A role for DIS3L2 over natural nonsense-mediated mRNA decay targets in human cells.

The nonsense-mediated decay (NMD) pathway selectively degrades mRNAs carrying a premature translation-termination codon but also regulates the abundance of a large number of physiological mRNAs that encode full-length proteins. In human cells, NMD-targeted mRNAs are degraded by endonucleolytic cleavage and exonucleolytic degradation from both 5-' and 3'-ends. This is done by a process not yet completely understood that recruits decapping and 5'-to-3' exonuclease activities, as well as deadenylating and 3'-to-5' exonuclease exosome activities. In yeast, DIS3/Rrp44 protein is the catalytic subunit of the exosome, but in humans, there are three known paralogues of this enzyme: DIS3, DIS3L1, and DIS3L2. However, little is known about their role in NMD. Here, we show that some NMD-targets are DIS3L2 substrates in human cells. In addition, we observed that DIS3L2 acts over full-length transcripts, through a process that also involves UPF1. Moreover, DIS3L2-mediated decay is dependent on the activity of the terminal uridylyl transferases Zcchc6/11 (TUT7/4). Together, our findings establish a role for DIS3L2 and uridylation in NMD.

Experimental supporting data on DIS3L2 over nonsense-mediated mRNA decay targets in human cells.

In this article, we present supportive data related to the research article "A role for DIS3L2 over natural nonsense-mediated mRNA decay targets in human cells" [1], where interpretation of the data presented here is available. Indeed, here we analyze the impact of the DIS3L2 exoribonuclease over nonsense-mediated mRNA decay (NMD)-targets. Specifically, we present data on: a) the expression of various reporter human ß-globin mRNAs, monitored by Northern blot and RT-qPCR, before and after altering DIS3L2 levels in HeLa cells, and b) the gene expression levels of deregulated transcripts generated by re-analyzing publicly available data from UPF1-depleted HeLa cells that were further cross-referenced with a dataset of transcripts upregulated in DIS3L2-depleted cells. These analyses revealed that DIS3L2 regulates the levels of a subset of NMD-targets. These data can be valuable for researchers interested in the NMD mechanism.

Constant-pH MD Simulations of DMPA/DMPC Lipid Bilayers.

Current constant-pH molecular dynamics (CpHMD) simulations provide a proper treatment of pH effects on the structure and dynamics of soluble biomolecules like peptides and proteins. However, addressing such effects on lipid membrane assemblies has remained problematic until now, despite the important role played by lipid ionization at physiological pH in a plethora of biological processes. Modeling (de)protonation events in these systems requires a proper consideration of the physicochemical features of the membrane environment, including a sound treatment of solution ions. Here, we apply our recent CpHMD-L method to the study of pH effects on a 25% DMPA/DMPC bilayer membrane model, closely reproducing the correct lipid phases of this system, namely, gel-fluid coexistence at pH 4 and a fluid phase at pH 7. A significant transition is observed for the membrane ionization and mechanical properties at physiological pH, providing a molecular basis for the well-established role of phosphatidic acid (PA) as a key player in the regulation of many cellular events. Also, as reported experimentally, we observed pH-induced PA-PA lipid aggregation at acidic pH. By including the titration of anionic phospholipids, the current methodology makes possible to simulate lipid bilayers with increased realism. To the best of our knowledge, this is the first simulation study dealing with a continuous phospholipid bilayer with pH titration of all constituent lipids.

Treatment of Ionic Strength in Biomolecular Simulations of Charged Lipid Bilayers.

Biological membranes are complex systems that have recently attracted a significant scientific interest. Due to the presence of many different anionic lipids, these membranes are usually negatively charged and sensitive to pH. The protonation states of lipids and the ion distribution close to the bilayer are two of the main challenges in biomolecular simulations of these systems. These two problems have been circumvented by using ionized (deprotonated) anionic lipids and enough counterions to preserve the electroneutrality. In this work, we propose a method based on the Poisson-Boltzmann equation to estimate the counterion and co-ion concentration close to a lipid bilayer that avoids the need for neutrality at this microscopic level. The estimated number of ions was tested in molecular dynamics simulations of a 25% DMPA/DMPC lipid bilayer at different ionization levels. Our results show that the system neutralization represents an overestimation of the number of counterions. Consequently, the resulting lipid bilayer becomes too ordered and practically insensitive to ionization. On the other hand, our proposed approach is able to correctly model the ionization dependent isothermal phase transition of the bilayer observed experimentally. Furthermore, our approach is not too computationally expensive and can easily be used to model diverse charged biomolecular systems in molecular dynamics simulations.

Conformational study of GSH and GSSG using constant-pH molecular dynamics simulations.

Glutathione is a small peptide with a crucial role in living organisms. This molecule is found in Nature in both reduced (GSH) and oxidized (GSSG) forms and a high GSH/GSSG ratio is essential to the cell. Glutathione is also present in several enzymatic reactions and can be found in many protein structures. As small peptides, these molecules do not have a defined structure in solution and are able to sample a broad conformational space. In addition, both molecules have several titration sites (four in GSH and six in GSSG) and their conformational space is inevitably influenced by pH. Here, we present a detailed conformational study of GSH and GSSG in a range of pH values, together with a full pH titration of these molecules. We performed constant-pH MD simulations of GSH and GSSG at 24 pH values in a total of 14.4 µs (300 ns per pH value). We obtained the two titration curves and the pKa values for all titrable groups with good agreement with experimental data. We also observed that GSH and GSSG have a large conformational variability in solution and their structural preferences are not significantly affected upon binding to proteins. Some exceptions were found and investigated in detail.