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.

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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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