HnRNP A1 regulates many alternative splicing events by the recognition of splicing silencer elements. Here, we provide the solution structures of its two RNA recognition motifs (RRMs) in complex with short RNA. In addition, we show by NMR that both RRMs of hnRNP A1 can bind simultaneously to a single bipartite motif of the human intronic splicing silencer ISS-N1, which controls survival of motor neuron exon 7 splicing. RRM2 binds to the upstream motif and RRM1 to the downstream motif. Combining the insights from the structure with in cell splicing assays we show that the architecture and organization of the two RRMs is essential to hnRNP A1 function. The disruption of the inter-RRM interaction or the loss of RNA binding capacity of either RRM impairs splicing repression by hnRNP A1. Furthermore, both binding sites within the ISS-N1 are important for splicing repression and their contributions are cumulative rather than synergistic.DOI: http://dx.doi.org/10.7554/eLife.25736.001

Coupling of spliceosome complexity to intron diversityHighlights d Phylogenetic analysis reveals the ancestral spliceosome was complex d Human spliceosomal protein orthologs lost in S. cerevisiae are found in C. neoformans d Functional analysis in C. neoformans demonstrates roles in splicing fidelity d Proteomic and genetic analysis reveals functional modules

The polypyrimidine tract binding protein (PTB) is a multi-domain protein involved in alternative splicing, mRNA localization, stabilization, polyadenylation and translation initiation from internal ribosome entry sites (IRES). In this latter process, PTB promotes viral translation by interacting extensively with complex structured regions in the 5′-untranslated regions of viral RNAs at pyrimidine-rich targets located in single strand and hairpin regions. To better understand how PTB recognizes structured elements in RNA targets, we solved the solution structure of the N-terminal RNA recognition motif (RRM) in complex with an RNA hairpin embedding the loop sequence UCUUU, which is frequently found in IRESs of the picornovirus family. Surprisingly, a new three-turn α3 helix C-terminal to the RRM, folds upon binding the RNA hairpin. Although α3 does not mediate any contacts to the RNA, it acts as a sensor of RNA secondary structure, suggesting a role for RRM1 in detecting pyrimidine tracts in the context of structured RNA. Moreover, the degree of helix formation depends on the RNA loop sequence. Finally, we show that the α3 helix region, which is highly conserved in vertebrates, is crucial for PTB function in enhancing Encephalomyocarditis virus IRES activity.

Cross-linking coupled with mass spectrometry is an increasingly popular methodology for elucidating structural information from biological complexes. Whilst protein-protein cross-linking workflows are widely used and well characterised, adoption of protein-RNA cross-linking workflows for structural studies is less widespread, and data produced from such experiments remains less well understood. The cross-linking of stable isotope labelled RNA coupled to mass spectrometry (CLIR-MS) workflow uses isotope labelled RNA to simultaneously confirm that peptides are cross-linked to RNA and aid cross-link localisation in an RNA sequence. For broader application of CLIR-MS as part of the structural analysis of ribonucleoproteins, the method must be sensitive, robust, and its reaction products need to be well characterised. We enhanced our previously published workflow, improving coverage and sensitivity. We used it to infer common properties of protein-RNA cross-links such as cross-linking distance, and to assess the impact of substitution of uracil with 4-thio-uracil in structural proteomics experiments. We profiled the compositional diversity of RNA-derived peptide modifications, and subsequently defined a more inclusive data analysis approach which more than doubles the number of cross-link spectrum matches compared with our past work. We defined distance restraints from these cross-links, and with the aid of visualisation software, demonstrated that on their own they provide sufficient information to localise an RNA chain to the correct position on the surface of a protein. We applied our enhanced workflow and understanding to characterise the binding interface of several protein-RNA complexes containing classical and uncommon RNA binding domains. The enhanced sensitivity and understanding demonstrated here underpin a wider adoption of protein-RNA cross-linking in structural biology.