Molecular basis for AU-rich element recognition and dimerization by the HuR C-terminal RRM.

Human antigen R (HuR) is a key regulator of cellular mRNAs containing adenylate/uridylate-rich elements (AU-rich elements; AREs). These are a major class of cis elements within 3' untranslated regions, targeting these mRNAs for rapid degradation. HuR contains three RNA recognition motifs (RRMs): a tandem RRM1 and 2, followed by a flexible linker and a C-terminal RRM3. While RRM1 and 2 are structurally characterized, little is known about RRM3. Here we present a 1.9-Å-resolution crystal structure of RRM3 bound to different ARE motifs. This structure together with biophysical methods and cell-culture assays revealed the mechanism of RRM3 ARE recognition and dimerization. While multiple RNA motifs can be bound, recognition of the canonical AUUUA pentameric motif is possible by binding to two registers. Additionally, RRM3 forms homodimers to increase its RNA binding affinity. Finally, although HuR stabilizes ARE-containing RNAs, we found that RRM3 counteracts this effect, as shown in a cell-based ARE reporter assay and by qPCR with native HuR mRNA targets containing multiple AUUUA motifs, possibly by competing with RRM12.

The Xist RNA A-repeat comprises a novel AUCG tetraloop fold and a platform for multimerization.

X-chromosome inactivation (XCI) in female mammals depends on the noncoding RNA X inactivation specific transcript (Xist). The mechanism of chromosome-wide silencing by Xist is poorly understood. While it is established that the 5' region of Xist RNA, comprising the A-repeats and holding 7.5-8.5 copies of a conserved 26-mer sequence, is essential for Xist-mediated silencing, high-resolution structural information for the A-repeats is not available. Here, we report the three-dimensional solution structure of a 14-mer hairpin in the 5' region of a human A-repeat. This hairpin is remarkably stable and adopts a novel AUCG tetraloop fold, the integrity of which is required for silencing. We show that, contrary to previous predictions, the 3' region of single or tandem A-repeats mediates duplex formation in vitro. Significantly, mutations in this region disrupt the inter-repeat duplex formation in vitro and abrogate the silencing function of Xist A-repeats in vivo. Our data suggest that the complete A-repeat region may be stabilized by inter-repeat duplex formation and, as such, may provide a platform for multimerization and specific recognition of the AUCG tetraloops by trans-acting factors.

Efficient detection of hydrogen bonds in dynamic regions of RNA by sensitivity-optimized NMR pulse sequences.

Improved Sensitivity: Efficient NMR experiments are presented for determining the secondary structure in large and dynamic RNAs using J-couplings across hydrogen bonds. The experiments provide up to eight-fold improved sensitivity and thus enable detection of base pairs in dynamic regions even in large RNAs.

RNA recognition by Npl3p reveals U2 snRNA-binding compatible with a chaperone role during splicing.

The conserved SR-like protein Npl3 promotes splicing of diverse pre-mRNAs. However, the RNA sequence(s) recognized by the RNA Recognition Motifs (RRM1 & RRM2) of Npl3 during the splicing reaction remain elusive. Here, we developed a split-iCRAC approach in yeast to uncover the consensus sequence bound to each RRM. High-resolution NMR structures show that RRM2 recognizes a 5´-GNGG-3´ motif leading to an unusual mille-feuille topology. These structures also reveal how RRM1 preferentially interacts with a CC-dinucleotide upstream of this motif, and how the inter-RRM linker and the region C-terminal to RRM2 contribute to cooperative RNA-binding. Structure-guided functional studies show that Npl3 genetically interacts with U2 snRNP specific factors and we provide evidence that Npl3 melts U2 snRNA stem-loop I, a prerequisite for U2/U6 duplex formation within the catalytic center of the Bact spliceosomal complex. Thus, our findings suggest an unanticipated RNA chaperoning role for Npl3 during spliceosome active site formation.

The solution structure of Dead End bound to AU-rich RNA reveals an unusual mode of tandem RRM-RNA recognition required for mRNA regulation.

Dead End (DND1) is an RNA-binding protein essential for germline development through its role in post-transcriptional gene regulation. The molecular mechanisms behind selection and regulation of its targets are unknown. Here, we present the solution structure of DND1's tandem RNA Recognition Motifs (RRMs) bound to AU-rich RNA. The structure reveals how an NYAYUNN element is specifically recognized, reconciling seemingly contradictory sequence motifs discovered in recent genome-wide studies. RRM1 acts as a main binding platform, including atypical extensions to the canonical RRM fold. RRM2 acts cooperatively with RRM1, capping the RNA using an unusual binding pocket, leading to an unusual mode of tandem RRM-RNA recognition. We show that the consensus motif is sufficient to mediate upregulation of a reporter gene in human cells and that this process depends not only on RNA binding by the RRMs, but also on DND1's double-stranded RNA binding domain (dsRBD), which is dispensable for binding of a subset of targets in cellulo. Our results point to a model where DND1 target selection is mediated by a non-canonical mode of AU-rich RNA recognition by the tandem RRMs and a role for the dsRBD in the recruitment of effector complexes responsible for target regulation.

A NMR strategy to unambiguously distinguish nucleic acid hairpin and duplex conformations applied to a Xist RNA A-repeat.

All RNA sequences that fold into hairpins possess the intrinsic potential to form intermolecular duplexes because of their high self-complementarity. The thermodynamically more stable duplex conformation is favored under high salt conditions and at high RNA concentrations, posing a challenging problem for structural studies of small RNA hairpin conformations. We developed and applied a novel approach to unambiguously distinguish RNA hairpin and duplex conformations for the structural analysis of a Xist RNA A-repeat. Using a combination of a quantitative HNN-COSY experiment and an optimized double isotope-filtered NOESY experiment we could define the conformation of the 26-mer A-repeat RNA. In contrast to a previous secondary structure prediction of a double hairpin structure, the NMR data show that only the first predicted hairpin is formed, while the second predicted hairpin mediates dimerization of the A-repeat by duplex formation with a second A-repeat. The strategy employed here will be generally applicable to identify and quantify populations of hairpin and duplex conformations and to define RNA folding topology from inter- and intra-molecular base-pairing patterns.

¹H, ¹³C, ¹5N and ³¹P chemical shift assignments of a human Xist RNA A-repeat tetraloop hairpin essential for X-chromosome inactivation.

Initiation of X-chromosome inactivation in female mammals depends on the non-coding RNA Xist. We have solved the NMR structure of a 14-nucleotide hairpin with a novel AUCG tetraloop fold from a Xist A-repeat that is essential for silencing. The (1)H, (13)C, (15)N and (31)P chemical shift assignments are reported.