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Thermodynamic stability of hnRNP A1 low complexity domain revealed by high-pressure NMR.

ABSTRACT: We have investigated the pressure- and temperature-induced conformational changes associated with the low complexity domain of hnRNP A1, an RNA-binding protein able to phase separate in response to cellular stress. Solution NMR spectra of the hnRNP A1 low-complexity domain fused with protein-G B1 domain were collected from 1 to 2500 bar and from 268 to 290 K. While the GB1 domain shows the typical pressure-induced and cold temperature-induced unfolding expected for small globular domains, the low-complexity domain of hnRNP A1 exhibits unusual pressure and temperature dependences. We observed that the low-complexity domain is pressure sensitive, undergoing a major conformational transition within the prescribed pressure range. Remarkably, this transition has the inverse temperature dependence of a typical folding-unfolding transition. Our results suggest the presence of a low-lying extended and fully solvated state(s) of the low-complexity domain that may play a role in phase separation. This study highlights the exquisite sensitivity of solution NMR spectroscopy to observe subtle conformational changes and illustrates how pressure perturbation can be used to determine the properties of metastable conformational ensembles.

SUBMITTER: Levengood JD

PROVIDER: S-EPMC9122033 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Thermodynamic and phylogenetic insights into hnRNP A1 recognition of the HIV-1 exon splicing silencer 3 element.

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Project description:This experiment identifies hnRNP A1 binding sites transcriptome-wide in Hela cells. HeLa cells with inducible expression of T7-tagged hnRNP A1 were grown to approximately 90% confluence and then subject to iCLIP analysis (following the protocol from Huppertz et al. 2014 (iCLIP: protein-RNA interactions at nucleotide resolution)). The iCLIP library was sequenced using Illumina's HighSeq 1500

2016-06-23 | E-MTAB-3612 | biostudies-arrayexpress

hnRNP A1 iCLIP

Project description:hnRNP A1 iCLIP

| PRJEB9546 | ENA

The hnRNP-like Nab3 termination factor can employ heterologous prion-like domains in place of its own essential low complexity domain.

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Project description:Heat shock protein (Hsp) 90 an emerging and attracting target in the anti-HIV drug discovery process due to the key role it plays in the pathogenicity of HIV-1 virus. In this research study, long-range all-atom molecular dynamics simulations were engaged for the bound and the unbound proteins to enhance the understanding of the molecular mechanisms of the Hsp90 dimerization and inhibition. Results evidently showed that coumermycin A1 (C-A1), a recently discovered Hsp90 inhibitor, binds at the dimer's active site of the Hsp90 protein and leads to a substantial parting between dimeric opposed residues, which include Arg591.B, Lys594.A, Ser663.A, Thr653.B, Ala665.A, Thr649.B, Leu646.B and Asn669.A. Significant differences in magnitudes were observed in radius of gyration, root-mean-square deviation and root-mean-square fluctuation, which confirms a reasonably more flexible state in the apo conformation associated with it dimerization. In contrast, the bound conformer of Hsp90 showed less flexibility. This visibly highpoints the inhibition process resulting from the binding of the ligand. These findings were further validated by principal component analysis. We believe that the detailed dynamic analyses of Hsp90 presented in this study, would give an imperative insight and better understanding to the function and mechanisms of inhibition. Furthermore, information obtained from the binding mode of the inhibitor would be of great assistance in the design of more potent inhibitors against the HIV target Hsp90.

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Project description:Human hnRNP A1 is a versatile single-stranded nucleic acid-binding protein that functions in various aspects of mRNA maturation and in telomere length regulation. The crystal structure of UP1, the amino-terminal domain of human hnRNP A1 containing two RNA-recognition motifs (RRMs), bound to a 12-nucleotide single-stranded telomeric DNA has been determined at 2.1 A resolution. The structure of the complex reveals the basis for sequence-specific recognition of the single-stranded overhangs of human telomeres by hnRNP A1. It also provides insights into the basis for high-affinity binding of hnRNP A1 to certain RNA sequences, and for nucleic acid binding and functional synergy between the RRMs. In the crystal structure, a UP1 dimer binds to two strands of DNA, and each strand contacts RRM1 of one monomer and RRM2 of the other. The two DNA strands are antiparallel, and regions of the protein flanking each RRM make important contacts with DNA. The extensive protein-protein interface seen in the crystal structure of the protein-DNA complex and the evolutionary conservation of the interface residues suggest the importance of specific protein-protein interactions for the sequence-specific recognition of single-stranded nucleic acids. Models for regular packaging of telomere 3' overhangs and for juxtaposition of alternative 5' splice sites are proposed.

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An intron element modulating 5' splice site selection in the hnRNP A1 pre-mRNA interacts with hnRNP A1.

Project description:The hnRNP A1 pre-mRNA is alternatively spliced to yield the A1 and A1b mRNAs, which encode proteins differing in their ability to modulate 5' splice site selection. Sequencing a genomic portion of the murine A1 gene revealed that the intron separating exon 7 and the alternative exon 7B is highly conserved between mouse and human. In vitro splicing assays indicate that a conserved element (CE1) from the central portion of the intron shifts selection toward the distal donor site when positioned in between the 5' splice sites of exon 7 and 7B. In vivo, the CE1 element promotes exon 7B skipping. A 17-nucleotide sequence within CE1 (CE1a) is sufficient to activate the distal 5' splice site. RNase T1 protection/immunoprecipitation assays indicate that hnRNP A1 binds to CE1a, which contains the sequence UAGAGU, a close match to the reported optimal A1 binding site, UAGGGU. Replacing CE1a by different oligonucleotides carrying the sequence UAGAGU or UAGGGU maintains the preference for the distal 5' splice site. In contrast, mutations in the AUGAGU sequence activate the proximal 5' splice site. In support of a direct role of the A1-CE1 interaction in 5'-splice-site selection, we observed that the amplitude of the shift correlates with the efficiency of A1 binding. Whereas addition of SR proteins abrogates the effect of CE1, the presence of CE1 does not modify U1 snRNP binding to competing 5' splice sites, as judged by oligonucleotide-targeted RNase H protection assays. Our results suggest that hnRNP A1 modulates splice site selection on its own pre-mRNA without changing the binding of U1 snRNP to competing 5' splice sites.

| S-EPMC232024 | biostudies-other

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