Summary FMRP loss-of-function causes Fragile X Syndrome (FXS) and autistic features. FMRP is a polyribosome-associated neuronal RNA-binding protein, suggesting that it plays a key role in regulating neuronal translation, but there has been little consensus regarding either its RNA targets or mechanism of action. Here we use high throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) to identify FMRP interactions with mouse brain polyribosomal mRNAs. FMRP interacts with the coding region of transcripts encoding pre- and postsynaptic proteins, and transcripts implicated in autism spectrum disorders (ASD). We developed a brain polyribosome-programmed translation system, revealing that FMRP reversibly stalls ribosomes specifically on its target mRNAs. Our results indicate that loss of a translational brake on the synthesis of a subset of synaptic proteins may contribute to FXS. In addition, they provide insight into the molecular basis of the cognitive and allied defects in FXS and ASD, and suggest multiple targets for clinical intervention.

SummaryProtein-RNA interactions play critical roles in all aspects of gene expression. Here we develop a genome-wide means of mapping protein-RNA binding sites in vivo, by high throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). HITS-CLIP analysis of the neuron-specific splicing factor Nova2 revealed extremely reproducible RNA binding maps in multiple mouse brains. These maps provide genome-wide in vivo biochemical footprints confirming the previous prediction that the position of Nova binding determines the outcome of alternative splicing; moreover, they are sufficiently powerful to predict Nova action de novo. HITS-CLIP revealed a large number of Nova-RNA interactions in 3′ UTRs, leading to the discovery that Nova regulates alternative polyadenylation in the brain. HITS-CLIP, therefore, provides a robust, unbiased means to identify functional protein-RNA interactions in vivo.

We adapted UV CLIP (cross-linking immunoprecipitation) to accurately locate tens of thousands of m 6 A residues in mammalian mRNA with single-nucleotide resolution. More than 70% of these residues are present in the 3 ′ -most (last) exons, with a very sharp rise (sixfold) within 150-400 nucleotides of the start of the last exon. Two-thirds of last exon m 6 A and >40% of all m 6 A in mRNA are present in 3 ′ untranslated regions (UTRs); contrary to earlier suggestions, there is no preference for location of m 6 A sites around stop codons. Moreover, m 6 A is significantly higher in noncoding last exons than in next-to-last exons harboring stop codons. We found that m 6 A density peaks early in the 3 ′ UTR and that, among transcripts with alternative polyA (APA) usage in both the brain and the liver, brain transcripts preferentially use distal polyA sites, as reported, and also show higher proximal m 6 A density in the last exons. Furthermore, when we reduced m6A methylation by knocking down components of the methylase complex and then examined 661 transcripts with proximal m6A peaks in last exons, we identified a set of 111 transcripts with altered (approximately two-thirds increased proximal) APA use. Taken together, these observations suggest a role of m 6 A modification in regulating proximal alternative polyA choice.

The SecA adenosine triphosphatase (ATPase) mediates extrusion of the amino termini of secreted proteins from the eubacterial cytosol based on cycles of reversible binding to the SecYEG translocon. We have determined the crystal structure of SecA with and without magnesium-adenosine diphosphate bound to the high-affinity ATPase site at 3.0 and 2.7 angstrom resolution, respectively. Candidate sites for preprotein binding are located on a surface containing the SecA epitopes exposed to the periplasm upon binding to SecYEG and are thus positioned to deliver preprotein to SecYEG. Comparisons with structurally related ATPases, including superfamily I and II ATP-dependent helicases, suggest that the interaction geometry of the tandem motor domains in SecA is modulated by nucleotide binding, which is shown by fluorescence anisotropy experiments to reverse an endothermic domain-dissociation reaction hypothesized to gate binding to SecYEG.

Understanding the biologic role of N 6 -methyladenosine (m 6 A) RNA modifications in mRNA requires an understanding of when and where in the life of a pre-mRNA transcript the modifications are made. We found that HeLa cell chromatin-associated nascent pre-mRNA (CA-RNA) contains many unspliced introns and m 6 A in exons but very rarely in introns. The m 6 A methylation is essentially completed upon the release of mRNA into the nucleoplasm. Furthermore, the content and location of each m 6 A modification in steady-state cytoplasmic mRNA are largely indistinguishable from those in the newly synthesized CA-RNA or nucleoplasmic mRNA. This result suggests that quantitatively little methylation or demethylation occurs in cytoplasmic mRNA. In addition, only ∼10% of m 6 As in CA-RNA are within 50 nucleotides of 5 ′ or 3 ′ splice sites, and the vast majority of exons harboring m 6 A in wild-type mouse stem cells is spliced the same in cells lacking the major m 6 A methyltransferase Mettl3. Both HeLa and mouse embryonic stem cell mRNAs harboring m 6 As have shorter half-lives, and thousands of these mRNAs have increased half-lives (twofold or more) in Mettl3 knockout cells compared with wild type. In summary, m 6 A is added to exons before or soon after exon definition in nascent pre-mRNA, and while m 6 A is not required for most splicing, its addition in the nascent transcript is a determinant of cytoplasmic mRNA stability. Studying nascent RNA synthesis in cultured cells using very brief pulse labeling with radioactive nucleosides allowed a number of advances in understanding premRNA synthesis and processing in the era before rapid nucleic acid sequencing. Examples include polyA addition on pre-mRNA before completion of mRNA processing and cytoplasmic entry (Darnell et al. 1971;Edmonds et al. 1971) and locating the first known boundaries of eukaryotic polymerase II transcription units through studying labeled nascent adenovirus transcripts (Bachenheimer and Darnell 1975;Evans et al. 1977;Weber et al. 1977).These early experiments were joined by a cell fractionation technique originated by Wuarin and Schibler (1994) that uses a 1 M urea solution to liberate a "chromatin" fraction from nuclei. This fraction provides a stringent purification of growing nascent pre-mRNA chains, isolated as a chromatin-associated RNA fraction (referred to as CA-RNA), along with RNA polymerase II plus all nuclear DNA and associated histones. Using specific labeled DNA probes, Wuarin and Schibler (1994) demonstrated removal in liver cell nuclei of some, but not all, introns from two specific nascent pre-mRNAs: a transcription factor pre-mRNA involved in circadian rhythm and the HMG coA reductase pre-mRNA. Recently, Pandya-Jones and Black (2009) adapted this procedure to study the extent and order of intron removal in cultured human carcinoma cell nuclei, again showing that many, but not all, introns are removed in CA-RNA.

microRNAs (miRNAs) act as sequence-specific guides for Argonaute (AGO) proteins, which mediate posttranscriptional silencing of target messenger RNAs. Despite their importance in many biological processes, rules governing AGO–miRNA targeting are only partially understood. Here we report a modified AGO HITS-CLIP strategy termed CLEAR (covalent ligation of endogenous Argonaute-bound RNAs)-CLIP, which enriches miRNAs ligated to their endogenous mRNA targets. CLEAR-CLIP mapped ∼130,000 endogenous miRNA–target interactions in mouse brain and ∼40,000 in human hepatoma cells. Motif and structural analysis define expanded pairing rules for over 200 mammalian miRNAs. Most interactions combine seed-based pairing with distinct, miRNA-specific patterns of auxiliary pairing. At some regulatory sites, this specificity confers distinct silencing functions to miRNA family members with shared seed sequences but divergent 3′-ends. This work provides a means for explicit biochemical identification of miRNA sites in vivo, leading to the discovery that miRNA 3′-end pairing is a general determinant of AGO binding specificity.

The control of RNA alternative splicing is critical for generating biological diversity. Despite emerging genome-wide technologies to study RNA complexity, reliable and comprehensive RNA-regulatory networks have not been defined. Here we used Bayesian networks to probabilistically model diverse datasets and predict the target networks of specific regulators. We applied this strategy to identify ~700 alternative splicing events directly regulated by the neuron-specific factor Nova in the mouse brain, integrating RNA-binding data, splicing microarray data, Nova-binding motifs, and evolutionary signatures. The resulting integrative network revealed combinatorial regulation by Nova and the neuronal splicing factor Fox, interplay between phosphorylation and splicing, and potential links to neurologic disease. Thus we have developed a general approach to understanding mammalian RNA regulation at the systems level.

Summary Hepatitis C virus uniquely requires the liver specific microRNA-122 for replication, yet global effects on endogenous miRNA targets during infection are unexplored. Here, high-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (Ago) during HCV infection showed robust Ago binding on the HCV 5′UTR, at known and predicted miR-122 sites. On the human transcriptome, we observed reduced Ago binding and functional mRNA de-repression of miR-122 targets during virus infection. This miR-122 “sponge” effect was relieved and redirected to miR-15 targets by swapping the miRNA tropism of the virus. Single-cell expression data from reporters containing miR-122 sites showed significant de-repression during HCV infection depending on expression level and site number. We describe a quantitative mathematical model of HCV induced miR-122 sequestration and propose that such miR-122 inhibition by HCV RNA may result in global de-repression of host miR-122 targets, providing an environment fertile for the long-term oncogenic potential of HCV.