Dynamic stacking of an expected branch point adenosine in duplexes containing pseudouridine-modified or unmodified U2 snRNA sites.

The pre-mRNA branch point sequence (BPS) anneals with a pseudouridine-modified region of the U2 small nuclear (sn)RNA, and offers a 2' hydroxyl group of a bulged adenosine as the nucleophile for the first catalytic step of pre-mRNA splicing. To increase our structural understanding of branch site selection, we characterized a duplex containing a BPS sequence that is common among multicellular eukaryotes (5'-UACUGAC-3') and the complementary U2 snRNA site using NMR. A major conformation of the expected branch site adenosine stacked within the duplex and paired with the conserved pseudouridine of the U2 snRNA strand. In contrast, the guanosine preceding the branch site appeared flexible and had weak contacts with the surrounding nucleotides. Pseudouridine-modified and unmodified U2 snRNA-BPS-containing duplexes remained structurally similar. These results highlight the importance of auxiliary factors to achieve the active bulged conformation of the branch site nucleophile for the first step of pre-mRNA splicing.

Three RNA recognition motifs participate in RNA recognition and structural organization by the pro-apoptotic factor TIA-1.

T-cell intracellular antigen-1 (TIA-1) regulates developmental and stress-responsive pathways through distinct activities at the levels of alternative pre-mRNA splicing and mRNA translation. The TIA-1 polypeptide contains three RNA recognition motifs (RRMs). The central RRM2 and C-terminal RRM3 associate with cellular mRNAs. The N-terminal RRM1 enhances interactions of a C-terminal Q-rich domain of TIA-1 with the U1-C splicing factor, despite linear separation of the domains in the TIA-1 sequence. Given the expanded functional repertoire of the RRM family, it was unknown whether TIA-1 RRM1 contributes to RNA binding as well as documented protein interactions. To address this question, we used isothermal titration calorimetry and small-angle X-ray scattering to dissect the roles of the TIA-1 RRMs in RNA recognition. Notably, the fas RNA exhibited two binding sites with indistinguishable affinities for TIA-1. Analyses of TIA-1 variants established that RRM1 was dispensable for binding AU-rich fas sites, yet all three RRMs were required to bind a polyU RNA with high affinity. Small-angle X-ray scattering analyses demonstrated a "V" shape for a TIA-1 construct comprising the three RRMs and revealed that its dimensions became more compact in the RNA-bound state. The sequence-selective involvement of TIA-1 RRM1 in RNA recognition suggests a possible role for RNA sequences in regulating the distinct functions of TIA-1. Further implications for U1-C recruitment by the adjacent TIA-1 binding sites of the fas pre-mRNA and the bent TIA-1 shape, which organizes the N- and C-termini on the same side of the protein, are discussed.