Functional association of human Ki-1 ⁄ 57 with pre-mRNA splicing events Gustavo C. Bressan 1,2 , Alexandre J. C. Quaresma 1 , Eduardo C. Moraes 1,2 , Adriana O. Manfiolli 3 , Dario O. Passos 1 , Marcelo D. Gomes 3 and Jo ¨ rg Kobarg 1,2 1 Laborato ´ rio Nacional de Luz Sı ´ ncrotron, Campinas, SP, Brasil 2 Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brasil 3 Departamento de Bioquı ´ mica e Imunologia, Faculdade de Medicina de Ribeira˜o Preto da Universidade de Sa˜ o Paulo, Ribeira˜ o Preto, Brasil Keywords Arg methylation; nuclear bodies; protein–protein interaction; RNA binding; splicing Correspondence J. Kobarg, Laborato ´ rio Nacional de Luz Sı ´ ncrotron, Centro de Biologia Molecular Estrutural, Rua Giuseppe Ma ´ ximo Scolfaro 10.000, C.P. 6192, 13084-971 Campinas, SP, Brasil Fax: +55 19 3512 1006 Tel: +55 19 3512 1125 E-mail: jkobarg@lnls.br (Received 12 April 2009, revised 8 May 2009, accepted 13 May 2009) doi:10.1111/j.1742-4658.2009.07092.x The cytoplasmic and nuclear protein Ki-1 ⁄ 57 was first identified in malig- nant cells from Hodgkin’s lymphoma. Despite studies showing its phos- phorylation, arginine methylation, and interaction with several regulatory proteins, the functional role of Ki-1 ⁄ 57 in human cells remains to be determined. Here, we investigated the relationship of Ki-1 ⁄ 57 with RNA functions. Through immunoprecipitation assays, we verified the associa- tion of Ki-1 ⁄ 57 with the endogenous splicing proteins hnRNPQ and SFRS9 in HeLa cell extracts. We also found that recombinant Ki-1 ⁄ 57 was able to bind to a poly-U RNA probe in electrophoretic mobility shift assays. In a classic splicing test, we showed that Ki-1 ⁄ 57 can modify the splicing site selection of the adenoviral E1A minigene in a dose-dependent manner. Further confocal and fluorescence microscopy analysis revealed the localization of enhanced green fluorescent protein–Ki-1 ⁄ 57 to nuclear bodies involved in RNA processing and or small nuclear ribonucleo- protein assembly, depending on the cellular methylation status and its N-terminal region. In summary, our findings suggest that Ki-1 ⁄ 57 is probably involved in cellular events related to RNA functions, such as pre-mRNA splicing. Structured digital abstract l MINT-7041074: Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with SF2P32 (uni- protkb: Q07021)bytwo hybrid (MI:0018) l MINT-7041232: Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with SFRS9 (uni- protkb: Q13242)bypull down (MI:0096) l MINT-7041203: P80-Coilin (uniprotkb:P38432) and Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) colocalize ( MI:0403)byfluorescence microscopy (MI:0416) l MINT-7041217: SMN (uniprotkb:Q16637) and Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) colocalize ( MI:0403)byfluorescence microscopy (MI:0416) l MINT-7041189: SC-35 (uniprotkb:Q01130) and Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) colocalize ( MI:0403)byfluorescence microscopy (MI:0416) l MINT-7041169: NPM (uniprotkb:P06748) and Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) colocalize ( MI:0403)byfluorescence microscopy (MI:0416) l MINT-7041249: Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with SFRS9 (uni- protkb: O60506)bypull down (MI:0096) Abbreviations Adox, adenosine-2¢,3¢-dialdehyde; EGFP, enhanced green fluorescent protein; EMSA, electrophoretic mobility shift assay; GEMS, Gemini of coiled bodies; GST, glutathione S-transferase; hnRNP, heterogeneous nuclear ribonucleoprotein; SMN, survival of motor neurons; snRNP, small nuclear ribonucleoprotein; SR protein, Ser ⁄ Arg protein. 3770 FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS Introduction Ki-1 was the first monoclonal antibody used in the specific detection of the malignant Hodgkin and Sternberg–Reed cells in Hodgkin lymphoma [1]. It has been demonstrated that Ki-1 binds to the 120 kDa lymphocyte costimulatory molecule CD30 on the Hodgkin cell’s surface [1,2]. However, it has been noticed that there is a cross-reaction of Ki-1 with a functionally and structurally uncharacterized intracellular phosphoprotein antigen of 57 kDa, termed Ki-1 ⁄ 57 [3]. Although its relationship with Hodgkin disease has not been confirmed, initial stud- ies revealed that Ki-1 ⁄ 57 is associated with Ser ⁄ Thr protein kinase activity when isolated from tumor cells [4] and localizes to both the cytoplasm and the nucleus, where it could be found at the nuclear pores and in several nuclear structures [2]. Ki-1 ⁄ 57 was found to associate with intracellular hyaluronic acid and other negatively charged molecules in vitro, and was therefore also named hyaluronic acid-binding protein 4 [5]. Another human protein, CGI-55, shares 40.7% iden- tity and 67.4% similarity with Ki-1 ⁄ 57, suggesting that they could be paralogs and have similar or redundant functions in human cells. CGI-55 is also a nucleus ⁄ cytoplasmic shuttling protein [6] and, because it was described as a protein able to bind to the 3¢-UTR region of the mRNA encoding the type 1 plas- minogen activator inhibitor, it was also named plas- minogen activator inhibitor RNA-binding protein 1 [7]. We have recently found that Ki-1 ⁄ 57 and CGI-55 have overlapping interacting protein partners. Among them are the chromatin remodeling factor chromo-heli- case DNA-binding domain protein 3 [8], DAXX, and Topors [6,9]. This suggests that the nuclear functions of both proteins may be related to transcriptional activity. Despite the fact that these proteins share reasonable sequence similarity, Ki-1 ⁄ 57, but not CGI- 55, interacts with the transcription factor MEF2C [10], p53 [9], and the signaling ⁄ scaffold receptor of activated protein kinase C (RACK1) [11,12]. Both Ki-1 ⁄ 57 and CGI-55 mRNAs show ubiquitous expression in all human tissues tested, and elevated expression in the heart, muscle, and liver [8]. Ki-1 ⁄ 57 is also expressed at higher levels in the brain [8]. Both Ki-1 ⁄ 57 and CGI-55 interact with and are methylated by the protein arginine methyltransferase PRMT1 [13]. This enzyme is responsible for the meth- ylation of more than 85% of the cellular protein substrates [14], and targets the arginines embedded in typical Arg ⁄ Gly-rich motifs (RG ⁄ RGG ⁄ RXR) [15]. These are conserved motifs in many RNA-binding pro- teins, and have been reported to mediate RNA binding [16,17]. In previous yeast two-hybrid screenings, we found the interaction of Ki-1 ⁄ 57 with several RNA-binding proteins [9] (unpublished observations). Functionally, most of these identified RNA-binding proteins are involved in pre-mRNA splicing regulation, pointing to a role of Ki-1 ⁄ 57 in pre-mRNA splicing. Here, we show the first functional signatures for Ki-1⁄ 57 in human cells, mainly those concerning its possible involvement in mechanisms of splice regulation. Results Protein–protein association analysis In yeast two-hybrid analyses using Ki-1⁄ 57 and PRMT1 as baits, we found common RNA-binding proteins that are functionally associated with each other in the context of pre-mRNA splicing regulation (Fig. 1). The splicing proteins SF2p32, YB-1 [9] and SFRS9 (unpublished observation) were found as posi- tive prey clones when we used the N-terminus of Ki-1 ⁄ 57 as bait in our screens (Fig. 1). On the basis of the functional interconnections between the Ki-1 ⁄ 57- interacting and PRMT1-interacting proteins (Fig. 1), we reasoned that heterogeneous nuclear ribonucleo- protein hnRNPQ could also be functionally related to Ki-1 ⁄ 57. hnRNPQ has been reported to be associated with the regulation of pre-mRNA splicing [18], and has been previously found to be a novel interacting l MINT-7041065: Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with SFRS9 (uni- protkb: Q13242)bytwo hybrid (MI:0018) l MINT-7041069: Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with YB1 (uni- protkb: P67809)bytwo hybrid (MI:0018) l MINT-7041079: Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with HNRPQ (uniprotkb: O60506)bytwo hybrid (MI:0018) l MINT-7041087: Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) physically interacts (MI:0218) with HNRPQ3 (uniprotkb: O60506-1), HNRPQ2 (uniprotkb:O60506-2) and HNRPQ-1 (uniprotkb:O60506-3) by anti bait coimmunoprecipitation ( MI:0006) G. C. Bressan et al. Human Ki-1 ⁄ 57 and pre-mRNA splicing FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS 3771 partner and target for Arg methylation by PRMT1 [13,19]. Aiming to confirm the endogenous association of Ki-1 ⁄ 57 with proteins involved in splicing regulation, we performed immunoprecipitation assays from HeLa cell extracts. We confirmed such an association when we immunoprecipitated Ki-1⁄ 57 (and detected SFRRS1 ⁄ 9 and hnRNPQ isoforms) (Fig. 2A), and also when we immunoprecipitated SFRS1 ⁄ 9 or hnRNPQ isoforms (and detected Ki-1 ⁄ 57) (Fig. 2B,C), suggest- ing that these proteins might form complexes in vivo. The pan-antibody against hnRNPQ recognizes the isoforms hnRNPQ1, hnRNPQ2, and hnRNPQ3, and the antibody against SFRS1 ⁄ 9 recognizes the splicing factors SFRS9 and SFRS1. The latter is also known as SF2 ⁄ ASF, and its regulatory subunit, called SF2p32 [20], was also found to be a Ki-1 ⁄ 57-interacting part- ner (Fig. 1) [9]. Next, we performed pull-down assays with recom- binant proteins to test the interaction of Ki-1 ⁄ 57 with SFRS9 and hnRNPQ in vitro. We found that the bacu- lovirus 6· His–SFRS9 was pulled down by the bacterial glutathione S-transferase (GST)–Ki-1⁄ 57 (Fig. 2D). Similarly, the bacterial GST–hnRNPQ (1–443) was also pulled down by the bacterial 6· His– Ki-1 ⁄ 57 (Fig. 2E). These results suggest that the inter- action of Ki-1 ⁄ 57 with these splicing proteins occurs directly and specifically, as no interaction was observed with GST alone. Moreover, we also detected the three hnRNPQ iso- forms when we immunoprecipitated SFRS1 ⁄ 9, although we did not observe direct in vitro binding activity between these proteins in our experimental conditions in pull-down assays (data not shown). This suggests that these proteins may form part of the same Ki-1 ⁄ 57-associated complex, although they might not all interact directly with each other. Yeast two-hybrid mapping assays Next, we were interested in knowing the regions of Ki-1 ⁄ 57 necessary for its interaction with splicing regulatory proteins. Several N-terminal and C-terminal Ki-1 ⁄ 57 truncated forms fused to the LexA DNA- binding domain (Fig. 2F) were cotransformed with constructs encoding the Ki-1⁄ 57-interacting proteins fused to a GAL4 activation domain, to test their abil- ity to interact with each other. Only the full-length and N-terminal Ki-1 ⁄ 57 constructs were able to inter- act with the splicing proteins SFRS9, SF2p32, and YB-1 (Fig. 2G, columns 1–3). This suggests that the interaction of Ki-1 ⁄ 57 with these molecules may occur predominantly through its N-terminal region. This pattern was not verified for hnRNPQ (Fig. 2G, col- umn 4), as we only observed its interaction with the full-length Ki-1 ⁄ 57 construct. On the other hand, this finding may explain why we were not able to identify hnRNPQ in our yeast two-hybrid screens, where only the truncated forms of the N-terminus and C-terminus of Ki-1 ⁄ 57 were used as ‘baits’. RNA-binding activity of Ki-1 ⁄ 57 in vitro Although Ki-1 ⁄ 57 does not have any classic RNA-bind- ing domains in its amino acid sequence, it has several Arg ⁄ Gly-rich clusters (RGG-box) (Fig. 3A). The RGG motif’s importance for the interaction of many RNA-binding proteins with RNA has already been reported [16,17]. The two major Ki-1 ⁄ 57 RGG-boxes located at its C-terminal region are highly similar to those of its putative paralog, CGI-55 (Fig. 3A). The exact role of CGI-55 in human cells also remains unknown, but it was found to bind to the 3¢-UTR of type 1 plasminogen activator inhibitor mRNA [7]. To test whether Ki-1 ⁄ 57 also binds RNA, we per- formed electrophoretic mobility shift assays (EMSAs). We found that the recombinant GST–Ki-1 ⁄ 57 bound Fig. 1. Functional interconnections of Ki-1 ⁄ 57 with splicing regula- tory proteins through direct physical interactions or participation in common protein complexes. Black bold lines: experiments described in this article (see Results for details). Dotted lines: previ- ously published findings; PRMT1 is found in the same complex with SF2p32 [48], SFRS9 is associated with SF2p32 [20], and YB-1 and SFRS9 interact with each other [21]. Thin dotted lines: YB-1 and hnRNPQ are functionally related, as both interact with hnRNPD ⁄ AUF1 [42]; SFRS9 is highly similar in amino acid sequence to SFRS1 (SF2 ⁄ ASF). Numbers in square brackets indi- cate the respective references. The database accession numbers for the proteins shown are (in UniProt code): Ki-1 ⁄ 57, Q5JVS0; SFRS9, Q13242; hnRNPQ, O60506; YB-1, P67809; SFRS1, Q07955; SF2P32, Q07021; PRMT-1, Q99873. Human Ki-1 ⁄ 57 and pre-mRNA splicing G. C. Bressan et al. 3772 FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS to a U-rich RNA probe (Fig. 3B, lanes 3–6). This was relatively specific, as we also tested other RNA homo- polymer probes (poly-A, poly-C, and poly-G), and found no significant binding activity (data not shown). These observations suggest that the binding of Ki-1 ⁄ 57 to its putative cellular RNA targets may involve U-rich regions, instead of A-rich regions as reported for CGI-55 [7]. As we found the N-terminus to be an important region for the interaction of Ki-1 ⁄ 57 with its protein A B C E H G F D Fig. 2. Confirmation of the protein–protein interactions among Ki-1 ⁄ 57 and proteins involved in pre-mRNA splicing. (A–C) Immunoprecipita- tion assays (IP) of endogenous proteins. HeLa cell extracts were immunoprecipitated with G-Sepharose beads and the indicated antibodies. The obtained protein complexes were analyzed by western blot (WB) as indicated in the figure panels. Arrows indicate the positions of ana- lyzed proteins. WL, whole cell lysate. Immunoprecipitation with the indicated control antibodies is shown on the right side. (D, E) In vitro pull-down assays. Recombinant proteins from bacteria [GST, GST–Ki-1 ⁄ 57, GST–hnRNPQ(1–443), 6· His–Ki-1 ⁄ 57] or baculovirus (6· His–SFRS9) were loaded onto Ni 2+ –nitrilotriacetic acid (6· His-fusion) or glutathione–Sepharose beads (GST-fusion) and incubated with supernatants of cell lysates as indicated. Arrows indicate the detected proteins. Arrowheads point to the position of the control protein GST. The additional bands observed correspond to proteolysis degradation products. (F–H) Yeast two-hybrid mapping of Ki-1 ⁄ 57 regions (F) that interact with the indicated splicing proteins. Black boxes in the diagrams represent the RGG-box motifs present in the sequence of Ki-1 ⁄ 57 (see also Fig. 3A). L40 yeast cells were cotransformed with the plasmids encoding several Ki-1 ⁄ 57 truncated constructs fused to LexA and the plasmids encoding the prey proteins fused to the GAL4-activating domain (G). Protein–protein interactions were checked through analysis of reporter gene activation: b-galactosidase activity or capacity to grow on selective minimal medium (in the absence of the amino acids Trp, Leu, and His) (not shown). (H) Autoactivation control: inability of full-length Ki-1 ⁄ 57 to activate reporter genes in the absence of its interacting partners. G. C. Bressan et al. Human Ki-1 ⁄ 57 and pre-mRNA splicing FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS 3773 partners involved in splicing regulation (Fig. 2G), we further investigated which regions of Ki-1⁄ 57 were involved in binding to the poly-U RNA. We observed that the two smaller RGG-box-containing constructs 6· His–Ki-1 ⁄ 57(151–260) and 6· His–Ki-1 ⁄ 57(261– 413) were able to bind, although weakly, to the poly- U probe; however, only the larger C-terminal Ki-1 ⁄ 57(122–413) construct could achieve binding as strong as that of full-length GST–Ki-1 ⁄ 57. Hence, the C-terminal region Ki-1 ⁄ 57(122–413) seems to be necessary and sufficient for efficient interaction with the RNA poly-U (Fig. 3B, lanes 7–10). The observed ‘supershifted’ bands seemed to be stronger upon the incubation of poly-U with increasing quan- tities of Ki-1 ⁄ 57(1–413) and Ki-1 ⁄ 57(122–413) (Fig. 3B, lanes 4–10). A 25-mer poly-U molecule is large enough to bind more than one molecule of Ki-1 ⁄ 57. Owing to its low expression yield and low stability in solution, an N-terminal construct, 6· His–Ki-1 ⁄ 57(1–222), could not be tested in these EMSA experiments. Influence of Ki-1 ⁄ 57 on E1A pre-mRNA splicing in vivo The association of Ki-1 ⁄ 57 with splicing proteins pointed to a possible functional role in pre-mRNA splicing regulation. Therefore, we investigated whether Ki-1 ⁄ 57 could modulate the splicing site selection of the adenoviral E1A test minigene, previously explored for the Ki-1 ⁄ 57-interacting proteins SFRS9 and YB-1 [21]. Depending on the 5¢-splice site selection, the E1A pre-mRNA may generate five isoforms: 13S, 12S, 11S, 10S, and 9S (Fig. 4A) [22]. These isoforms can be monitored by RT-PCR followed by agarose gel analy- sis, where the intensity of each band in the gel directly correlates with the splicing site selection, which, in turn, reflects the positive or negative influence of regu- latory proteins [22,23]. We transiently cotransfected the encoding E1A mini- gene plasmid with increasing amounts of vectors expressing the recombinant enhanced green fluorescent protein (EGFP)–Ki-1 ⁄ 57 in COS7 cells. We observed a significant effect of EGFP–Ki-1 ⁄ 57 in modifying the pattern of splicing of E1A mRNA in comparison with empty pEGFP vector (Fig. 4B). Expression of EGFP– Ki-1 ⁄ 57 leads to formation of the 10S and 9S iso- forms, concomitantly with a reduction of 13S isoform formation, in a dose-dependent way (Fig. 4B, lanes 2–4). This finding strongly suggests the functional involvement of Ki-1 ⁄ 57 in regulatory mechanisms of pre-mRNA splicing. Although we also observed a significant modification of the E1A mRNA splicing pattern by Ki-1 ⁄ 57(1–222) and Ki-1 ⁄ 57(122–413), respectively, it only occurred at the highest plasmid concentrations used (Fig. 4C,D). Moreover, the effects seemed to be isoform specific for each Ki-1 ⁄ 57 region, as the formation of 10S mRNA was only increased by the C-terminal region of Ki-1 ⁄ 57 although with a lower efficiency in compari- son with the full length protein (Fig. 4C–E). The influ- ence of the C-terminal construct Ki-1 ⁄ 57(122–413) on the generation of the 9S mRNA was also more pronounced (Fig. 4F). A B Fig. 3. GST–Ki-1 ⁄ 57 binds poly-U RNA in vitro. (A) Schematic view of the RGG-box motif localization in the sequence of Ki-1 ⁄ 57 and its paralog CGI-55 (database accession number: Q8NC51). (B, C) EMSA results. (B) Interaction of Ki-1 ⁄ 57 with poly-U RNA. On top of the panel: the three different truncated versions of recombinant Ki-1 ⁄ 57 used in the mapping experiments. Increasing concentra- tions (0.5, 1, 2 and 4 m M, respectively) of the recombinant protein GST–Ki-1 ⁄ 57 and the three 6· His-fused truncated versions of Ki-1 ⁄ 57 (122–413, 151–260, and 261–413) were incubated with the 32 P-labeled poly-U probe (25-mer) and subjected to native polyacryl- amide gel (10% gel, with a 29:1 acrylamide:bis-acrylamide ratio) electrophoresis. The arrow indicates shifted bands and the dashed arrowheads indicate supershifted bands. Human Ki-1 ⁄ 57 and pre-mRNA splicing G. C. Bressan et al. 3774 FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS A B CE F D Fig. 4. Ki-1 ⁄ 57 influences the splicing pattern of the E1A pre-mRNA. (A) Diagram showing the splicing events that generate the 13S, 12S, 10S and 9S mRNAS of the E1A reporter gene [22,23]. (B–D, G) In vivo splicing assays. COS7 cells were transiently cotransfected with an E1A minigene encoding plasmid, an empty pEGFP vector and increasing amounts (1·,5lg; 2·,10lg; 3·,15lg) of pEGFP-Ki-1 ⁄ 57 (full length) or pEGFP–Ki-1 ⁄ 57(1–222) and pEGFP–Ki-1 ⁄ 57(122–413) vectors. The empty pEGFP vector was used to keep constant the DNA con- centration in each transfection. Splicing activity quantization was performed as described in Experimental procedures. The displayed figures are representative of at least three independent experiments. Vertical bars in the graphs indicate ± standard deviation. Wherever it exists, the significance of the difference relative to the control (empty pEGFP vector alone; line 1) is indicated by *P < 0.05. (B–D) Influence of the overexpression of full-length Ki-1 ⁄ 57 (B) and its N-terminal (C) or C-terminal (D) constructs on E1A splice site selection. Essentially the same results were obtained in HEK293 cells and when we used a flag-tagged construct of Ki-1 ⁄ 57 (data not shown). (E, F) Treatment ⁄ control band intensity ratios – comparison of the splicing site selection efficiency of Ki-1 ⁄ 57 and its N-terminal or C-terminal truncated forms. The average of band intensity values obtained for the isoforms 10S (E) or 9S (F) in (B), (C) and (D) in comparison to the average of the intensities in the control samples were plotted in the graphs, and represent the fold induction of each isoform in relation to the control. We achieved approxi- mately 60% transfection efficiency in all experiments performed. Open circles, unspliced pre-mRNA; M, marker. G. C. Bressan et al. Human Ki-1 ⁄ 57 and pre-mRNA splicing FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS 3775 Effect of SFRS9 on E1A pre-mRNA splicing in the presence of Ki-1 ⁄ 57 SFRS9 and many other Ser ⁄ Arg proteins (SR proteins) are well known as regulators of E1A pre-mRNA splic- ing [21,24]. Seeking for a possible functional influence of Ki-1 ⁄ 57 on SFRS9 splicing activity, we performed splicing assays in which both proteins were coex- pressed in COS-7 cells. When we cotransfected the construct EGFP–SFRS9 alone with the pMTE1A vector, we observed a strong inhibitory effect on the formation of the 12S and 10S mRNAs (Fig. 5, lanes 2 and 3), but, similarly to what was found for EGFP–Ki-1 ⁄ 57, we also observed stimu- latory activity in generating the 9S isoform (Fig. 5, lanes 2 and 3). This finding may suggest that although both proteins may act together in selecting the most distal splice site region that generates the 9S isoform, they can also be involved in different regulatory splic- ing mechanisms, as EGFP–Ki-1 ⁄ 57 has an opposite stimulatory activity in generating the 10S isoform in comparison with SFRS9, which is inhibitory. Interest- ingly, upon adding increasing amounts of EGFP– Ki-1 ⁄ 57 we consistently observed that the inhibitory effect of EGFP–SFRS9 in selecting the 10S isoform can be partially reversed. Colocalization analysis of Ki-1 ⁄ 57 to nucleoli and splicing speckles We have previously shown that, upon treatment with the inhibitor of methylation adenosine-2¢,3¢-dialdehyde (Adox), the endogenous Ki-1 ⁄ 57, instead of showing a uniform nuclear ⁄ cytoplasmic distribution, relocalizes predominantly to the nucleus, where it appears as nuclear dots [13]. As the results that we present here pointed to involvement of Ki-1 ⁄ 57 with RNA-binding proteins related to RNA ⁄ mRNA processing, and as most of the so far characterized nuclear subdomains are sites for RNA maturation and processing [25], we decided to investigate, through confocal microscopy analysis, the identity of the nuclear substructures where Ki-1 ⁄ 57 is present in Adox-treated cells. We tested two lineages of adherent cells, COS7 and HEK293, and found, in both of them, that upon Adox treatment the recombinant EGFP–Ki-1⁄ 57 displayed similar nuclear relocalization, at several dots, as displayed by the endogenous Ki-1 ⁄ 57 in HeLa cells [13] (data not shown). We then decided to use the recombinant EGFP-fused form of Ki-1 ⁄ 57 in our con- focal analysis, mainly because of the insufficient qual- ity of the images obtained by labeling the endogenous Ki-1 ⁄ 57 with monoclonal antibodies. We noticed that Fig. 5. Effect of Ki-1 ⁄ 57 on SFRS9 activity. COS7 cells were transiently cotransfected with an E1A minigene-encoding plasmid [21,23], an empty EGFP vector, and increasing amounts of pEGFP vectors encoding full-lengths constructs for SFRS9 or Ki-1 ⁄ 57 (1·,4lg; 2·,8lg; 3·, 12 lg). The empty pEGFP vector was used to keep constant the DNA concentration in each transfection. Splicing activity quantization was performed as described in Experimental procedures. The displayed figures are representative of at least three independent experiments. Ver- tical bars in the graphs indicate ± standard deviation. Wherever it exists, the significance of the difference relative to the control (empty pEGFP vector alone; line 1) is indicated by *P < 0.05. Lanes 2 and 3 display the activity of SFRS9 alone, whereas lines 4–6 (darker gray bars) show the effect of the increasing amounts of Ki-1 ⁄ 57. The white triangle indicates that the value plotted for the 10S isoform in line 6 is dif- ferent (P < 0.05) to that in line 4. We achieved approximately 60% transfection efficiency in all experiments performed. Open circles, unsp- liced pre-mRNA; M, marker. The expression of cotransfected Ki-1 ⁄ 57 and SFRS9 was controlled by RT-PCR and is shown in Fig. S1. Human Ki-1 ⁄ 57 and pre-mRNA splicing G. C. Bressan et al. 3776 FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS the most evident dot-forming Ki-1 ⁄ 57 in the nuclei of Adox-treated cells seemed to be related to nucleoli, mainly because of the well-known large area that this structure occupies in the cell nucleus. Although EGFP–Ki-1 ⁄ 57 shows a diffuse distribu- tion throughout the nucleus, it showed a stronger signal that colocalizes with the staining of the nucleoli marker nucleophosmin (Fig. 6Aiii) in Adox-treated cells. This suggests that the methylation status of Ki-1 ⁄ 57 is important for its colocalization to this nuclear subcompartment. Besides the larger, nucleolar-associated bodies, we also observed, in the Adox-treated cell nuclei, several small dot-forming Ki-1 ⁄ 57 domains. Owing to the interaction of Ki-1 ⁄ 57 with proteins associated with pre-mRNA splicing, a plausible hypothesis would be that these regions corresponded to nuclear speckles, which are nuclear substructures known to be enriched in small nuclear ribonucleoprotein (snRNP) and many other transcription-related and pre-mRNA splicing- related proteins [25,26]. To investigate this possibility, we studied Ki-1 ⁄ 57’s colocalization with the SR pro- tein SC-35, a marker protein for splicing speckles [27]. Despite the diffuse distribution of Ki-1 ⁄ 57 in the nucleus, we saw partial colocalization with the SC-35 dots in nontreated cells (Fig. 6Bjjj). In Adox-treated cells, we noticed, however, a juxtaposition of the EGFP–Ki-1 ⁄ 57 and SC-35 nuclear substructures (Fig. 6Biii). This is an indication that the cellular methylation status has specific effects on the localiza- tion of EGFP–Ki-1 ⁄ 57 among different subnuclear compartments. Colocalization of EGFP–Ki-1 ⁄ 57 with Cajal and Gemini of coiled bodies (GEMS) nuclear bodies The partial colocalization of EGFP–Ki-1 ⁄ 57 with splicing speckles in untreated control cells has led us to test antibodies against molecular marker proteins for Cajal bodies and GEMS (Gemini of coiled bodies), both of which are considered to be nuclear compart- ments involved in snRNP storage and ⁄ or in the assem- bly of pre-mRNA splicing complexes [25]. Interestingly, we found, through confocal analyses, that EGFP–Ki-1 ⁄ 57 was again localized in a diffusive fashion throughout the nucleoplasm, but showed stronger spotted staining that colocalized with the Cajal body protein marker p80-coilin in the nucleus of HEK293 cells treated with Adox (Fig. 7Ai–iii). This finding may, in addition, strengthen the hypothesis of the involvement of Ki-1 ⁄ 57 in pre-mRNA processing events. The GEMS are regions enriched with the survival of motor neurons (SMN) protein complexes and are con- sidered to be Cajal body-like domains [25]. Although they may be found as distinct structures, they can also be found colocalized. This suggests that they are functionally related [28,29]. However, an interesting particularity of the SMN protein is its demand for the presence of Arg ⁄ Gly-rich regions in most of its inter- Fig. 6. Localization of Ki-1 ⁄ 57 to nucleoli and splicing speckles. HEK293 cells were transfected with EGFP–Ki-1 ⁄ 57 and treated or not treated with the methylation inhibitor Adox. After fixation, the cells were immuno- stained with the antibodies against the nuclear proteins nucleophosmin (NPM; marker protein of nucleoli) or SC-35 (marker protein of speckles), and analyzed by laser- scanning confocal microscopy. (A) Partial colocalization of EGFP–Ki-1 ⁄ 57 to nucleoli (nucleophosmin) in Adox-treated cells (Aiii), but not in the control cells (Ajjj). (B) Partial colocalization of EGFP–Ki-1 ⁄ 57 to speckles (SC-35) only in the control cells (Bjjj). The Adox treatment seems to cause only a close juxtaposition between the speckles and the Ki-1 ⁄ 57-associated substructure (Biii, inset). Bars: 5 lm. Figure insets emphasize colocalizations or close juxtaposi- tion of structures. G. C. Bressan et al. Human Ki-1 ⁄ 57 and pre-mRNA splicing FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS 3777 acting partners [30], suggesting the possible requirement of Arg methylation for these protein–protein associa- tions. Through confocal analyses, we observed the partial colocalization of Ki-1⁄ 57 with SMN ⁄ GEMS in Adox-treated cells (Fig. 7Biii). Despite the diffuse nuclear distribution of EGFP–Ki-1 ⁄ 57, the majority of the red GEMS spots coincide with spots of brighter EGFP–Ki-1 ⁄ 57 staining (Fig. 7Bi–ii). This finding unveils a novel nuclear body with which Ki-1 ⁄ 57 is associated in HEK293 cells treated with Adox. We, like others before us, observed also that Cajal bodies colocalize with nucleoli in some cells [31] (not shown). Subcellular localization of Ki-1 ⁄ 57 truncated forms In order to obtain further clues on the functions of different regions of the Ki-1 ⁄ 57 amino acid sequence in human cells, we fused to EGFP the various trun- cated forms used in the mapping studies described before (Figs 2G, 3B and 4C–D). Through fluorescence microscopy analysis of transfected HEK293 cells, we observed that all tested C-terminal constructs showed similar nuclear and cytoplasmic localization as observed for full-length Ki-1 ⁄ 57 (Fig. 8D–F). In turn, the N-terminal construct displayed an exclusively nuclear localization that, after careful analysis, could be found in a few regions consisting of nuclear bodies (Fig. 8C). This may suggest that the targeting of Ki-1 ⁄ 57 to nuclear subdomains requires its N-terminal region. On the other hand, when we treated the HEK293 cells with Adox, we observed a small but sig- nificant change in the localization of the C-terminal construct. In the majority of analyzed cells Ki-1 ⁄ 57(122–413) was seen more predominantly in the nuclear compartment, in contrast to the diffusely nuclear ⁄ cytoplasmic distribution observed in control cells (compare panels I and D in Fig. 8). It is interest- ing to observe that, upon Adox treatment, the N-ter- minal construct showed pronounced relocalization from the nucleoplasm to several well-defined nuclear bodies (Fig. 8H), as observed for the full-length Ki-1 ⁄ 57 construct (Fig. 8G). More than 90% and 98% of the cells transfected with full-length EGFP–Ki-1 ⁄ 57 (Figs 6 and 7) and with EGFP–Ki-1 ⁄ 57(1–222) (not shown), respectively, were found in the nucleus at nuclear substructures upon Adox treatment (Fig. 8L). This suggests that the C-terminus of Ki-1 ⁄ 57 is not a required region for its association with these nuclear subdomains, and therefore suggests that some signal for localization control may exist at the N-terminus. This localization may occur via protein–protein inter- actions, as suggested by our mapping results with the Ki-1 ⁄ 57-interacting proteins involved in pre-mRNA splicing (Fig. 2G). Discussion We here describe Ki-1 ⁄ 57 as a novel human protein that is functionally related to regulatory events of pre- mRNA processing. From a wider point of view, the Fig. 7. EGFP–Ki-1 ⁄ 57 is found in Cajal bodies and GEMS upon Adox treatment. HEK293 cells transfected with EGFP– Ki-1 ⁄ 57 were treated or not treated with the inhibitor of methylation Adox. The fixed cells were stained with antibodies against p80-coilin, which label Cajal bodies (A), or against SMN, a marker for GEMS (B), and analyzed by laser-scanning confocal microscopy. Bars: 5 lm. Figure insets focus on colocalizations or point out high- magnification images of the selected structures. Human Ki-1 ⁄ 57 and pre-mRNA splicing G. C. Bressan et al. 3778 FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS involvement of Ki-1⁄ 57 in modulating the splicing site selection of the E1A pre-mRNA reported here may unify its roles in the two main functional ‘worlds’ of the nuclear context: RNA metabolism and trans- criptional regulation. It is well known that in eukary- otes, transcription and pre-mRNA maturation events (5¢-capping, splicing, 3¢-end processing and polyadeny- lation) occur cotranscriptionally and that the machin- eries responsible for these activities are functionally and physically associated [31,32]. Furthermore, it could be speculated that not only the processing ⁄ maturation, but also the expression, of some subsets of mRNA may be regulated by Ki-1 ⁄ 57 in a defined cellular context. Several of the identified Ki-1 ⁄ 57-interacting proteins are involved in transcriptional control, such as CHD3, RACK1, p53 and others p53-associated proteins [8,9,11], thereby reinforcing a putative tran- scriptional regulation role for Ki-1 ⁄ 57. Here, as well as in a previous study, Ki-1 ⁄ 57 has been observed as dot-like structures in the cell nucleus [2,13]. Growing evidence points to important roles of these nuclear subdomains, not only as storage spaces but also as dynamic structures involved in RNA tran- scription, processing, and maturation [25,27,33,34]. We further showed the importance of methylation for its localization at distinct nuclear ‘spots’, and showed that in control cells, but not in Adox-treated cells, EGFP– Ki-1 ⁄ 57 partially localizes to nuclear speckles, whereas in Adox-treated cells it partially localizes to GEMS, Cajal bodies, and nucleoli. The nuclear speckles are known to be storage places for pre-mRNA splicing complexes and, in turn, Cajal bodies and GEMS are regions involved in snRNA modification, snRNP bio- genesis, and trafficking of small nucleolar RNPs (small nucleolar RNPs) ⁄ snRNPs to nucleoli or speckles, respectively [25]. Therefore, the methylation process may be an important step in the migration of Ki-1 ⁄ 57 in ‘assembled’ RNP complexes at Cajal ⁄ GEMS bodies to nuclear speckles, from where they could be recruited to transcriptionally active sites, which are regions that are likely to involve synchronous splicing activity. Interestingly, the methylation effects on snRNP assembly in both cytoplasmic and nuclear phases have been demonstrated in other studies. Gonsalvez et al. [35] have shown that in cells treated with the methyla- tion inhibitor 5¢-deoxy-5¢-(methylthio)adenosine, the process of snRNP assembly in the cytoplasmic compart- ment is disrupted. Similarly, the Arg methylation of the 100 50 0 Nuclear - with dots Number of adox treated cells (%) Adox + Control Nuclear/Cytopl. - without dots n = 3 Ki-1/57 Ki-1/57(1–222) Ki-1/57(1–413) B A L CDEF GH I JK Ki-1/57(1–222) Ki-1/57(122–413) Ki-1/57(151–263) Ki-1/57(264–413) Fig. 8. The localization of Ki-1 ⁄ 57 to nuclear bodies depends on its N-terminal region. (A) Schematic representations of the truncated Ki-1 ⁄ 57 constructs fused to EGFP, used for the localization assays in untreated or Adox- treated HEK293 cells. (B–F) Untreated cells. The full-length EGFP–Ki-1 ⁄ 57 and its C-ter- minal constructs (122–413), (151–263) and (264–413) show a diffuse nuclear and cyto- plasmic localization (B, D–F), whereas the N-terminal construct (1–222) shows an exclusively nuclear localization (C), at dis- crete nuclear dots (white arrowheads). (G–L) Adox-treated cells. The full-length EGFP–Ki- 1 ⁄ 57 and its N-terminal construct (1–222) predominantly show nuclear localization (G, H), at several nuclear bodies (white arrow- heads). A small but significant amount of nuclear relocalization can be observed for the C-terminal construct (122–413) (I). No changes were observed for the smaller C- terminal constructs (151–263) and (264–413) (J, K). (L) Proportion of Adox-treated cells containing nuclear bodies. More than 90% and 98% of the cells transfected with full- length EGFP–Ki-1 ⁄ 57 and EGFP–Ki-1 ⁄ 57 (1–222), respectively, were found in the nucleus at several dots. Approximately 100 cells were analyzed in each of three inde- pendent experiments. G. C. Bressan et al. Human Ki-1 ⁄ 57 and pre-mRNA splicing FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS 3779 [...]... N-terminus of Ki-1 ⁄ 57 seems to functionally mediate protein–protein 3780 interactions with splicing proteins and that the RG-box-containing C-terminus seems to mediate interactions with RNA is also very interesting; especially in light of our results obtained with the protein deletions in the splice assay The protein with the N-terminal deletion and that with the C-terminal deletion are less functional. . .Human Ki-1 ⁄ 57 and pre-mRNA splicing G C Bressan et al Sm proteins in the nucleus seemed to be important for subnuclear targeting of snRNPs or for the regulation of pre-mRNA splicing [36] Moreover, the methylation has been shown to be important for the subnuclear relocalization of other proteins not related to snRNPs, including MRE11, which... probably a novel human protein involved in mechanisms related to RNA metabolism, such as pre-mRNA splicing Further studies are necessary to dissect the molecular mechanisms underlying the regulatory effect of Ki-1 ⁄ 57 in pre-mRNA splicing, as well as to unveil its putative endogenous pre-mRNA targets Experimental procedures Plasmids and yeast two-hybrid interaction analysis Cloning of the complete... JF, Stamm S, Helfman DM & Krainer AR (1994) Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors Science 265, 1706–1709 ´ 24 Himmelspach M, Cavaloc Y, Chebli K, Stevenin J & Gattoni R (1995) Titration of serine ⁄ arginine (SR) splicing factors during adenoviral infection modulates E1A pre-mRNA alternative splicing RNA 1, 794–806 25 Handwerger KE & Gall JG (2006)... with the associated pre-mRNA splicing proteins SFRS9, SFp32, and YB-1 Analyzing the subcellular localizations of these truncated Ki-1 ⁄ 57 forms in HEK293 cells treated with the inhibitor of methylation Adox, we noticed that only the N-terminal construct was able to localize to nuclear bodies, similarly to what was observed with the full-length construct Therefore, the localization of the N-terminus... Production of a monoclonal antibody specific for Hodgkin and Sternberg-Reed cells of Hodgkin’s disease and a subset of normal lymphoid cells Nature 299, 65–67 2 Rohde D, Hansen H, Hafner M, Lange H, Mielke V, Hansmann ML & Lemke H (1992) Cellular localization and processing of the two molecular forms of the Hodg- FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS 3781 Human. .. Gelehrter TD (2001) Identification and cDNA cloning of a novel RNA-binding protein that interacts with the cyclic nucleotide responsive sequence in the type-1 plasminogen activator inhibitor mRNA J Biol Chem 276, 3341– 3347 Lemos TA, Passos DO, Nery FC & Kobarg J (2003) Characterization of a new family of proteins that interact with the C-terminal region of the chromatin-remodeling factor CHD-3 FEBS Lett... MD, USA) The intensities of all isoforms were summed, set as 100%, and used to normalize the intensity of each band Microscopy analyses For the subcellular localization assays, HEK293 cells were grown on glass coverslips with the required culture medium The cells were fixed with NaCl ⁄ Pi containing 2% paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked with NaCl ⁄ Pi ⁄ 2% BSA The primary... Cell Biol 6, 887–899 48 Yanagida M, Hayano T, Yamauchi Y, Shinkawa T, Natsume T, Isobe T & Takahashi N (2004) Human fibrillarin forms a sub-complex with splicing factor 2-associated p32, protein arginine methyltransferases, and tubulins alpha 3 and beta 1 that is independent of its association with preribosomal ribonucleoprotein complexes J Biol Chem 279, 1607–1614 Supporting information The following... G (2001) SMN interacts with a novel family of hnRNP and spliceosomal proteins EMBO J 20, 5443–5452 19 Passos DO, Quaresma AJC & Kobarg J (2006) The methylation of the C-terminal region of hnRNPQ (NSAP1) is important for its nuclear localization Biochem Biophys Res Commun 346, 517–525 ¨ 20 Petersen-Mahrt SK, Estmer C, Ohrmalm C, Matthews DA, Russell WC & Akusjarvi G (1999) The splicing ¨ factor-associated . experiments. Influence of Ki-1 ⁄ 57 on E1A pre-mRNA splicing in vivo The association of Ki-1 ⁄ 57 with splicing proteins pointed to a possible functional role in pre-mRNA splicing. Functional association of human Ki-1 ⁄ 57 with pre-mRNA splicing events Gustavo C. Bressan 1,2 , Alexandre J.