Global identification of hnRNP A1 binding sites for SSO-based splicing modulation

Bruun, Georg M.; Doktor, Thomas Koed; Borch-Jensen, Jonas; Matsubara, Akio; Krainer, Adrian R.; Ohno, Kinji; Andresen, Brage S.

doi:10.1186/s12915-016-0279-9

Cited by 66 publications

(75 citation statements)

References 96 publications

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“…Quantitative sequence specificity modeling of the resulting distribution of binding affinities allowed the intrinsic determinants of hnRNP A1 recognition to be identified. HnRNP A1 is generally characterized as nonspecific owing to its wide range of cellular functions; however, crosslinking immunoprecipitation (CLIP) and RNAcompete studies identified similar consensus patterns that center around a composite UAGG motif (11,24,34). Consistent with those results, the highest affinity SL3 ESS3 variant identified from HTS-EQ has a GUAGGAG sequence and 76% of the top 50 variants all contain UAG.…”

Section: Ess3supporting

confidence: 71%

“…Selection amplification experiment(s) (SELEX) identified a high-affinity motif 5′-UAGGGA/U-3′ that now serves as a benchmark for sequence-specific hnRNP A1 binding (23). Transcriptome-wide studies of functional hnRNP A1 binding sites using motif finder algorithms further indicate specificity for a 5′-UAG-3′ motif (11,24). In previous work, we determined a small angle X-ray scattering (SAXS) model of UP1 in complex with the HIV ESS3 stem loop (SL3 ESS3 ), which was guided by a 1.92-Å crystal structure of UP1 bound to a 5′-AGU-3′ oligomer (25).…”

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confidence: 99%

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Rules of RNA specificity of hnRNP A1 revealed by global and quantitative analysis of its affinity distribution

Jain

Lin

Morgan

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a multipurpose RNA-binding protein (RBP) involved in normal and pathological RNA metabolism. Transcriptome-wide mapping and in vitro evolution identify consensus hnRNP A1 binding motifs; however, such data do not reveal how surrounding RNA sequence and structural context modulate affinity. We determined the affinity of hnRNP A1 for all possible sequence variants (n = 16,384) of the HIV exon splicing silencer 3 (ESS3) 7-nt apical loop. Analysis of the affinity distribution identifies the optimal motif 5′-YAG-3′ and shows how its copy number, position in the loop, and loop structure modulate affinity. For a subset of ESS3 variants, we show that specificity is determined by association rate constants and that variants lacking the minimal sequence motif bind competitively with consensus RNA. Thus, the results reveal general rules of specificity of hnRNP A1 and provide a quantitative framework for understanding how it discriminates between alternative competing RNA ligands in vivo.hnRNP A1 | protein-RNA specificity | RNA structure | thermodynamics | binding kinetics G ene expression is regulated by an ensemble of protein-RNA complexes that assemble and disassemble throughout the lifetime of a transcript (1-5). Knowledge of the determinants of RNA-binding protein (RBP) specificity is therefore essential to understanding the relative affinity for sites of association within the transcriptome. A characteristic feature of many RBPs is their ability to elicit biological function by binding at sites in RNAs that vary significantly in sequence and local structure. This broad specificity of RBPs challenges the simplistic description of RNA binding as either specific or nonspecific. Global profiling methods provide consensus sequence motifs that represent preferential binding sites by RBPs in the transcriptome (6-11). Although powerful, such approaches are usually nonquantitative. Moreover, they do not readily provide information on how surrounding sequence identity or positioning of preferred sequences within higher order structure alters affinity. Methods such as high-throughput sequencing kinetics (HTS-KIN), RNA Bind-n-Seq, and RNA MaP have recently been developed that allow the affinities and reaction kinetics of thousands of RNAs to be measured simultaneously (6,(12)(13)(14)(15). These methods can potentially provide global information on the surrounding context of a given sequence motif; however, they have yet to see wide application for structure/function studies of RNA specificity.Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a diverse family of RBPs that are implicated at most stages of posttranscriptional gene regulation (16,17). The prototypical member of this family, hnRNP A1, regulates alternative splicing, nuclear export, translation, and other RNA processing events (17). HnRNP A1 has a modular domain organization consisting of tandem RNA recognition motifs (RRMs), collectively referred to as unwinding protein 1 (UP1), and an intrinsically di...

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Section: Ess3supporting

confidence: 71%

mentioning

confidence: 99%

Rules of RNA specificity of hnRNP A1 revealed by global and quantitative analysis of its affinity distribution

Jain

Lin

Morgan

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…First, both RRMs have a preference for the 5´-UAGG-3´ motif, which was indeed present in many RNAs found by SELEX or iCLIP (Burd and Dreyfuss, 1994; Bruun et al, 2016). Second, both RRMs are tolerant to accommodate a cytosine 5´ to the AG core, extending the recognition motif to 5´-U /C AGG-3´.…”

Section: Resultsmentioning

confidence: 90%

“…Interestingly, motifs of in vivo targets of hnRNP A1 can be fairly different (see for instance the ISS-N1 binding sites 5´-CAGCAU-3´ and 5´-UGAAAG-3´ in the context of SMN pre-mRNAs [Hua et al, 2008] or the binding sites in SKA2 exon 3 recently identified by iCLIP [Bruun et al, 2016]).…”

Section: Resultsmentioning

confidence: 99%

Tandem hnRNP A1 RNA recognition motifs act in concert to repress the splicing of survival motor neuron exon 7

Beusch

Barraud

Moursy

et al. 2017

eLife

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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

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“…A point mutation at position 6 in exon 7 of SMN2 eliminates an exonic splicing enhancer (ESE) recognized by serine/arginine-rich splicing factor 1 (SRSF1) (5,6) and creates an exonic splicing silencer (ESS) recognized by heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) (7). This ESS acts synergistically with other ESS motifs that bind to hnRNP A1 and Sam68, and repress exon 7 inclusion (8–11). This leads to the production of a truncated and unstable protein (12).…”

Section: Introductionmentioning

confidence: 99%

RNA-sequencing of a mouse-model of spinal muscular atrophy reveals tissue-wide changes in splicing of U12-dependent introns

et al. 2016

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Spinal Muscular Atrophy (SMA) is a neuromuscular disorder caused by insufficient levels of the Survival of Motor Neuron (SMN) protein. SMN is expressed ubiquitously and functions in RNA processing pathways that include trafficking of mRNA and assembly of snRNP complexes. Importantly, SMA severity is correlated with decreased snRNP assembly activity. In particular, the minor spliceosomal snRNPs are affected, and some U12-dependent introns have been reported to be aberrantly spliced in patient cells and animal models. SMA is characterized by loss of motor neurons, but the underlying mechanism is largely unknown. It is likely that aberrant splicing of genes expressed in motor neurons is involved in SMA pathogenesis, but increasing evidence indicates that pathologies also exist in other tissues. We present here a comprehensive RNA-seq study that covers multiple tissues in an SMA mouse model. We show elevated U12-intron retention in all examined tissues from SMA mice, and that U12-dependent intron retention is induced upon siRNA knock-down of SMN in HeLa cells. Furthermore, we show that retention of U12-dependent introns is mitigated by ASO treatment of SMA mice and that many transcriptional changes are reversed. Finally, we report on missplicing of several Ca2+ channel genes that may explain disrupted Ca2+ homeostasis in SMA and activation of Cdk5.

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Global identification of hnRNP A1 binding sites for SSO-based splicing modulation

Cited by 66 publications

References 96 publications

Rules of RNA specificity of hnRNP A1 revealed by global and quantitative analysis of its affinity distribution

Rules of RNA specificity of hnRNP A1 revealed by global and quantitative analysis of its affinity distribution

Tandem hnRNP A1 RNA recognition motifs act in concert to repress the splicing of survival motor neuron exon 7

RNA-sequencing of a mouse-model of spinal muscular atrophy reveals tissue-wide changes in splicing of U12-dependent introns

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