The recognition of
-regulatory RNA motifs in human transcripts by RNA binding proteins (RBPs) is essential for gene regulation. The molecular features that determine RBP specificity are often poorly ...understood. Here, we combined NMR structural biology with high-throughput iCLIP approaches to identify a regulatory mechanism for U2AF2 RNA recognition. We found that the intrinsically disordered linker region connecting the two RNA recognition motif (RRM) domains of U2AF2 mediates autoinhibitory intramolecular interactions to reduce nonproductive binding to weak Py-tract RNAs. This proofreading favors binding of U2AF2 at stronger Py-tracts, as required to define 3' splice sites at early stages of spliceosome assembly. Mutations that impair the linker autoinhibition enhance the affinity for weak Py-tracts result in promiscuous binding of U2AF2 along mRNAs and impact on splicing fidelity. Our findings highlight an important role of intrinsically disordered linkers to modulate RNA interactions of multidomain RBPs.
Splicing of pre-mRNAs critically contributes to gene regulation and proteome expansion in eukaryotes, but our understanding of the recognition and pairing of splice sites during spliceosome assembly ...lacks detail. Here, we identify the multidomain RNA-binding protein FUBP1 as a key splicing factor that binds to a hitherto unknown cis-regulatory motif. By collecting NMR, structural, and in vivo interaction data, we demonstrate that FUBP1 stabilizes U2AF2 and SF1, key components at the 3' splice site, through multivalent binding interfaces located within its disordered regions. Transcriptional profiling and kinetic modeling reveal that FUBP1 is required for efficient splicing of long introns, which is impaired in cancer patients harboring FUBP1 mutations. Notably, FUBP1 interacts with numerous U1 snRNP-associated proteins, suggesting a unique role for FUBP1 in splice site bridging for long introns. We propose a compelling model for 3' splice site recognition of long introns, which represent 80% of all human introns.
The recognition of cis-regulatory RNA motifs in human transcripts by RNA binding proteins (RBPs) is essential for gene regulation. The molecular features that determine RBP specificity are often ...poorly understood. Here, we combined NMR structural biology with high-throughput iCLIP approaches to identify a regulatory mechanism for U2AF2 RNA recognition. We found that the intrinsically disordered linker region connecting the two RNA recognition motif (RRM) domains of U2AF2 mediates autoinhibitory intramolecular interactions to reduce nonproductive binding to weak Py-tract RNAs. This proofreading favors binding of U2AF2 at stronger Py-tracts, as required to define 3′ splice sites at early stages of spliceosome assembly. Mutations that impair the linker autoinhibition enhance the affinity for weak Py-tracts result in promiscuous binding of U2AF2 along mRNAs and impact on splicing fidelity. Our findings highlight an important role of intrinsically disordered linkers to modulate RNA interactions of multidomain RBPs.
Reactive aldehydes are produced by normal cellular metabolism or after alcohol consumption, and they accumulate in human tissues if aldehyde clearance mechanisms are impaired. Their toxicity has been ...attributed to the damage they cause to genomic DNA and the subsequent inhibition of transcription and replication. However, whether interference with other cellular processes contributes to aldehyde toxicity has not been investigated. We demonstrate that formaldehyde induces RNA-protein crosslinks (RPCs) that stall the ribosome and inhibit translation in human cells. RPCs in the messenger RNA (mRNA) are recognized by the translating ribosomes, marked by atypical K6-linked ubiquitylation catalyzed by the RING-in-between-RING (RBR) E3 ligase RNF14, and subsequently resolved by the ubiquitin- and ATP-dependent unfoldase VCP. Our findings uncover an evolutionary conserved formaldehyde-induced stress response pathway that protects cells against RPC accumulation in the cytoplasm, and they suggest that RPCs contribute to the cellular and tissue toxicity of reactive aldehydes.
Innate immunity represents the first line of defense in animals. We report a genome-wide in vivo Drosophila RNA interference screen to uncover genes involved in susceptibility or resistance to ...intestinal infection with the bacterium Serratia marcescens. We first employed whole-organism gene suppression, followed by tissue-specific silencing in gut epithelium or hemocytes to identify several hundred genes involved in intestinal antibacterial immunity. Among the pathways identified, we showed that the JAK-STAT signaling pathway controls host defense in the gut by regulating stem cell proliferation and thus epithelial cell homeostasis. Therefore, we revealed multiple genes involved in antibacterial defense and the regulation of innate immunity.
Abstract Objective Compare morphological and functional coronary plaque markers derived from coronary CT angiography (CCTA) for their ability to detect lesion-specific ischemia. Materials and methods ...Data of patients who had undergone both dual-source CCTA and invasive fractional flow reserve (FFR) measurement within 3 months were retrospectively analyzed. Various quantitative stenosis markers were derived from CCTA: Corrected coronary opacification (CCO), transluminal attenuation gradient (TAG), remodeling index (RI), computational FFR (cFFR), lesion length (LL), vessel volume (VV), total plaque volume (TPV), and calcified and non-calcified plaque volume (CPV and NCPV). Discriminatory power of these markers for flow-limiting versus non-significant coronary stenosis was assessed against invasive FFR as the reference standard. Results The cohort included 37 patients (61±12 years, 68% male). Among 37 lesions, 11 were hemodynamically significant by FFR. On a per-lesion level, sensitivity and specificity of TPV, CPV, and NCPV for hemodynamically significant stenosis detection were 88% and 74%, 67% and 53%, and 92% and 81%, respectively. For CCO, TAG, RI, and cFFR these were 64% and 86%, 35% and 56%, 82% and 54%, and 100% and 90%, respectively. At ROC analysis, only TPV (0.78, p =0.013), NCPV (0.79, p =0.009), cFFR (0.85, p =0.003), and CCO (0.82, p =0.0003) showed discriminatory power for detecting hemodynamically significant stenosis. Conclusion TPV, NCPV, CCO, and cFFR derived from CCTA can aid detecting hemodynamically significant coronary lesions with cFFR showing the greatest discriminatory ability.
Innate immunity represents the first line of defense in animals. We report a genome-wide
in vivo Drosophila
RNA interference screen to uncover genes involved in susceptibility or resistance to ...intestinal infection with the bacterium
Serratia marcescens
. We employed first whole-organism gene suppression followed by tissue-specific silencing in gut epithelium or hemocytes to identify several hundred genes involved in intestinal anti-bacterial immunity. Among the pathways identified, we showed that the JAK-STAT signaling pathway controls host defense in the gut by regulating stem cell proliferation and thus epithelial cell homeostasis. Thus, we revealed multiple genes involved in anti-bacterial defense and the regulation of innate immunity.
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