Characterization of the RNA recognition mode of hnRNP G extends its role in SMN2 splicing regulation

Abstract

Regulation of SMN2 exon 7 splicing is crucial for the production of active SMN protein and the survival of Spinal Muscular Atrophy (SMA) patients. One of the most efficient activators of exon 7 inclusion is hnRNP G, which is recruited to the exon by Tra2-β1. We report that in addition to the C-terminal region of hnRNP G, the RNA Recognition Motif (RRM) and the middle part of the protein containing the Arg–Gly–Gly (RGG) box are important for this function. To better understand the mode of action of hnRNP G in this context we determined the structure of its RRM bound to an SMN2 derived RNA. The RRM interacts with a 5′-AAN-3′ motif and specifically recognizes the two consecutive adenines. By testing the effect of mutations in hnRNP G RRM and in its putative binding sites on the splicing of SMN2 exon 7, we show that it specifically binds to exon 7. This interaction is required for hnRNP G splicing activity and we propose its recruitment to a polyA tract located upstream of the Tra2-β1 binding site. Finally, our data suggest that hnRNP G plays a major role in the recruitment of the Tra2-β1/hnRNP G/SRSF9 trimeric complex to SMN2 exon 7.

One, Two, Three, Four! How Multiple RRMs Read the Genome Sequence

“…Similarly, the immediate neighborhood of Tra2-β1 in vivo binding sites was found to be enriched in adenines, leading to the extended consensus motif identified within sequences cross-linked by this protein (Grellscheid et al, 2011). In agreement with a proposed co-recruitment model of Tra2-β1 and hnRNP G regulating factors in alternative splicing (Hofmann & Wirth, 2002), a recent structural work with hnRNP G RRM (Moursy et al, 2014) indeed established this protein's preference for adenine repeats.…”

“…As discussed earlier as well, follow-up in vivo studies support the functional relevance of the structural information. Mutational analyses allowed to validate the intermolecular contacts observed in the complex structures and confirmed the role of RRMs in specific recognition of conserved sites regulating alternative splicing Daubner et al, 2012;Moursy et al, 2014) or translation (Afroz et al, 2014) . The conformational constraints in protein domain orientation found in the structures of Tra2-β1 RRM (Cléry et al, 2011) or PTB RRM34 (Lamichhane et al, 2010;Oberstrass et al, 2005) bound to RNA allowed to hypothesize about interdomain or interprotein interaction and to suggest models for splicing regulation mechanism.…”

“…In addition to stabilize the RNA molecule, they provide a large set of additional sidechain and main-chain contacts that are used to increase the repertoire of sequences recognized by RRMs. The N-terminus (CUGBP1 (Tsuda et al, 2009), CBP20 (Mazza et al, 2002)), the C-terminus (PTB (Oberstrass et al, 2005), PABP (Deo et al, 1999), RBMY (Skrisovska et al, 2007), DAZL ( Jenkins et al, 2011), SUP-12 (Amrane et al, 2014;Kuwasako et al, 2014), hnRNP G (Moursy et al, 2014), and Musashi1 (Ohyama et al, 2012)) or both extremities (Tra2-β1 (Cléry et al, 2011;Tsuda et al, 2011), SRSF2 (Daubner et al, 2012), and hnRNP C (Cienikova et al, 2014)) were used for this purpose in the respective complexes. Importantly, these domain extensions do not need to adopt a secondary structure to interact with RNA and the number of residues involved in RNA binding can be up to 10 amino acids as shown for CUGBP1 RRM3 N-terminus (Tsuda et al, 2009).…”

The role of short RNA loops in recognition of a single-hairpin exon derived from a mammalian-wide interspersed repeat

“…Future studies should therefore explore the role of proteins that recruit Tra2 to MIR RNA, including hnRNP G, which was recently shown to contact diadenosines in single-stranded regions. 66 As shown for SMN2 exon 7, 67 SSOs that bind exonic sequences and promote their inclusion in mRNA are relatively uncommon. For example, exon-promoting antisense targets were found only for 2/10 (20%) tested oligos in the initial walk across SMN2 exon 7.…”

“…As shown for group II self-splicing introns, 72 adoption of complex tertiary structures could remain very important also for splicing catalysis of their likely evolutionary descendants, i.e., human introns. Our model should therefore prove useful for evaluating the relative importance of diadenosine binding proteins 66 and intramolecular tertiary contacts in RNA processing. Ultimately, high resolution structures will help us understand how hairpins facilitate exon selection and alternative splicing, which should substantially improve future iterations of exonic and intronic splicing code, accurate prediction of phenotypic consequences of disease-causing splicing mutations and their correction by antisense strategies.…”