Biological Chemistry最新文献

Small non-coding RNAs (sncRNA) are involved in many steps of the gene expression cascade and regulate processing and expression of mRNAs by the formation of ribonucleoprotein complexes (RNP) such as the RNA-induced silencing complex (RISC). By analyzing small RNA Seq data sets, we identified a sncRNA annotated as piR-hsa-1254, which is likely derived from the 3'-end of 7SL RNA2 (RN7SL2), herein referred to as snc7SL RNA. The 7SL RNA is an abundant long non-coding RNA polymerase III transcript and serves as structural component of the cytoplasmic signal recognition particle (SRP). To evaluate a potential functional role of snc7SL RNA, we aimed to define its cellular localization by live cell imaging. Therefore, a Molecular Beacon (MB)-based method was established to compare the subcellular localization of snc7SL RNA with its precursor 7SL RNA. We designed and characterized several MBs in vitro and tested those by live cell fluorescence microscopy. Using a multiplex approach, we show that 7SL RNA localizes mainly to the endoplasmic reticulum (ER), as expected for the SRP, whereas snc7SL RNA predominately localizes to the nucleus. This finding suggests a fundamentally different function of 7SL RNA and its derivate snc7SL RNA.

Glucosinolates are plant thioglucosides, which act as chemical defenses. Upon tissue damage, their myrosinase-catalyzed hydrolysis yields aglucones that rearrange to toxic isothiocyanates. Specifier proteins such as thiocyanate-forming protein from Thlaspi arvense (TaTFP) are non-heme iron proteins, which capture the aglucone to form alternative products, e.g. nitriles or thiocyanates. To resolve the electronic state of the bound iron cofactor in TaTFP, we applied continuous wave electron paramagnetic resonance (CW EPR) spectroscopy at X-and Q-band frequencies (∼9.4 and ∼34 GHz). We found characteristic features of high spin and low spin states of a d ⁵ electronic configuration and local rhombic symmetry during catalysis. We monitored the oxidation states of bound iron during conversion of allylglucosinolate by myrosinase and TaTFP in presence and absence of supplemented Fe²⁺. Without added Fe²⁺, most high spin features of bound Fe³⁺ were preserved, while different g'-values of the low spin part indicated slight rearrangements in the coordination sphere and/or structural geometry. We also examined involvement of the redox pair Fe³⁺/Fe² in samples with supplemented Fe²⁺. The absence of any EPR signal related to Fe³⁺ or Fe²⁺ using an iron-binding deficient TaTFP variant allowed us to conclude that recorded EPR signals originated from the bound iron cofactor.

Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder with currently no cure. Central to the cellular dysfunction associated with this fatal proteinopathy is the accumulation of unfolded/misfolded superoxide dismutase 1 (SOD1) in various subcellular locations. The molecular mechanism driving the formation of SOD1 aggregates is not fully understood but numerous studies suggest that aberrant aggregation escalates with folding instability of mutant apoSOD1. Recent advances on combining organelle-targeting therapies with the anti-aggregation capacity of chemical chaperones have successfully reduce the subcellular load of misfolded/aggregated SOD1 as well as their downstream anomalous cellular processes at low concentrations (micromolar range). Nevertheless, if such local aggregate reduction directly correlates with increased folding stability remains to be explored. To fill this gap, we synthesized and tested here the effect of 9 ER-, mitochondria- and lysosome-targeted chemical chaperones on the folding stability of truncated monomeric SOD1 (SOD1_bar) mutants directed to those organelles. We found that compound ER-15 specifically increased the native state stability of ER-SOD1_bar-A4V, while scaffold compound FDA-approved 4-phenylbutyric acid (PBA) decreased it. Furthermore, our results suggested that ER15 mechanism of action is distinct from that of PBA, opening new therapeutic perspectives of this novel chemical chaperone on ALS treatment.

The activity of neuronal Kv7.2/Kv7.3 channels is critically dependent on PIP₂ and finely modulated by cholesterol. Here, we report the crosstalk between cholesterol and PIP₂ in the regulation of Kv7.2/Kv7.3 channels. Our results show that currents passing through Kv7.2/Kv7.3 channels in cholesterol-depleted cells, by acute application of methyl-β-cyclodextrin (MβCD), were less sensitive to PIP₂ dephosphorylation strategies than those of control cells, suggesting that cholesterol depletion enhances the Kv7.2/Kv7.3-PIP₂ interaction. In contrast, the sensitivity of Kv7.2/Kv7.3 channels to acute membrane cholesterol depletion by MβCD was not altered in mutant channels with different apparent affinities for PIP₂.

Mammalian genomes are extensively transcribed, producing a large number of coding and non-coding transcripts. A large fraction of the nuclear RNAs is physically associated with chromatin, functioning in gene activation and silencing, shaping higher-order genome organisation, such as involvement in long-range enhancer-promoter interactions, transcription hubs, heterochromatin, nuclear bodies and phase transitions. Different mechanisms allow the tethering of these chromatin-associated RNAs (caRNA) to chromosomes, including RNA binding proteins, the RNA polymerases and R-loops. In this review, we focus on the sequence-specific targeting of RNA to DNA by forming triple helical structures and describe its interplay with chromatin. It turns out that nucleosome positioning at triple helix target sites and the nucleosome itself are essential factors in determining the formation and stability of triple helices. The histone H3-tail plays a critical role in triple helix stabilisation, and the role of its epigenetic modifications in this process is discussed.

There is a growing interest in characterizing the structure and dynamics of large biomolecular assemblies and their interactions within the cellular environment. A diverse array of experimental techniques allows us to study biomolecular systems on a variety of length and time scales. These techniques range from imaging with light, X-rays or electrons, to spectroscopic methods, cross-linking mass spectrometry and functional genomics approaches, and are complemented by AI-assisted protein structure prediction methods. A challenge is to integrate all of these data into a model of the system and its functional dynamics. This review focuses on Bayesian approaches to integrative structure modeling. We sketch the principles of Bayesian inference, highlight recent applications to integrative modeling and conclude with a discussion of current challenges and future perspectives.

Splicing of precursor mRNAs is a hallmark of eukaryotic cells, performed by a huge macromolecular machine, the spliceosome. Four DEAH-box ATPases are essential components of the spliceosome, which play an important role in the spliceosome activation, the splicing reaction, the release of the spliced mRNA and intron lariat, and the disassembly of the spliceosome. An integrative approach comprising X-ray crystallography, single particle cryo electron microscopy, single molecule FRET, and molecular dynamics simulations provided deep insights into the structure, dynamics and function of the spliceosomal DEAH-box ATPases.

Cell viability largely depends on the surveillance of mRNA export and translation. Upon pre-mRNA processing and nuclear quality control, mature mRNAs are exported into the cytoplasm via Mex67-Mtr2 attachment. At the cytoplasmic site of the nuclear pore complex, the export receptor is displaced by the action of the DEAD-box RNA helicase Dbp5. Subsequent quality control of the open reading frame requires translation. Our studies suggest an involvement of Dbp5 in cytoplasmic no-go-and non-stop decay. Most importantly, we have also identified a key function for Dbp5 in translation termination, which identifies this helicase as a master regulator of mRNA expression.