Multipartite RNA recognition

RNA-binding proteins (RBPs) are involved in a diverse set of functions carried out only by a handful of RNA binding domains. Since RBPs usually contain multiple RNA binding modules, it is not surprising that they cater to this large functional diversity by employing a combination of modules. This way the RBPs achieve high affinity and specificity of target recognition. The relatively weak interactions of the individual domains make it easier for their regulation especially in cases where assembly and dis-assembly of complexes is required.

Tandem domains connected together via short linkers act as a classic example of formation of an extended RNA binding interface. Since the individual domains only provide base specific recognition of about 2-3 nucleotides, such a combination of closely positioned RNA binding domains may help in specific recognition of a longer RNA sequence (Shamoo, Abdul-Manan et al. 1995, Mackereth, Madl et al. 2011, Hennig, Militti et al. 2014). The presence of a long flexible linker between the RNA binding modules could also be advantageous in certain situations where the module specific nucleotides are positioned far apart from each other on the same or different RNAs (Braddock, Louis et al. 2002, Oberstrass, Auweter et al. 2005, Stefl, Xu et al. 2006). Another unique way of RNA recognition via dimerization of individual domains leading to a cooperative mode of RNA recognition has also been observed in certain proteins (Liu, Luyten et al. 2001, Ryder, Frater et al. 2004, Beuck, Szymczyna et al. 2010, Meyer, Tripsianes et al. 2010, Feracci, Foot et al. 2016). Additionally, dimerization of RBPs caused by structural rearrangements upon RNA binding can also play an important role in RNA binding (Varani, Gunderson et al. 2000, Chao, Lee et al. 2005, Lingel, Simon et al. 2005).

The inter-domain arrangement during RNA recognition is also quite important. It is frequently observed that domains acting as independent moieties undergo major structural

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organization upon RNA binding to behave as a single compact molecule as observed in the case of Sxl (Handa, Nureki et al. 1999), PABP (Deo, Bonanno et al. 1999), Hrp1 (Perez-Canadillas 2006) and TIA-1 (Wang, Hennig et al. 2014) proteins. Contrastingly, there exist other examples where a pre-formed arrangement of RNA binding domains is required for target recognition (Hudson, Martinez-Yamout et al. 2004). Another form of modulation of RNA recognition occurs via conformational selection. In the tandem RRM domains of the essential splicing factor U2AF65, an ensemble of active and inactive conformations of the tandem domains exist in the free form and a dynamic population shift from inactive to active conformation occurs in the presence of strong polypyrimidine tract (Mackereth, Madl et al.

2011, Huang, Warner et al. 2014, Voith von Voithenberg, Sanchez-Rico et al. 2016)

The linker length is also important. Theoretically, the affinity of a tandem domain protein for its RNA target could be obtained by multiplying that of the individual domains. But with increasing linker length (>50-60 residues), the domains behave as independent modules without affecting the overall affinity while in case of a short linker, the combined affinity can increase by 10-1000 fold (Shamoo, Abdul-Manan et al. 1995).

RBM5 involves a novel mode of RNA recognition where a stable, compact arrangement between the RRM1 and Zf1 domains is required. The crystal structure of RRM1-Zf1 reveals the tethering of the domains via a tri-partite mechanism where specific contacts between all three parts, i.e. RRM1, linker and Zf1 are essential. Consistently, ¹⁵N-relaxation data of the free and protein-RNA complex show that the two domains tumble together in solution in both free and RNA-bound forms , although the existence of a slightly extended conformation in the RNA bound form is indicated by both ¹⁵N-relaxation as well as SAXS data. Contrastingly, in case of RBM10, the RRM1 and Zf1 domains tumble independently of one another in the free form (Collins, Kainov et al. 2017), whereas they act as a single moiety upon RNA binding (Martin Ruebbelke, personal communication).

It is noteworthy that the length of the linker is quite short, spanning only 7 residues, possibly providing a pre-formed extended RNA-binding interface in RBM5. Upon RNA titration into the tandem domain construct, large chemical shift perturbations (CSP) are observed in Zf1 domain. This could either be only due to RNA binding as we know that Zf1 is the highest affinity domain or it could be indicative of a cumulative effect of RNA binding and possible domain re-orientation upon RNA binding. Consistent with the latter, SAXS analysis of the free and protein-RNA complex of the tandem domain show a slight increase in the

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maximum dimension of the protein upon RNA-binding, which would not be observed in case no domain re-organization occurs.

Additionally, the relatively longer linker between Zf1 and RRM2 compared to that between RRM1 and Zf1 could also be a suggestion of the presence of two separate RNA recognition entities- RRM1-Zf1 and RRM2 in RBM5. The ¹⁵N-relaxation data on the triple domain RRM1-Zf1-RRM2 C191G mutant indicate that the three domains tumble together in solution in both free and RNA bound forms. Intriguingly, the SAXS curve for the free protein is indicative of presence of multiple conformations in the free form while that of the RNA bound form seems to be a uniform curve. It is noteworthy that the ¹H,¹⁵N-HSQC spectra of both the free and RNA-bound RRM1-Zf1-RRM2 C191G mutant show considerable line-broadening in RRM1-Zf1 while the RRM2 signals are relatively sharp, indicative of additional exchange processes on the RRM1-Zf1 side.

Figure 74 Hypothetical model of RBM5 RNA binding domains

A hypothetical arrangement of the RNA binding domains of RBM5 in the free form (A) and in the presence of RNA (B). RRM1, Zf1 and RRM2 domains are color coded in blue, green and pink, while the RNA is shown in red. In the free form, RRM2 is able to sample multiple conformations while in complex with RNA, it stably achieves a single conformation.

Combining all these data, it is tempting to speculate that RRM1-Zf1 and RRM2 are partially uncoupled in the free form but they behave as a single entity in the RNA-bound form (Figure 74). PRE experiments planned in the future could provide more conclusive evidence of the conformation of the triple-domain RBM5 RNA binding region in the presence and absence of RNA. Interestingly, it was recently proposed that in case of RBM10, the RRM2 domain re-orients quasi-independently from RRM1-Zf1 in solution (Collins, Kainov et al.

2017). Such conformational dynamics in multi-domain proteins could be highly relevant in the biological context where the splicing factors need to scan multiple pre-mRNA targets to finally bind and regulate only a handful of them. This would point towards the possibility of

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recognition of distinct pre-mRNA targets by either individual domains or a specific combination of domains which may exist either in a pre-formed RNA binding conformation or may undergo structural re-arrangement upon RNA binding. In this way, the functional capacity of the splicing factors can greatly expand.