Structural basis for dual roles of Aar2p in U5 snRNP assembly

Yeast U5 small nuclear ribonucleoprotein particle (snRNP) is assembled via a cytoplasmic precursor that contains the U5-specific Prp8 protein but lacks the U5-specific Brr2 helicase. Instead, pre-U5 snRNP includes the Aar2 protein not found in mature U5 snRNP or spliceosomes. Aar2p and Brr2p bind competitively to a C-terminal region of Prp8p that comprises consecutive RNase H-like and Jab1/MPN-like domains. To elucidate the molecular basis for this competition, we determined the crystal structure of Aar2p in complex with the Prp8p RNase H and Jab1/MPN domains. Aar2p binds on one side of the RNase H domain and extends its C terminus to the other side, where the Jab1/MPN domain is docked onto a composite Aar2p–RNase H platform. Known Brr2p interaction sites of the Jab1/MPN domain remain available, suggesting that Aar2p-mediated compaction of the Prp8p domains sterically interferes with Brr2p binding. Moreover, Aar2p occupies known RNA-binding sites of the RNase H domain, and Aar2p interferes with binding of U4/U6 di-snRNA to the Prp8p C-terminal region. Structural and functional analyses of phospho-mimetic mutations reveal how phosphorylation reduces affinity of Aar2p for Prp8p and allows Brr2p and U4/U6 binding. Our results show how Aar2p regulates both protein and RNA binding to Prp8p during U5 snRNP assembly

Site-specific covalent labeling of His-tag fused proteins with N-acyl-N-alkyl sulfonamide reagent

The ability to incorporate a desired functionality into proteins of interest in a site-specific manner can provide powerful tools for investigating biological systems and creating therapeutic conjugates. However, there are not any universal methods that can be applied to all proteins, and it is thus important to explore the chemical strategy for protein modification. In this paper, we developed a new reactive peptide tag/probe pair system for site-specific covalent protein labeling. This method relies on the recognition-driven reaction of a peptide tag and a molecular probe, which comprises the lysine-containing short histidine tag (KH6 or H6K) and a binuclear nickel (II)- nitrilotriacetic acid (Ni²⁺-NTA) complex probe containing a lysine-reactive N-acyl-N-alkyl sulfonamide (NASA) group. The selective interaction of the His-tag and Ni²⁺–NTA propeles a rapid nucleophilic reaction between a lysine residue of the tag and the electrophilic NASA group of the probe by the proximity effect, resulting in the tag-site-specific functionalization of proteins. We characterized the reactive profile and site-specificity of this method using model peptides and proteins in vitro, and demonstrated the general utility for production of a nanobody-chemical probe conjugate without compromising its binding ability

Structural and functional investigation of the human snRNP assembly factor AAR2 in complex with the RNase H-like domain of PRPF8

Small nuclear ribonucleoprotein complexes (snRNPs) represent the main subunits of the spliceosome. While the assembly of the snRNP core particles has been well characterized, comparably little is known of the incorporation of snRNP-specific proteins and the mechanisms of snRNP recycling. U5 snRNP assembly in yeast requires binding of the the Aar2 protein to Prp8p as a placeholder to preclude premature assembly of the SNRNP200 helicase, but the role of the human AAR2 homolog has not yet been investigated in detail. Here, a crystal structure of human AAR2 in complex with the RNase H-like domain of the U5-specific PRPF8 (PRP8F RH) is reported, revealing a significantly different interaction between the two proteins compared with that in yeast. Based on the structure of the AAR2–PRPF8 RH complex, the importance of the interacting regions and residues was probed and AAR2 variants were designed that failed to stably bind PRPF8 in vitro. Protein-interaction studies of AAR2 with U5 proteins using size-exclusion chromatography reveal similarities and marked differences in the interaction patterns compared with yeast Aar2p and imply phosphorylation-dependent regulation of AAR2 reminiscent of that in yeast. It is found that in vitro AAR2 seems to lock PRPF8 RH in a conformation that is only compatible with the first transesterification step of the splicing reaction and blocks a conformational switch to the step 2-like, Mg2+-coordinated conformation that is likely during U5 snRNP biogenesis. These findings extend the picture of AAR2 PRP8 interaction from yeast to humans and indicate a function for AAR2 in the spliceosomal assembly process beyond its role as an SNRNP200 placeholder in yeast

Intrinsic Thermal Sensing Controls Proteolysis of Yersinia Virulence Regulator RovA

Pathogens, which alternate between environmental reservoirs and a mammalian host, frequently use thermal sensing devices to adjust virulence gene expression. Here, we identify the Yersinia virulence regulator RovA as a protein thermometer. Thermal shifts encountered upon host entry lead to a reversible conformational change of the autoactivator, which reduces its DNA-binding functions and renders it more susceptible for proteolysis. Cooperative binding of RovA to its target promoters is significantly reduced at 37°C, indicating that temperature control of rovA transcription is primarily based on the autoregulatory loop. Thermally induced reduction of DNA-binding is accompanied by an enhanced degradation of RovA, primarily by the Lon protease. This process is also subject to growth phase control. Studies with modified/chimeric RovA proteins indicate that amino acid residues in the vicinity of the central DNA-binding domain are important for proteolytic susceptibility. Our results establish RovA as an intrinsic temperature-sensing protein in which thermally induced conformational changes interfere with DNA-binding capacity, and secondarily render RovA susceptible to proteolytic degradation

IRS4, a novel modulator of BMP/Smad and Akt signalling during early muscle differentiation

Elaborate regulatory networks of the Bone Morphogenetic Protein (BMP) pathways ensure precise signalling outcome during cell differentiation and tissue homeostasis. Here, we identified IRS4 as a novel regulator of BMP signal transduction and provide molecular insights how it integrates into the signalling pathway. We found that IRS4 interacts with the BMP receptor BMPRII and specifically targets Smad1 for proteasomal degradation consequently leading to repressed BMP/Smad signalling in C2C12 myoblasts while concomitantly activating the PI3K/Akt axis. IRS4 is present in human and primary mouse myoblasts, the expression increases during myogenic differentiation but is downregulated upon final commitment coinciding with Myogenin expression. Functionally, IRS4 promotes myogenesis in C2C12 cells, while IRS4 knockdown inhibits differentiation of myoblasts. We propose that IRS4 is particularly critical in the myoblast stage to serve as a molecular switch between BMP/Smad and Akt signalling and to thereby control cell commitment. These findings provide profound understanding of the role of BMP signalling in early myogenic differentiation and open new ways for targeting the BMP pathway in muscle regeneration

Common and divergent features in transcriptional control of the homologous small RNAs GlmY and GlmZ in Enterobacteriaceae

Small RNAs GlmY and GlmZ compose a cascade that feedback-regulates synthesis of enzyme GlmS in Enterobacteriaceae. Here, we analyzed the transcriptional regulation of glmY/glmZ from Yersinia pseudotuberculosis, Salmonella typhimurium and Escherichia coli, as representatives for other enterobacterial species, which exhibit similar promoter architectures. The GlmY and GlmZ sRNAs of Y. pseudotuberculosis are transcribed from σ54-promoters that require activation by the response regulator GlrR through binding to three conserved sites located upstream of the promoters. This also applies to glmY/glmZ of S. typhimurium and glmY of E. coli, but as a difference additional σ70-promoters overlap the σ54-promoters and initiate transcription at the same site. In contrast, E. coli glmZ is transcribed from a single σ70-promoter. Thus, transcription of glmY and glmZ is controlled by σ54 and the two-component system GlrR/GlrK (QseF/QseE) in Y. pseudotuberculosis and presumably in many other Enterobacteria. However, in a subset of species such as E. coli this relationship is partially lost in favor of σ70-dependent transcription. In addition, we show that activity of the σ54-promoter of E. coli glmY requires binding of the integration host factor to sites upstream of the promoter. Finally, evidence is provided that phosphorylation of GlrR increases its activity and thereby sRNA expression

Correction: Contribution of the Cpx envelope stress system to metabolism and virulence regulation in Salmonella enterica serovar Typhimurium

[This corrects the article DOI: 10.1371/journal.pone.0211584.]

Cryo-electron tomography of NLRP3-activated ASC complexes reveals organelle co-localization.

NLRP3 induces caspase-1-dependent pyroptotic cell death to drive inflammation. Aberrant activity of NLRP3 occurs in many human diseases. NLRP3 activation induces ASC polymerization into a single, micron-scale perinuclear punctum. Higher resolution imaging of this signaling platform is needed to understand how it induces pyroptosis. Here, we apply correlative cryo-light microscopy and cryo-electron tomography to visualize ASC/caspase-1 in NLRP3-activated cells. The puncta are composed of branched ASC filaments, with a tubular core formed by the pyrin domain. Ribosomes and Golgi-like or endosomal vesicles permeate the filament network, consistent with roles for these organelles in NLRP3 activation. Mitochondria are not associated with ASC but have outer-membrane discontinuities the same size as gasdermin D pores, consistent with our data showing gasdermin D associates with mitochondria and contributes to mitochondrial depolarization

Structural basis for intrinsic thermosensing by the master virulence regulator RovA of Yersinia

Pathogens often rely on thermosensing to adjust virulence gene expression. In yersiniae, important virulence-associated traits are under the control of the master regulator RovA, which uses a built-in thermosensor to control its activity. Thermal upshifts encountered upon host entry induce conformational changes in the RovA dimer that attenuate DNA binding and render the protein more susceptible to proteolysis. Here, we report the crystal structure of RovA in the free and DNA-bound forms and provide evidence that thermo-induced loss of RovA activity is promoted mainly by a thermosensing loop in the dimerization domain and residues in the adjacent C-terminal helix. These determinants allow partial unfolding of the regulator upon an upshift to 37 °C. This structural distortion is transmitted to the flexible DNA-binding domain of RovA. RovA contacts mainly the DNA backbone in a low-affinity binding mode, which allows the immediate release of RovA from its operator sites. We also show that SlyA, a close homolog of RovA from Salmonella with a very similar structure, is not a thermosensor and remains active and stable at 37 °C. Strikingly, changes in only three amino acids, reflecting evolutionary replacements in SlyA, result in a complete loss of the thermosensing properties of RovA and prevent degradation. In conclusion, only minor alterations can transform a thermotolerant regulator into a thermosensor that allows adjustment of virulence and fitness determinants to their thermal environment

Structural and functional investigation of the human snRNP assembly factor AAR2 in complex with the RNase H like domain of PRPF8

Small nuclear ribonucleoprotein complexes snRNPs represent the main subunits of the spliceosome. While the assembly of the snRNP core particles has been well characterized, comparably little is known of the incorporation of snRNP specific proteins and the mechanisms of snRNP recycling. U5 snRNP assembly in yeast requires binding of the the Aar2 protein to Prp8p as a placeholder to preclude premature assembly of the SNRNP200 helicase, but the role of the human AAR2 homolog has not yet been investigated in detail. Here, a crystal structure of human AAR2 in complex with the RNase H like domain of the U5 specific PRPF8 PRP8F RH is reported, revealing a significantly different interaction between the two proteins compared with that in yeast. Based on the structure of the AAR2 PRPF8 RH complex, the importance of the interacting regions and residues was probed and AAR2 variants were designed that failed to stably bind PRPF8 in vitro. Protein interaction studies of AAR2 with U5 proteins using size exclusion chromatography reveal similarities and marked differences in the interaction patterns compared with yeast Aar2p and imply phosphorylation dependent regulation of AAR2 reminiscent of that in yeast. It is found that in vitro AAR2 seems to lock PRPF8 RH in a conformation that is only compatible with the first transesterification step of the splicing reaction and blocks a conformational switch to the step 2 like, Mg2 coordinated conformation that is likely during U5 snRNP biogenesis. These findings extend the picture of AAR2 PRP8 interaction from yeast to humans and indicate a function for AAR2 in the spliceosomal assembly process beyond its role as an SNRNP200 placeholder in yeas