Regulation and mechanisms of action of RNA helicases.

RNA helicases are an evolutionary conserved class of nucleoside triphosphate dependent enzymes found in all kingdoms of life. Their cellular functions range from transcription regulation up to maintaining genomic stability and viral defence. As dysregulation of RNA helicases has been shown to be involved in several cancers and various diseases, RNA helicases are potential therapeutic targets.

Structural basis of RNA-induced autoregulation of the DExH-type RNA helicase maleless.

RNA unwinding by DExH-type helicases underlies most RNA metabolism and function. It remains unresolved if and how the basic unwinding reaction of helicases is regulated by auxiliary domains. We explored the interplay between the RecA and auxiliary domains of the RNA helicase maleless (MLE) from Drosophila using structural and functional studies.

Structural basis for specific RNA recognition by the alternative splicing factor RBM5.

The RNA-binding motif protein RBM5 belongs to a family of multi-domain RNA binding proteins that regulate alternative splicing of genes important for apoptosis and cell proliferation and have been implicated in cancer. RBM5 harbors structural modules for RNA recognition, such as RRM domains and a Zn finger, and protein-protein interactions such as an OCRE domain. Here, we characterize binding of the RBM5 RRM1-ZnF1-RRM2 domains to cis-regulatory RNA elements.

Upstream of N-Ras C-terminal cold shock domains mediate poly(A) specificity in a novel RNA recognition mode and bind poly(A) binding protein.

RNA binding proteins (RBPs) often engage multiple RNA binding domains (RBDs) to increase target specificity and affinity. However, the complexity of target recognition of multiple RBDs remains largely unexplored. Here we use Upstream of N-Ras (Unr), a multidomain RBP, to demonstrate how multiple RBDs orchestrate target specificity.

A KLK6 Activity-Based Probe Reveals a Role for KLK6 Activity in Pancreatic Cancer Cell Invasion.

Pancreatic cancer has the lowest survival rate of all common cancers due to late diagnosis and limited treatment options. Serine hydrolases are known to mediate cancer progression and metastasis through initiation of signaling cascades and cleavage of extracellular matrix proteins, and the kallikrein-related peptidase (KLK) family of secreted serine proteases have emerging roles in pancreatic ductal adenocarcinoma (PDAC). However, the lack of reliable activity-based probes (ABPs) to profile KLK activity has hindered progress in validation of these enzymes as potential targets or biomarkers.

Correction: Scalable synthesis and structural characterization of reversible KLK6 inhibitors.

[This corrects the article DOI: 10.1039/D2RA04670A.].

Scalable synthesis and structural characterization of reversible KLK6 inhibitors.

Scalable asymmetric syntheses of two kallikrein-related protease 6 (KLK6) inhibitors are reported. The inhibitors are assembled by linking enantiomerically enriched fragments amide bond formation, followed by conversion of a cyano group to an amidine. One fragment, an amine, was prepared using the Ellman auxiliary, and a lack of clarity in the literature regarding the stereochemical outcome of this reaction was solved X-ray crystallographic analysis of two derivatives.

Structure and dynamics of the quaternary hunchback mRNA translation repression complex.

A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. This suppression is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development.

Molecular basis of mRNA transport by a kinesin-1-atypical tropomyosin complex.

Kinesin-1 carries cargos including proteins, RNAs, vesicles, and pathogens over long distances within cells. The mechanochemical cycle of kinesins is well described, but how they establish cargo specificity is not fully understood. Transport of mRNA to the posterior pole of the oocyte is mediated by kinesin-1, also called kinesin heavy chain (Khc), and a putative cargo adaptor, the atypical tropomyosin, Tm1.

Divergent evolution toward sex chromosome-specific gene regulation in .

The dosage compensation complex (DCC) of identifies its X-chromosomal binding sites with exquisite selectivity. The principles that assure this vital targeting are known from the model: DCC-intrinsic specificity of DNA binding, cooperativity with the CLAMP protein, and noncoding roX2 RNA transcribed from the X chromosome. We found that in , a species separated from by 40 million years of evolution, all principles are active but contribute differently to X specificity.

Identification of phenothiazine derivatives as UHM-binding inhibitors of early spliceosome assembly.

Interactions between U2AF homology motifs (UHMs) and U2AF ligand motifs (ULMs) play a crucial role in early spliceosome assembly in eukaryotic gene regulation. UHM-ULM interactions mediate heterodimerization of the constitutive splicing factors U2AF65 and U2AF35 and between other splicing factors that regulate spliceosome assembly at the 3' splice site, where UHM domains of alternative splicing factors, such as SPF45 and PUF60, contribute to alternative splicing regulation. Here, we performed high-throughput screening using fluorescence polarization assays with hit validation by NMR and identified phenothiazines as general inhibitors of UHM-ULM interactions.

Transcriptional regulation of the N -fructoselysine metabolism in Escherichia coli by global and substrate-specific cues.

Thermally processed food is an important part of the human diet. Heat-treatment, however, promotes the formation of so-called Amadori rearrangement products, such as fructoselysine. The gut microbiota including Escherichia coli can utilize these compounds as a nutrient source.

Integrative Structural Biology of Protein-RNA Complexes.

Ribonucleoprotein complexes (RNPs) are central to all processes in the cell. One of the prerequisites to understand how RNPs work is to determine their high-resolution structures. With the recent revolution in cryoelectron microscopy this task has become easier for large RNP machines, such as ribosomes, spliceosomes, and polymerases.

Switching the Post-translational Modification of Translation Elongation Factor EF-P.

Tripeptides with two consecutive prolines are the shortest and most frequent sequences causing ribosome stalling. The bacterial translation elongation factor P (EF-P) relieves this arrest, allowing protein biosynthesis to continue. A seven amino acids long loop between beta-strands β3/β4 is crucial for EF-P function and modified at its tip by lysylation of lysine or rhamnosylation of arginine.

Selective Inhibitors of FKBP51 Employ Conformational Selection of Dynamic Invisible States.

The recently discovered SAFit class of inhibitors against the Hsp90 co-chaperone FKBP51 show greater than 10 000-fold selectivity over its closely related paralogue FKBP52. However, the mechanism underlying this selectivity remained unknown. By combining NMR spectroscopy, biophysical and computational methods with mutational analysis, we show that the SAFit molecules bind to a transient pocket in FKBP51.

Structure, dynamics and roX2-lncRNA binding of tandem double-stranded RNA binding domains dsRBD1,2 of Drosophila helicase Maleless.

Maleless (MLE) is an evolutionary conserved member of the DExH family of helicases in Drosophila. Besides its function in RNA editing and presumably siRNA processing, MLE is best known for its role in remodelling non-coding roX RNA in the context of X chromosome dosage compensation in male flies. MLE and its human orthologue, DHX9 contain two tandem double-stranded RNA binding domains (dsRBDs) located at the N-terminal region.

Mechanism of Mg-Accompanied Product Release in Sugar Nucleotidyltransferases.

The nucleotidyl transfer reaction, catalyzed by sugar nucleotidyltransferases (SNTs), is assisted by two active site Mg ions. While studying this reaction using X-ray crystallography, we captured snapshots of the pyrophosphate (product) as it exits along a pocket. Surprisingly, one of the active site Mg ions remains coordinated to the exiting pyrophosphate.

Structural Basis for EarP-Mediated Arginine Glycosylation of Translation Elongation Factor EF-P.

Glycosylation is a universal strategy to posttranslationally modify proteins. The recently discovered arginine rhamnosylation activates the polyproline-specific bacterial translation elongation factor EF-P. EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP.

Segmental, Domain-Selective Perdeuteration and Small-Angle Neutron Scattering for Structural Analysis of Multi-Domain Proteins.

Multi-domain proteins play critical roles in fine-tuning essential processes in cellular signaling and gene regulation. Typically, multiple globular domains that are connected by flexible linkers undergo dynamic rearrangements upon binding to protein, DNA or RNA ligands. RNA binding proteins (RBPs) represent an important class of multi-domain proteins, which regulate gene expression by recognizing linear or structured RNA sequence motifs.

Rational Design of Cyclic Peptide Inhibitors of U2AF Homology Motif (UHM) Domains To Modulate Pre-mRNA Splicing.

U2AF homology motifs (UHMs) are atypical RNA recognition motif domains that mediate critical protein-protein interactions during the regulation of alternative pre-mRNA splicing and other processes. The recognition of UHM domains by UHM ligand motif (ULM) peptide sequences plays important roles during early steps of spliceosome assembly. Splicing factor 45 kDa (SPF45) is an alternative splicing factor implicated in breast and lung cancers, and splicing regulation of apoptosis-linked pre-mRNAs by SPF45 was shown to depend on interactions between its UHM domain and ULM motifs in constitutive splicing factors.

Structure, dynamics and RNA binding of the multi-domain splicing factor TIA-1.

Alternative pre-messenger ribonucleic acid (pre-mRNA) splicing is an essential process in eukaryotic gene regulation. The T-cell intracellular antigen-1 (TIA-1) is an apoptosis-promoting factor that modulates alternative splicing of transcripts, including the pre-mRNA encoding the membrane receptor Fas. TIA-1 is a multi-domain ribonucleic acid (RNA) binding protein that recognizes poly-uridine tract RNA sequences to facilitate 5' splice site recognition by the U1 small nuclear ribonucleoprotein (snRNP).

Crystal structures identify an atypical two-metal-ion mechanism for uridyltransfer in GlmU: its significance to sugar nucleotidyl transferases.

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU), exclusive to prokaryotes, is a bifunctional enzyme that synthesizes UDP-GlcNAc-an important component of the cell wall of many microorganisms. Uridyltransfer, one of the reactions it catalyzes, involves binding GlcNAc-1-P, UTP and Mg(2+) ions; however, whether one or two ions catalyze this reaction remains ambiguous. Here, we resolve this using biochemical and crystallographic studies on GlmU from Mycobacterium tuberculosis (GlmU(Mtb)) and identify a two-metal-ion mechanism (mechanism-B).