Diverse functionalities of RBM5/6/10 proteins

The conservation of domain organization as well as a high degree of sequence similarity are indicative of overlapping functions between RBM5/6/10 proteins. To ascertain the effects of RBMs in alternative splicing regulation, a large-scale splicing-sensitive microarray analysis was performed whereby each of the individual RBMs was silenced and the effects on splicing events were quantified (Bechara, Sebestyen et al. 2013). Interestingly, depletion of RBM5 affected expression of only 281 transcripts while RBM10 and RBM6 had a more wide-spread effect on 1294 and 1202 transcripts, respectively. Moreover, only 20% overlap between alternative splicing events regulated by RBM5/6/10 was observed suggesting more distinct than overlapping functions of these proteins.

RBM10 OCRE domain is required for AS regulation of Fas pre-mRNA

In this thesis, the solution NMR structure of RBM10 OCRE domain is presented. It is a globular domain consisting of six antiparallel β-strands which tyrosine residues exposed on either surface of the domain. Recently, Martin et al. also published the solution NMR structure of the RBM10 OCRE domain (Martin, Serrano et al. 2016) (PDB ID: 2MXW). The domain boundaries used in their study extend from residues 558-646 as opposed to those presented in this thesis (residues 562-621). The C-terminal extension in their construct is completely disordered, as seen in Figure 73A. Consistently, most of the NOE-based distance restraints were obtained between the residues in the central core of RBM10 OCRE domain.

The superposition of a single representative structure from NMR ensembles of RBM10 OCRE domain determined in this thesis and that from the previously published structure (PDB ID: 2MXW) clearly indicates that the two structures are essentially the same (Figure 73B).

The backbone RMSD between the two NMR ensembles is 0.813 Å, calculated using residues 563-619 for RBM10 OCRE and residues 564-618 for 2MXW with SuperPose v1.0 webserver (Maiti, Van Domselaar et al. 2004). The arrangement of the surface exposed side-chains of tyrosine residues is also conserved between the two structures, as shown in Figure 73B.

164 Figure 73 Comparison of RBM10 OCRE domain structures

(A) Solution NMR ensemble of RBM10 OCRE domain (PDB ID: 2MXW). The N- and extended C-termini are unstructured and do not converge in the NMR ensemble upon superposition for the best fit of the central core. (B) Superposition of single representative structures from the NMR ensembles of RBM10 OCRE domain determined in this thesis (black) with that of 2MXW (teal) is presented and the side chains of surface exposed tyrosine residues are denoted.

OCRE domains of RBM5/10 are structurally conserved and both can possibly bind to the core spliceosomal machinery in a similar manner as well, as indicated by similar binding affinities of RBM5/10 OCRE domains for SmN/B/B’ C-terminal poly-proline rich tails.

Furthermore, in vivo splicing assays were done on Fas minigene reporter to ascertain similarity between effects of RBM5/10 OCRE domain deletions on AS regulation of Fas.

As expected, RBM10 OCRE domain is required for Fas exon 6 skipping, consistent with effects of RBM5 OCRE domain. Together with structural and binding data, there are strong indications of the existence of a similar mechanism of Fas pre-mRNA AS regulation by RBM10.

A few years ago, Inoue et al. also studied the effects of RBM10 on AS regulation of Fas pre-mRNA (Inoue, Yamamoto et al. 2014). Interestingly, they found out that depletion of RBM10 alone was sufficient to observe a significant decrease in Fas exon 6 skipping (using RNAi mediated knockdown in HeLa and HLE cells), in contrast to the previous observation by our collaborators (Bonnal, Martinez et al. 2008) where simultaneous depletion of RBM5/6/10 was necessary, possibly due to partially redundant functional activities (using RNAi mediated knockdown in HeLa cells). Additionally, they suggested a likely mechanism by which RBM10 regulates AS of Fas pre-mRNA. Briefly, RBM10 binds to the 5’ splice site

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of Fas exon 6 that is rich in U- and G- nucleotides (uuguuuggG|GUaaguucuu) possibly via its Zf1 domain which specifically recognizes AGGUAA RNA motifs (Nguyen, Mansfield et al.

2011) with high affinity. Due to this direct protein-RNA interaction, the 5’ splice site of exon 6 would be blocked and therefore unavailable for splicing, leading to exon 6 skipping.

Furthermore, it was suggested that in case of Bcl-x pre-mRNA, RBM10 promotes internal 5’

splice site selection by possibly binding to a similar G/U rich sequence (GG|GUAAG) on the 5’ splice site on exon 2. It is noteworthy that their model is primarily based on similarity of sequences at the blocked splice sites between Fas and Bcl-x pre-mRNAs and they did not present any experimental data to support their model.

The mechanism of action of RBM10 in Fas AS regulation suggested by Inoue et al. is contrasting to that suggested in this thesis whereby the OCRE domain is shown to be responsible.

RBM6 modulates AS of Fas pre-mRNA

In this thesis I revealed that RBM6 OCRE domain is a truncated OCRE domain that lacks the ability to recognize the SmN/B/B’ poly-proline rich tails. It is noteworthy that RBM6 is still able to regulate the alternative splicing of Fas pre-mRNA, although with opposite outcome compared to that of RBM5/10, by promoting exon 6 inclusion. Interestingly, this function of RBM6 is OCRE independent further supporting the data that RBM6 OCRE domain is unable to bind SmN/B/B’ tails. It is therefore possible that the AS regulation of Fas pre-mRNA by RBM6 protein is conferred via a different mechanism which could perhaps involve another domain in the multi-domain RBM6.

The inability of RBM6 OCRE domain to bind SmN/B/B’ C-terminal tails implies that either the domain is just non-functional or it has another function which has not yet been discovered. In addition, the RNA binding domains (RRM1 and Zf1) of RBM6 also lack most of the canonical RNA binding residues and RRM1 also has an unusually low pI of 4.32. Initial NMR experiments involving RNA titrations derived from the NUMB RNA (Bechara, Sebestyen et al. 2013) into RRM1 and RRM1-Zf1 domains of RBM6 showed minimal chemical shifts although RRM2 can still bind RNA. Contrastingly, RBM5 and RBM10 (data from another doctoral student in the lab) RNA binding domains can bind their respective target RNA motifs with high affinity.

Another hint for distinct possible functionalities of RBMs came a few years ago, when Heath et al. (Heath, Sablitzky et al. 2010) suggested the involvement of RBM6 in

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transciptional packaging. They also demonstrated the ability of RBM6 to be targeted to splicing speckles, a function which was attributed to its N-terminal multimerization RGG domain. This repeat region is quite short or even absent in both RBM5 and RBM10 while it spans ~22 kDa in the case of RBM6.

In light of these data, it is possible that RBM6 has evolved to perform a diverse set of functions and is possibly involved in protein-protein and protein-RNA interactions via distinct mechanisms from RBM5/10.