The information required to produce proteins is encoded within genes. In the first step of creating a protein, its gene is “transcribed” to form a pre-messenger RNA molecule (called pre-mRNA for short). Both the gene and the pre-mRNA contain regions called exons that code for protein, and regions called introns that do not. The pre-mRNA therefore undergoes a process called splicing to remove the introns and join the exons together into a final mRNA molecule that is “translated” to make the protein.

Many pre-mRNAs can be spliced in several different ways to include different combinations of exons in the final mRNA molecule. This process of “alternative splicing” allows different versions of a protein to be produced from the same gene. Changes that alter the pattern of alternative splicing in a cell affect various cellular and developmental processes and have been linked to diseases such as cancer.

The pre-mRNA transcribed from a gene called FAS can be alternatively spliced so that it either does or does not contain an exon that enables the protein to embed itself in the cell membrane. The protein produced from mRNA that includes this exon generates a cell response that leads to cell death. By contrast, protein produced from mRNA that lacks this exon is released from cells and promotes their survival. A splicing factor called RBM5 promotes the removal of this exon from FAS pre-mRNA.

RBM5 binds to some of the proteins that make up the molecular machine that splices pre-mRNA molecules. Mourão, Bonnal, Soni, Warner et al. have now used a technique called nuclear magnetic resonance spectroscopy to solve the three-dimensional structure formed when RBM5 binds to one of these proteins, called SmN. Further experiments introduced specific mutations to the proteins to investigate their effects in human cells. This revealed that mutations that impaired the association between RBM5 and SmN compromised the activity of RBM5 to regulate the alternative splicing of FAS pre-mRNA molecules.

Future research could examine how RBM5 associates with pre-mRNAs and other components of the splicing machinery, and investigate whether proteins that are closely related to RBM5 act in similar ways.

An essential step during the regulation of eukaryotic gene expression is the removal of non-coding intron sequences from pre-mRNA transcripts through the process of pre-mRNA splicing. The catalytic steps of pre-mRNA splicing are carried out by the spliceosome, a large and dynamic assembly of five small nuclear ribonucleoprotein (snRNP) complexes and more than 150 additional splicing factor proteins (Wahl et al., 2009). Many splicing factors are involved in early steps of the assembly of the spliceosome through the recognition of short regulatory RNA motifs and/or through protein-protein interactions. Alternative splicing is the mechanism by which particular intronic or exonic regions are included or excluded to produce diverse mRNAs from the same gene (Blencowe, 2006). It is thought that more than 90% of human multi-exon genes undergo alternative splicing (Pan et al., 2008; Wang et al., 2008). The genomic diversity of eukaryotic gene expression is thus greatly expanded by alternative splicing of mRNA transcripts. Often, the protein products of alternative splicing have antagonistic roles in cellular functions and are implicated in human diseases (Cooper et al., 2009). Notably, mutations in splicing factors that modulate alternative splicing decisions have been implicated in cancer (David and Manley, 2010; Bonnal et al., 2012).

A biologically important example of alternative splicing is found in the FAS gene (also known as CD95 or APO-1). The FAS gene encodes a transmembrane signaling protein that stimulates a pro-apoptotic signaling cascade upon binding of the FAS ligand at the cell surface (Krammer, 2000). Alternatively spliced FAS transcripts that exclude exon 6 encode a soluble Fas isoform that lacks the transmembrane domain. This soluble isoform can be secreted outside of the cell where it sequesters the FAS ligand and inhibits downstream activation of apoptosis (Cheng et al., 1994; Cascino et al., 1995). Thus, regulation of the alternative splicing of FAS can either stimulate or inhibit cell survival. The pro-apoptotic Fas protein plays an important role during T-lymphocyte maturation (Liu et al., 1995; Papoff et al., 1996; Van Parijs et al., 1998; Roesler et al., 2005) and additional evidence implicates this isoform in the proliferation of cancer cells (Chen et al., 2010).

A number of splicing factors have been shown to modulate FAS alternative splicing, including RBM5. The multi-domain RNA-binding protein 5 (RBM5) regulates FAS splicing by promoting skipping of exon 6 (Bonnal et al., 2008). RBM5 is a 92 kDa, multi-domain protein with an arginine-serine (RS)-rich region, two RNA Recognition Motifs (RRM1 and RRM2), two Zinc-Finger domains (ZF1 and ZF2), a C-terminal OCtamer REpeat (OCRE) domain (Callebaut and Mornon, 2005) in addition to a glycine patch, and KEKE (lysine/glutamate) repeats (Figure 1). It belongs to the family of RNA Binding Motif (RBM) proteins, including RBM6 and RBM10, which share a similar domain organization with RBM5 and have 30% and 50% amino acid identity, respectively (Sutherland et al., 2005). The RBM5 (also known as H37 and LUCA-15) and RBM6 genes are located in a chromosomal region 3p21.3, which is frequently deleted in heavy smokers and lung cancers (Oh et al., 2002; Zabarovsky et al., 2002). RBM5 is known to regulate the alternative splicing of apoptosis–related genes, such as FAS and Caspase-2 (Bonnal et al., 2008; Fushimi et al., 2008). It has also been reported to suppress metastasis by modulating the expression of Rac1, β-catenin, collagen and laminin (Oh et al., 2010). However, RBM5 has also been found to be up-regulated in some aggressive forms of breast and ovarian cancers (Oh et al., 1999; Rintala-Maki et al., 2007). The possible dual role of RBM5 in cancer progression may be linked to its various activities as splicing regulator, including differential recognition of its pre-mRNA targets.

In the regulation of FAS alternative splicing, RBM5 inhibits mature spliceosome formation by blocking association of the U4/U6.U5 tri-snRNP complex (in complex B) after the splice sites flanking exon 6 have been recognized by U1 and U2 snRNPs (Bonnal et al., 2008). RBM5 therefore appears to influence the pairing between the splice sites after complex A formation and thus promote tri-snRNP assembly at the distal splice sites, leading to exon 6 skipping (Bonnal et al., 2008). Moreover, it was shown that the OCRE domain of RBM5 interacts with components of the tri-snRNP and is essential for the function of RBM5 in regulating FAS splicing (Bonnal et al., 2008).

OCRE domains, which are present in different proteins in various organisms (Xiao et al., 2013), were identified by bioinformatic sequence analysis as a tandem of five imperfect octameric repeat sequences with triplets of aromatic residues (Callebaut and Mornon, 2005), expected to predominantly adopt β-strand secondary conformation. Due to the presence of OCRE domain in RNA binding proteins such as RBM5, they were presumed to facilitate RNA binding (Callebaut and Mornon, 2005). However, at least in the case of RBM5, the OCRE domain appears to play a role in mediating protein-protein interactions (Bonnal et al., 2008).

In order to provide molecular insights into the function of the OCRE domain in splicing regulation, we have combined structural and biochemical experiments with mutational analyses in vitro and in vivo. We show that the RBM5 OCRE domain directly binds to the spliceosomal SmN/B/B’ proteins. Sm proteins are core components of most spliceosomal snRNPs and form a heptameric ring composed of SmD1, SmD2, SmF, SmE, SmG, SmD3 and SmN/B/B’, which jointly recognize the uridine-rich Sm site RNA motif in U1, U2, U4 and U5 snRNAs (Kambach et al., 1999; Will and Lührmann, 2001; Pomeranz Krummel et al., 2009; Weber et al., 2010; Leung et al., 2011; Matera and Wang, 2014; Kondo et al., 2015). SmB and SmB' are encoded by two transcript variants from the SNRPB gene, while a different gene, highly expressed in brain and heart, encodes the homologous protein SmN (Schmauss et al., 1992). SmN/B/B’, SmD1, and SmD3 have C-terminal extensions that include symmetrically dimethylated RG repeats (Brahms et al., 2001; Tripsianes et al., 2011). The SmN/B/B’ C-terminal tails contain additional proline-rich sequences, where these regions in SmN, SmB and SmB’ are 93% homologous (van Dam et al., 1989).

Here, we show that the RBM5 OCRE domain binds to the C-terminal proline-rich motifs present in SmB and SmN. The structure of the RBM5 OCRE domain adopts a unique β-sheet fold that recognizes the proline-rich C-terminal tails of the SmN/B/B' proteins through aromatic-CH interactions. We demonstrate that disruption of these interactions by mutations in the OCRE domain or in the proline-rich motifs of its ligands decreases the affinity between SmN/B/B’ and RBM5 in vitro and affects alternative splicing regulation of the FAS gene. Our results demonstrate that OCRE is a novel protein-protein interaction domain that mediates interactions with the core spliceosome in alternative splicing regulation.

The Octamer Repeat (OCRE) domain of RBM5 adopts a unique three-dimensional fold with a large number of conserved tyrosine residues. Although the primary sequence signature could suggest a linear sequence of octamer repeats, our structural analysis clearly reveals that the OCRE domain adopts a small globular fold. We show that the twisted β-sheet of the OCRE domain exposes conserved tyrosine residues that provide a platform for the recognition of a proline rich motif (PRM). This interaction is based upon a network of tyrosine residues as well as anchoring residues located in the loops of the β-strands. The SmN PRM is flanked by a positively charged arginine residue preceding the four consecutive prolines, followed by a hydrophobic residue and another arginine. Taking into account the sequence conservation, binding studies (Figure 3), and the details of molecular recognition of the SmN peptide (Figure 4), we propose that the OCRE domain recognizes a RPPP(P)GϕR consensus motif. A key feature of the motif is a central PPII helix, which is recognized by a network of parallel and aromatic T-stacking of Tyr470, Tyr479, Tyr482, Tyr488 and Tyr495 with the proline side chains in the PPII helix. These tyrosines are exposed at one side of the OCRE β-sheet and collectively embed the PPII helix. Specific contacts that define the orientation of the bound peptide are mediated by two flanking arginine residues, with a stronger contribution of the N-terminal arginine (SmN Arg221, Figure 3B). The glycine residue may enable an unusual backbone conformation such that hydrophobic residues (ϕ, Ile/Met/Val flanking different PRMs in the SmN/B/B’ C-terminal tails, Figure 3A) can shield the central hydrophobic PPII motif from solvent exposure. Notably, unbound PRMs harboring 3–4 consecutive prolines already adopt (at least partially) a preformed PPII conformation and thus reduce the entropy loss associated with binding to the OCRE domain. The somewhat reduced affinity for peptides with only three prolines may reflect a smaller propensity of forming PPII conformation in the free ligands and the lack of a preceding arginine, which is consistently present in all four proline PRMs.

The tertiary fold and the mode of PRM recognition by the OCRE domain is distinct from other PRM binding domains, such as GYF, SH3, and WW domains (Figure 7A). Although – as a common feature – aromatic residues are used to interact with proline residues and to recognize the PPII helix (Ball et al., 2005), different secondary structure elements are found in GYF (α-helical), SH3 (α/β fold), WW (β-sheet) and OCRE domains (β-sheet). Interestingly, a somewhat related PRM motif in the CD2 protein (PPPPGHR) was reported to interact with the GYF domain of the CD2BP2 protein (Freund et al., 2002). The CD2BP2 GYF domain and the FBP21 WW domain have been previously implicated in binding to the PRM sequences of SmB, suggesting that interactions between PRMs and these factors may be critical for the function of these proteins in spliceosome regulation (Bedford et al., 1998; Klippel et al., 2011).

Figure 7

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An interesting aspect of the OCRE/Sm interaction is that the presence of multiple PRM motifs greatly enhances the overall affinity ranging from K_D ≈ mM for a single PRM peptide to K_D = 20–40 µM for complete Sm tails, and thus comparable to other PRM binding domains. The relatively weak affinity of a single PRM found in the Sm tails argues that avidity effects due to the presence of multiple motifs play an important role for high affinity binding by increasing the local concentration of PRM motifs available for OCRE binding. We note that avidity effects provided by multiple binding sites have also been observed for other PRM binding domains (Varani et al., 2000; Klippel et al., 2011), and that this feature is reminiscent of the recognition of dimethyl-arginine residues in the Sm tails by Tudor domains (Tripsianes et al., 2011). On the other hand, the weak binding affinity of a single motif indicates that the interaction between RBM5 and SmN/B/B’ may have been selected to be transient, as expected for a regulatory interaction that needs to promote interactions with the spliceosome but also needs to be disrupted at later steps of the splicing reaction.

Previously, we have shown that the splicing regulation by RBM5 acts at the stage of spliceosomal B complex formation (Bonnal et al., 2008). The role of RBM5 and its OCRE domain in this context could be to attract the U4/U6.U5 tri-snRNP to the splice sites. The fact that the SmN/B/B’ tails are intrinsically disordered and extend very far away from the seven-membered Sm core ring in U snRNPs (Figure 7B,C), is consistent with such an activity. In fact, the recent EM structure of the tri-snRNP (Nguyen et al., 2015) revealed that the Sm cores of U4 and U5, as well as the LSm ring of U6 are located at the outside of this large RNP (Figure 7C). Thus, the C-terminal tails of SmN/B/B’ of the U4 and U5 components are clearly accessible and could scan for possible binding partners of their proline-rich motifs, such as the RBM5 OCRE domain. This could help to localize the tri-snRNP to the splice sites that are regulated by RBM5.

SmB was described to participate in alternative splicing of its own pre-mRNA and many additional genes, including the FAS gene (Saltzman et al., 2011). Recently, mutations in SmB were linked to cerebro-costo-mandibular syndrome (Bacrot et al., 2015). The multi-domain protein RBM5 promotes FAS exon 6 skipping and it has been proposed that for this activity the protein modulates splice site pairing after the competing 5’ and 3’ splice sites have been recognized by U1 and U2 snRNPs, respectively (Bonnal et al., 2008). This model is based upon the observation that RBM5 inhibits the transition from A (U2 snRNP binding) to B (U4/U6.U5 tri-snRNP binding) complex formation in the introns flanking exon 6. It is conceivable that interactions mediated by the C-terminal tails of SmN/B/B’, which are present in U1, U2 and the tri-snRNP, facilitate the transition from A to B complex and that binding of the OCRE domain of RBM5 prevents these interactions and therefore the progression of spliceosome assembly. Direct or indirect association of RBM5 with particular regions of the pre-mRNA – likely involving other regions of the protein including RRM or zinc finger domains – may prevent A to B transition of pre-spliceosomes assembled on the flanking introns of exon 6, but fail to prevent tri-snRNP assembly on the distal splice sites, thus facilitating exon skipping. Alternatively, or in addition, RBM5 interaction with the C-terminal tails of SmN/B/B’ may lead to a general decrease in snRNP function, possibly more acute for U2 snRNP. As decreased activity of U2 snRNP – e.g. by individual knock down of its protein components – is known to result in increased FAS exon skipping (Papasaikas et al., 2015), RBM5-mediated reduction in U2 function could potentially explain at least part of the effects of the protein on FAS splicing regulation.

Taken together, our study provides a molecular understanding of how the OCRE domain of RBM5 interacts with proline-rich sequences of the SmN/B/B' tail and thus identifies a key interaction essential for regulation of alternative splicing.

It is interesting to note that, based on primary sequence alignments, the OCRE domains of RBM5 and RBM10 are expected to adopt highly similar structures and possibly functions. In fact, the recently reported structure of the RBM10 OCRE domain is highly similar to RBM5 OCRE (backbone coordinate r.m.s.d. 1.1Å) (Martin et al., 2016). Interestingly, mutation of a conserved tyrosine residue (corresponding to Tyr470 in RBM5, where it contributes to the recognition of the PRMs by the OCRE domain) is frequently found associated with lung carcinoma (Imielinski et al., 2012). It is thus tempting to speculate that splicing defects that are linked to impaired interactions with PRM in SmN/B/B’ proteins contribute to the pathogenesis.

The atomic coordinates for the NMR ensembles of the RBM5 OCRE domain and the complex with the SmN (219–229) are deposited in the Protein Data Bank under accession numbers 5MFY and 5MF9, respectively. The chemical shift assignments have been deposited in the Biological Magnetic Resonance Data Bank under accession numbers 34068 and 34067.

1. 1. Bacrot S
   2. Doyard M
   3. Huber C
   4. Alibeu O
   5. Feldhahn N
   6. Lehalle D
   7. Lacombe D
   8. Marlin S
   9. Nitschke P
   10. Petit F
   11. Vazquez MP
   12. Munnich A
   13. Cormier-Daire V

   (2015) Mutations in SNRPB, encoding components of the core splicing machinery, cause cerebro-costo-mandibular syndrome

   Human Mutation 36:187–190.

   https://doi.org/10.1002/humu.22729

   • PubMed
   • Google Scholar
2. 1. Ball LJ
   2. Kühne R
   3. Schneider-Mergener J
   4. Oschkinat H

   (2005) Recognition of proline-rich motifs by protein-protein-interaction domains

   Angewandte Chemie 44:2852–2869.

   https://doi.org/10.1002/anie.200400618

   • PubMed
   • Google Scholar
3. 1. Bedford MT
   2. Reed R
   3. Leder P

   (1998) WW domain-mediated interactions reveal a spliceosome-associated protein that binds a third class of proline-rich motif: the proline glycine and methionine-rich motif

   PNAS 95:10602–10607.

   https://doi.org/10.1073/pnas.95.18.10602

   • PubMed
   • Google Scholar
4. 1. Bhattacharya A
   2. Tejero R
   3. Montelione GT

   (2007) Evaluating protein structures determined by structural genomics consortia

   Proteins 66:778–795.

   https://doi.org/10.1002/prot.21165

   • PubMed
   • Google Scholar
5. 1. Blencowe BJ

   (2006) Alternative splicing: new insights from global analyses

   Cell 126:37–47.

   https://doi.org/10.1016/j.cell.2006.06.023

   • PubMed
   • Google Scholar
6. 1. Bonnal S
   2. Martínez C
   3. Förch P
   4. Bachi A
   5. Wilm M
   6. Valcárcel J

   (2008) RBM5/Luca-15/H37 regulates Fas alternative splice site pairing after exon definition

   Molecular Cell 32:81–95.

   https://doi.org/10.1016/j.molcel.2008.08.008

   • PubMed
   • Google Scholar
7. 1. Bonnal S
   2. Vigevani L
   3. Valcárcel J

   (2012) The spliceosome as a target of novel antitumour drugs

   Nature Reviews Drug Discovery 11:847–859.

   https://doi.org/10.1038/nrd3823

   • PubMed
   • Google Scholar
8. 1. Brahms H
   2. Meheus L
   3. de Brabandere V
   4. Fischer U
   5. Lührmann R

   (2001) Symmetrical dimethylation of arginine residues in spliceosomal Sm protein B/B' and the Sm-like protein LSm4, and their interaction with the SMN protein

   RNA 7:1531–1542.

   https://doi.org/10.1017/S135583820101442X

   • PubMed
   • Google Scholar
9. 1. Brown AM
   2. Zondlo NJ

   (2012) A propensity scale for type II polyproline helices (PPII): aromatic amino acids in proline-rich sequences strongly disfavor PPII due to proline-aromatic interactions

   Biochemistry 51:5041–5051.

   https://doi.org/10.1021/bi3002924

   • PubMed
   • Google Scholar
10. 1. Brunger AT
    2. Adams PD
    3. Clore GM
    4. DeLano WL
    5. Gros P
    6. Grosse-Kunstleve RW
    7. Jiang JS
    8. Kuszewski J
    9. Nilges M
    10. Pannu NS
    11. Read RJ
    12. Rice LM
    13. Simonson T
    14. Warren GL

    (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination

    Acta Crystallographica Section D 54:905–921.

    https://doi.org/10.1107/S0907444998003254

    • Google Scholar
11. 1. Callebaut I
    2. Mornon JP

    (2005) OCRE: a novel domain made of imperfect, aromatic-rich octamer repeats

    Bioinformatics 21:699–702.

    https://doi.org/10.1093/bioinformatics/bti065

    • PubMed
    • Google Scholar
12. 1. Cascino I
    2. Fiucci G
    3. Papoff G
    4. Ruberti G

    (1995)

    Three functional soluble forms of the human apoptosis-inducing Fas molecule are produced by alternative splicing

    Journal of Immunology 154:2706–2713.

    • Google Scholar
13. 1. Cazalla D
    2. Sanford JR
    3. Cáceres JF

    (2005) A rapid and efficient protocol to purify biologically active recombinant proteins from mammalian cells

    Protein Expression and Purification 42:54–58.

    https://doi.org/10.1016/j.pep.2005.03.035

    • PubMed
    • Google Scholar
14. 1. Chen L
    2. Park SM
    3. Tumanov AV
    4. Hau A
    5. Sawada K
    6. Feig C
    7. Turner JR
    8. Fu YX
    9. Romero IL
    10. Lengyel E
    11. Peter ME

    (2010) CD95 promotes tumour growth

    Nature 465:492–496.

    https://doi.org/10.1038/nature09075

    • PubMed
    • Google Scholar
15. 1. Cheng J
    2. Zhou T
    3. Liu C
    4. Shapiro JP
    5. Brauer MJ
    6. Kiefer MC
    7. Barr PJ
    8. Mountz JD

    (1994) Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule

    Science 263:1759–1762.

    https://doi.org/10.1126/science.7510905

    • PubMed
    • Google Scholar
16. 1. Cooper TA
    2. Wan L
    3. Dreyfuss G

    (2009) RNA and disease

    Cell 136:777–793.

    https://doi.org/10.1016/j.cell.2009.02.011

    • PubMed
    • Google Scholar
17. 1. David CJ
    2. Manley JL

    (2010) Alternative pre-mRNA splicing regulation in cancer: pathways and programs unhinged

    Genes & Development 24:2343–2364.

    https://doi.org/10.1101/gad.1973010

    • PubMed
    • Google Scholar
18. 1. Delaglio F
    2. Grzesiek S
    3. Vuister GW
    4. Zhu G
    5. Pfeifer J
    6. Bax A

    (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes

    Journal of Biomolecular NMR 6:277–293.

    https://doi.org/10.1007/bf00197809

    • PubMed
    • Google Scholar
19. 1. Doreleijers JF
    2. Sousa da Silva AW
    3. Krieger E
    4. Nabuurs SB
    5. Spronk CA
    6. Stevens TJ
    7. Vranken WF
    8. Vriend G
    9. Vuister GW

    (2012) CING: an integrated residue-based structure validation program suite

    Journal of Biomolecular NMR 54:267–283.

    https://doi.org/10.1007/s10858-012-9669-7

    • PubMed
    • Google Scholar
20. 1. Drake AF
    2. Siligardi G
    3. Gibbons WA

    (1988) Reassessment of the electronic circular dichroism criteria for random coil conformations of poly(L-lysine) and the implications for protein folding and denaturation studies

    Biophysical Chemistry 31:143–146.

    https://doi.org/10.1016/0301-4622(88)80019-X

    • PubMed
    • Google Scholar
21. 1. Fields S
    2. Song O

    (1989) A novel genetic system to detect protein-protein interactions

    Nature 340:245–246.

    https://doi.org/10.1038/340245a0

    • PubMed
    • Google Scholar
22. 1. Freund C
    2. Kühne R
    3. Yang H
    4. Park S
    5. Reinherz EL
    6. Wagner G

    (2002) Dynamic interaction of CD2 with the GYF and the SH3 domain of compartmentalized effector molecules

    The EMBO Journal 21:5985–5995.

    https://doi.org/10.1093/emboj/cdf602

    • PubMed
    • Google Scholar
23. 1. Fromont-Racine M
    2. Rain JC
    3. Legrain P

    (1997) Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens

    Nature Genetics 16:277–282.

    https://doi.org/10.1038/ng0797-277

    • PubMed
    • Google Scholar
24. 1. Fushimi K
    2. Ray P
    3. Kar A
    4. Wang L
    5. Sutherland LC
    6. Wu JY

    (2008) Up-regulation of the proapoptotic caspase 2 splicing isoform by a candidate tumor suppressor, RBM5

    PNAS 105:15708–15713.

    https://doi.org/10.1073/pnas.0805569105

    • Google Scholar
25. 1. Förch P
    2. Puig O
    3. Kedersha N
    4. Martínez C
    5. Granneman S
    6. Séraphin B
    7. Anderson P
    8. Valcárcel J

    (2000) The apoptosis-promoting factor TIA-1 is a regulator of alternative pre-mRNA splicing

    Molecular Cell 6:1089–1098.

    https://doi.org/10.1016/s1097-2765(00)00107-6

    • PubMed
    • Google Scholar
26. 1. Güntert P

    (2009) Automated structure determination from NMR spectra

    European Biophysics Journal 38:129–143.

    https://doi.org/10.1007/s00249-008-0367-z

    • PubMed
    • Google Scholar
27. 1. Holm L
    2. Kääriäinen S
    3. Rosenström P
    4. Schenkel A

    (2008) Searching protein structure databases with DaliLite v.3

    Bioinformatics 24:2780–2781.

    https://doi.org/10.1093/bioinformatics/btn507

    • PubMed
    • Google Scholar
28. 1. Imielinski M
    2. Berger AH
    3. Hammerman PS
    4. Hernandez B
    5. Pugh TJ
    6. Hodis E
    7. Cho J
    8. Suh J
    9. Capelletti M
    10. Sivachenko A
    11. Sougnez C
    12. Auclair D
    13. Lawrence MS
    14. Stojanov P
    15. Cibulskis K
    16. Choi K
    17. de Waal L
    18. Sharifnia T
    19. Brooks A
    20. Greulich H
    21. Banerji S
    22. Zander T
    23. Seidel D
    24. Leenders F
    25. Ansén S
    26. Ludwig C
    27. Engel-Riedel W
    28. Stoelben E
    29. Wolf J
    30. Goparju C
    31. Thompson K
    32. Winckler W
    33. Kwiatkowski D
    34. Johnson BE
    35. Jänne PA
    36. Miller VA
    37. Pao W
    38. Travis WD
    39. Pass HI
    40. Gabriel SB
    41. Lander ES
    42. Thomas RK
    43. Garraway LA
    44. Getz G
    45. Meyerson M

    (2012) Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing

    Cell 150:1107–1120.

    https://doi.org/10.1016/j.cell.2012.08.029

    • PubMed
    • Google Scholar
29. 1. Izquierdo JM
    2. Majós N
    3. Bonnal S
    4. Martínez C
    5. Castelo R
    6. Guigó R
    7. Bilbao D
    8. Valcárcel J

    (2005) Regulation of Fas alternative splicing by antagonistic effects of TIA-1 and PTB on exon definition

    Molecular Cell 19:475–484.

    https://doi.org/10.1016/j.molcel.2005.06.015

    • PubMed
    • Google Scholar
30. 1. Johnson BA
    2. Blevins RA

    (1994) NMR View: A computer program for the visualization and analysis of NMR data

    Journal of Biomolecular NMR 4:603–614.

    https://doi.org/10.1007/BF00404272

    • PubMed
    • Google Scholar
31. 1. Kambach C
    2. Walke S
    3. Nagai K

    (1999) Structure and assembly of the spliceosomal small nuclear ribonucleoprotein particles

    Current Opinion in Structural Biology 9:222–230.

    https://doi.org/10.1016/S0959-440X(99)80032-3

    • PubMed
    • Google Scholar
32. 1. Kelly MA
    2. Chellgren BW
    3. Rucker AL
    4. Troutman JM
    5. Fried MG
    6. Miller AF
    7. Creamer TP

    (2001) Host-guest study of left-handed polyproline II helix formation

    Biochemistry 40:14376–14383.

    https://doi.org/10.1002/prot.10477

    • PubMed
    • Google Scholar
33. 1. Klippel S
    2. Wieczorek M
    3. Schümann M
    4. Krause E
    5. Marg B
    6. Seidel T
    7. Meyer T
    8. Knapp EW
    9. Freund C

    (2011) Multivalent binding of formin-binding protein 21 (FBP21)-tandem-WW domains fosters protein recognition in the pre-spliceosome

    Journal of Biological Chemistry 286:38478–38487.

    https://doi.org/10.1074/jbc.M111.265710

    • PubMed
    • Google Scholar
34. 1. Kondo Y
    2. Oubridge C
    3. van Roon AM
    4. Nagai K

    (2015) Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5' splice site recognition

    eLife 4:e04986.

    https://doi.org/10.7554/eLife.04986

    • PubMed
    • Google Scholar
35. 1. Krammer PH

    (2000) CD95's deadly mission in the immune system

    Nature 407:789–795.

    https://doi.org/10.1038/35037728

    • PubMed
    • Google Scholar
36. 1. Krissinel E
    2. Henrick K

    (2004) Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions

    Acta Crystallographica Section D Biological Crystallography 60:2256–2268.

    https://doi.org/10.1107/S0907444904026460

    • PubMed
    • Google Scholar
37. 1. Laskowski RA
    2. Rullmannn JA
    3. MacArthur MW
    4. Kaptein R
    5. Thornton JM

    (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR

    Journal of Biomolecular NMR 8:477–486.

    https://doi.org/10.1007/BF00228148

    • PubMed
    • Google Scholar
38. 1. Leung AK
    2. Nagai K
    3. Li J

    (2011) Structure of the spliceosomal U4 snRNP core domain and its implication for snRNP biogenesis

    Nature 473:536–539.

    https://doi.org/10.1038/nature09956

    • PubMed
    • Google Scholar
39. 1. Linge JP
    2. O'Donoghue SI
    3. Nilges M

    (2001) Automated assignment of ambiguous nuclear overhauser effects with ARIA

    Methods in Enzymology 339:71–90.

    https://doi.org/10.1016/s0076-6879(01)39310-2

    • PubMed
    • Google Scholar
40. 1. Linge JP
    2. Williams MA
    3. Spronk CA
    4. Bonvin AM
    5. Nilges M

    (2003) Refinement of protein structures in explicit solvent

    Proteins 50:496–506.

    https://doi.org/10.1002/prot.10299

    • PubMed
    • Google Scholar
41. 1. Liu C
    2. Cheng J
    3. Mountz JD

    (1995) Differential expression of human Fas mRNA species upon peripheral blood mononuclear cell activation

    Biochemical Journal 310:957–963.

    https://doi.org/10.1042/bj3100957

    • PubMed
    • Google Scholar
42. 1. Mackereth CD
    2. Madl T
    3. Bonnal S
    4. Simon B
    5. Zanier K
    6. Gasch A
    7. Rybin V
    8. Valcárcel J
    9. Sattler M

    (2011) Multi-domain conformational selection underlies pre-mRNA splicing regulation by U2AF

    Nature 475:408–U174.

    https://doi.org/10.1038/nature10171

    • PubMed
    • Google Scholar
43. 1. Martin BT
    2. Serrano P
    3. Geralt M
    4. Wüthrich K

    (2016) Nuclear magnetic resonance structure of a novel globular domain in RBM10 containing OCRE, the octamer repeat sequence motif

    Structure 24:158–164.

    https://doi.org/10.1016/j.str.2015.10.029

    • PubMed
    • Google Scholar
44. 1. Matera AG
    2. Wang Z

    (2014) A day in the life of the spliceosome

    Nature Reviews Molecular Cell Biology 15:108–121.

    https://doi.org/10.1038/nrm3742

    • PubMed
    • Google Scholar
45. 1. Mouaikel J
    2. Narayanan U
    3. Verheggen C
    4. Matera AG
    5. Bertrand E
    6. Tazi J
    7. Bordonné R

    (2003) Interaction between the small-nuclear-RNA cap hypermethylase and the spinal muscular atrophy protein, survival of motor neuron

    EMBO Reports 4:616–622.

    https://doi.org/10.1038/sj.embor.embor863

    • PubMed
    • Google Scholar
46. 1. Nguyen TH
    2. Galej WP
    3. Bai XC
    4. Savva CG
    5. Newman AJ
    6. Scheres SH
    7. Nagai K

    (2015) The architecture of the spliceosomal U4/U6.U5 tri-snRNP

    Nature 523:47–52.

    https://doi.org/10.1038/nature14548

    • PubMed
    • Google Scholar
47. 1. Oh JJ
    2. Grosshans DR
    3. Wong SG
    4. Slamon DJ

    (1999) Identification of differentially expressed genes associated with HER-2/neu overexpression in human breast cancer cells

    Nucleic Acids Research 27:4008–4017.

    https://doi.org/10.1093/nar/27.20.4008

    • PubMed
    • Google Scholar
48. 1. Oh JJ
    2. Taschereau EO
    3. Koegel AK
    4. Ginther CL
    5. Rotow JK
    6. Isfahani KZ
    7. Slamon DJ

    (2010) RBM5/H37 tumor suppressor, located at the lung cancer hot spot 3p21.3, alters expression of genes involved in metastasis

    Lung Cancer 70:253–262.

    https://doi.org/10.1016/j.lungcan.2010.02.012

    • PubMed
    • Google Scholar
49. 1. Oh JJ
    2. West AR
    3. Fishbein MC
    4. Slamon DJ

    (2002)

    A candidate tumor suppressor gene, H37, from the human lung cancer tumor suppressor locus 3p21.3

    Cancer Research 62:3207–3213.

    • PubMed
    • Google Scholar
50. 1. Pan Q
    2. Shai O
    3. Lee LJ
    4. Frey BJ
    5. Blencowe BJ

    (2008) Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing

    Nature Genetics 40:1413–1415.

    https://doi.org/10.1038/ng.259

    • PubMed
    • Google Scholar
51. 1. Papasaikas P
    2. Tejedor JR
    3. Vigevani L
    4. Valcárcel J

    (2015) Functional splicing network reveals extensive regulatory potential of the core spliceosomal machinery

    Molecular Cell 57:7–22.

    https://doi.org/10.1016/j.molcel.2014.10.030

    • PubMed
    • Google Scholar
52. 1. Papoff G
    2. Cascino I
    3. Eramo A
    4. Starace G
    5. Lynch DH
    6. Ruberti G

    (1996)

    An N-terminal domain shared by Fas/Apo-1 (CD95) soluble variants prevents cell death in vitro

    Journal of Immunology 156:4622–4630.

    • PubMed
    • Google Scholar
53. 1. Petrella EC
    2. Machesky LM
    3. Kaiser DA
    4. Pollard TD

    (1996) Structural requirements and thermodynamics of the interaction of proline peptides with profilin

    Biochemistry 35:16535–16543.

    https://doi.org/10.1021/bi961498d

    • PubMed
    • Google Scholar
54. 1. Pomeranz Krummel DA
    2. Oubridge C
    3. Leung AK
    4. Li J
    5. Nagai K

    (2009) Crystal structure of human spliceosomal U1 snRNP at 5.5 A resolution

    Nature 458:475–480.

    https://doi.org/10.1038/nature07851

    • PubMed
    • Google Scholar
55. 1. Rintala-Maki ND
    2. Goard CA
    3. Langdon CE
    4. Wall VE
    5. Traulsen KE
    6. Morin CD
    7. Bonin M
    8. Sutherland LC

    (2007) Expression of RBM5-related factors in primary breast tissue

    Journal of Cellular Biochemistry 100:1440–1458.

    https://doi.org/10.1002/jcb.21134

    • PubMed
    • Google Scholar
56. 1. Roesler J
    2. Izquierdo JM
    3. Ryser M
    4. Rösen-Wolff A
    5. Gahr M
    6. Valcarcel J
    7. Lenardo MJ
    8. Zheng L

    (2005) Haploinsufficiency, rather than the effect of an excessive production of soluble CD95 (CD95{Delta}TM), is the basis for ALPS Ia in a family with duplicated 3' splice site AG in CD95 intron 5 on one allele

    Blood 106:1652–1659.

    https://doi.org/10.1182/blood-2004-08-3104

    • PubMed
    • Google Scholar
57. 1. Saltzman AL
    2. Pan Q
    3. Blencowe BJ

    (2011) Regulation of alternative splicing by the core spliceosomal machinery

    Genes & Development 25:373–384.

    https://doi.org/10.1101/gad.2004811

    • PubMed
    • Google Scholar
58. 1. Schmauss C
    2. Brines ML
    3. Lerner MR

    (1992)

    The gene encoding the small nuclear ribonucleoprotein-associated protein N is expressed at high levels in neurons

    Journal of Biological Chemistry 267:8521–8529.

    • PubMed
    • Google Scholar
59. 1. Sharpe NG
    2. Wall W
    3. Latchman DS

    (1990) Expression of the tissue specific RNA splicing protein SmN in HeLa cells

    Nucleic Acids Research 18:3425–3426.

    https://doi.org/10.1093/nar/18.11.3425

    • PubMed
    • Google Scholar
60. 1. Shen Y
    2. Delaglio F
    3. Cornilescu G
    4. Bax A

    (2009) TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts

    Journal of Biomolecular NMR 44:213–223.

    https://doi.org/10.1007/s10858-009-9333-z

    • PubMed
    • Google Scholar
61. 1. Sutherland LC
    2. Rintala-Maki ND
    3. White RD
    4. Morin CD

    (2005) RNA binding motif (RBM) proteins: a novel family of apoptosis modulators?

    Journal of Cellular Biochemistry 94:5–24.

    https://doi.org/10.1002/jcb.20204

    • PubMed
    • Google Scholar
62. 1. Tripsianes K
    2. Madl T
    3. Machyna M
    4. Fessas D
    5. Englbrecht C
    6. Fischer U
    7. Neugebauer KM
    8. Sattler M

    (2011) Structural basis for dimethylarginine recognition by the Tudor domains of human SMN and SPF30 proteins

    Nature Structural & Molecular Biology 18:1414–1420.

    https://doi.org/10.1038/nsmb.2185

    • PubMed
    • Google Scholar
63. 1. van Dam A
    2. Winkel I
    3. Zijlstra-Baalbergen J
    4. Smeenk R
    5. Cuypers HT

    (1989) Cloned human snRNP proteins B and B' differ only in their carboxy-terminal part

    The EMBO Journal 8:3853–3860.

    https://doi.org/10.1007/978-3-642-46681-6_7

    • PubMed
    • Google Scholar
64. 1. Van Parijs L
    2. Biuckians A
    3. Abbas AK

    (1998)

    Functional roles of Fas and Bcl-2-regulated apoptosis of T lymphocytes

    Journal of Immunology 160:2065–2071.

    • PubMed
    • Google Scholar
65. 1. Varani L
    2. Gunderson SI
    3. Mattaj IW
    4. Kay LE
    5. Neuhaus D
    6. Varani G

    (2000) The NMR structure of the 38 kDa U1A protein - PIE RNA complex reveals the basis of cooperativity in regulation of polyadenylation by human U1A protein

    Nature Structural Biology 7:329–335.

    https://doi.org/10.1038/74101

    • PubMed
    • Google Scholar
66. 1. Wahl MC
    2. Will CL
    3. Lührmann R

    (2009) The spliceosome: design principles of a dynamic RNP machine

    Cell 136:701–718.

    https://doi.org/10.1016/j.cell.2009.02.009

    • PubMed
    • Google Scholar
67. 1. Wang ET
    2. Sandberg R
    3. Luo S
    4. Khrebtukova I
    5. Zhang L
    6. Mayr C
    7. Kingsmore SF
    8. Schroth GP
    9. Burge CB

    (2008) Alternative isoform regulation in human tissue transcriptomes

    Nature 456:470–476.

    https://doi.org/10.1038/nature07509

    • PubMed
    • Google Scholar
68. 1. Weber G
    2. Trowitzsch S
    3. Kastner B
    4. Lührmann R
    5. Wahl MC

    (2010) Functional organization of the Sm core in the crystal structure of human U1 snRNP

    The EMBO Journal 29:4172–4184.

    https://doi.org/10.1038/emboj.2010.295

    • PubMed
    • Google Scholar
69. 1. Will CL
    2. Lührmann R

    (2001) Spliceosomal UsnRNP biogenesis, structure and function

    Current Opinion in Cell Biology 13:290–301.

    https://doi.org/10.1016/s0955-0674(00)00211-8

    • PubMed
    • Google Scholar
70. 1. Wingfield PT
    2. Palmer I
    3. Liang SM

    (2001) Folding and purification of insoluble (inclusion body) proteins from Escherichia coli

    Current Protocols in Protein Science Chapter 6:6–5.

    https://doi.org/10.1002/0471140864.ps0605s00

    • PubMed
    • Google Scholar
71. 1. Xiao SJ
    2. Wang LY
    3. Kimura M
    4. Kojima H
    5. Kunimoto H
    6. Nishiumi F
    7. Yamamoto N
    8. Nishio K
    9. Fujimoto S
    10. Kato T
    11. Kitagawa S
    12. Yamane H
    13. Nakajima K
    14. Inoue A

    (2013) S1-1/RBM10: multiplicity and cooperativity of nuclear localisation domains

    Biology of the Cell 105:162–174.

    https://doi.org/10.1111/boc.201200068

    • PubMed
    • Google Scholar
72. 1. Zabarovsky ER
    2. Lerman MI
    3. Minna JD

    (2002) Tumor suppressor genes on chromosome 3p involved in the pathogenesis of lung and other cancers

    Oncogene 21:6915–6935.

    https://doi.org/10.1038/sj.onc.1205835

    • PubMed
    • Google Scholar

Author details

1. André Mourão

   1. Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany
   2. Biomolecular NMR and Center for Integrated Protein Science Munich, Department Chemie, Technische Universität München, Garching, Germany

   Contribution

   Analysis and interpretation of data, Drafting or revising the article, Conception and design, Acquisition of data

   Contributed equally with

   Sophie Bonnal, Komal Soni and Lisa Warner

   Competing interests

   No competing interests declared

2. Sophie Bonnal

   Barcelona Institute of Science and Technology and Universitat Pompeu Fabra, Centre de Regulació Genòmica, Barcelona, Spain

   Contribution

   Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   André Mourão, Komal Soni and Lisa Warner

   Competing interests

   No competing interests declared

3. Komal Soni

   1. Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany
   2. Biomolecular NMR and Center for Integrated Protein Science Munich, Department Chemie, Technische Universität München, Garching, Germany

   Contribution

   Acquisition of data, Analysis and interpretation of data

   Contributed equally with

   André Mourão, Sophie Bonnal and Lisa Warner

   Competing interests

   No competing interests declared

4. Lisa Warner

   1. Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany
   2. Biomolecular NMR and Center for Integrated Protein Science Munich, Department Chemie, Technische Universität München, Garching, Germany

   Contribution

   Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   André Mourão, Sophie Bonnal and Komal Soni

   Competing interests

   No competing interests declared

5. Rémy Bordonné

   Institut de Génétique Moléculaire de Montpellier, CNRS-UMR5535, Université de Montpellier, Montpellier, France

   Contribution

   Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared

6. Juan Valcárcel

   1. Barcelona Institute of Science and Technology and Universitat Pompeu Fabra, Centre de Regulació Genòmica, Barcelona, Spain
   2. Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain

   Contribution

   Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   juan.valcarcel@crg.es

   Competing interests

   Reviewing editor, eLife

7. Michael Sattler

   1. Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany
   2. Biomolecular NMR and Center for Integrated Protein Science Munich, Department Chemie, Technische Universität München, Garching, Germany

   Contribution

   Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   sattler@helmholtz-muenchen.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1594-0527

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