:Q9NYL9 Ubiquitous tropomodulin SWISS‐PROT:P11172 UMP synthase Unc‐112 related protein 1, Kindlerin SWISS‐PROT:Q9BQL6 Component of focal adhesions (2) SWISS‐PROT:Q9H0A0 UPF0202 protein Nuclear and nucleolar localisation SWISS‐PROT:P50552 VASP (1) Focal adhesion protein; binds actin and profiling; microfilament‐ associated SWISS‐PROT:Q00341 Vigilin (2) Functions in cell sterol metabolism SWISS‐PROT:P08670 Vimentin (2) SWISS‐PROT:Q9BV38 WD‐repeat protein 18 (2) TREMBL:Q96P11 Williams Beuren syndrome critical region 20A (2) TREMBL:Q96SQ6 WIZ protein (1) Nucleic acid and zinc ion binding TREMBL:O95001 WUGSC:H_267D11.3 protein (1) SWISS‐PROT:P49750 ZAP3 Contains PP1 binding motif (RvxF). Potential regulator of protein phosphatase activity

Supplementary Table legends

Table I

Proteins involved in gene expression. All the proteins marked (1) in the Protein Name column of this table were identified only in the peak fractions from the anti-SKIP antibody column and the proteins marked (2) were identified in multi-protein complexes purified from the anti-SKIP antibody and the anti-CDC5L antibody columns. Unmarked proteins were identified only in the fractions from the anti-CDC5L antibody column.

(A) proteins involved in RNA processing and hnRNPs

(B) proteins involved in gene transcription

(C) proteins involved in protein translation

Table II

Proteins that function in the cell division cycle. The proteins marked (1) in the Protein

Name column were identified only in the peak fractions from the anti-SKIP antibody column whereas the proteins marked (2) were identified in protein complexes purified from the anti-SKIP antibody and the anti-CDC5L antibody columns. Unmarked proteins were identified only in the fractions from the anti-CDC5L antibody column.

Table III

Proteins involved in intracellular transport. The proteins marked (1) in the Protein Name column were identified only in the peak fractions from the anti-SKIP antibody column and the proteins marked (2) were identified in protein complexes purified from the anti-

SKIP antibody and the anti-CDC5L antibody columns. Unmarked proteins were identified only in the fractions from the anti-CDC5L antibody column.

Table IV

Proteins with other cellular functions. The proteins marked (1) in the Protein Name column of the table were identified only in the peak fractions from the anti-SKIP antibody column and the proteins marked (2) were identified in complexes purified from the anti-SKIP antibody and the anti-CDC5L antibody columns. Unmarked proteins were identified only in the fractions from the anti-CDC5L antibody column.

(A) DExH/DexD helicases

(B) Proteins involved in DNA damage repair

(C) Proteins that function in gene replication

(D) Proteins involved with in protein ubiquitination and the cellular proteasome

machinery

Table V

Other proteins identified in the proteomic analysis of nuclear complexes containing

CDC5L and SKIP. The proteins marked (1) in the Protein Name column of this table were identified only in the peak fractions from the anti-SKIP antibody column and the proteins marked (2) in the same column were identified in complexes purified from the anti-SKIP antibody and the anti-CDC5L antibody columns. Unmarked proteins were identified only in the fractions from the anti-CDC5L antibody column.