HuR Promotes the Differentiation of Goat Skeletal Muscle Satellite Cells by Regulating Myomaker mRNA Stability.

Human antigen R (HuR) is an RNA-binding protein that contributes to a wide variety of biological processes and diseases. HuR has been demonstrated to regulate muscle growth and development, but its regulatory mechanisms are not well understood, especially in goats. In this study, we found that HuR was highly expressed in the skeletal muscle of goats, and its expression levels changed during longissimus dorsi muscle development in goats. The effects of HuR on goat skeletal muscle development were explored using skeletal muscle satellite cells (MuSCs) as a model. The overexpression of HuR accelerated the expression of myogenic differentiation 1 (MyoD), Myogenin (MyoG), myosin heavy chain (MyHC), and the formation of myotubes, while the knockdown of HuR showed opposite effects in MuSCs. In addition, the inhibition of HuR expression significantly reduced the mRNA stability of MyoD and MyoG. To determine the downstream genes affected by HuR at the differentiation stage, we conducted RNA-Seq using MuSCs treated with small interfering RNA, targeting HuR. The RNA-Seq screened 31 upregulated and 113 downregulated differentially expressed genes (DEGs) in which 11 DEGs related to muscle differentiation were screened for quantitative real-time PCR (qRT-PCR) detection. Compared to the control group, the expression of three DEGs (Myomaker, CHRNA1, and CAPN6) was significantly reduced in the siRNA-HuR group (p < 0.01). In this mechanism, HuR bound to Myomaker and increased the mRNA stability of Myomaker. It then positively regulated the expression of Myomaker. Moreover, the rescue experiments indicated that the overexpression of HuR may reverse the inhibitory impact of Myomaker on myoblast differentiation. Together, our findings reveal a novel role for HuR in promoting muscle differentiation in goats by increasing the stability of Myomaker mRNA.

METTL3 Promotes the Differentiation of Goat Skeletal Muscle Satellite Cells by Regulating MEF2C mRNA Stability in a m6A-Dependent Manner.

The development of mammalian skeletal muscle is a highly complex process involving multiple molecular interactions. As a prevalent RNA modification, N6-methyladenosine (m6A) regulates the expression of target genes to affect mammalian development. Nevertheless, it remains unclear how m6A participates in the development of goat muscle. In this study, methyltransferase 3 (METTL3) was significantly enriched in goat longissimus dorsi (LD) tissue. In addition, the global m6A modification level and differentiation of skeletal muscle satellite cells (MuSCs) were regulated by METTL3. By performing mRNA-seq analysis, 8050 candidate genes exhibited significant changes in expression level after the knockdown of METTL3 in MuSCs. Additionally, methylated RNA immunoprecipitation sequencing (MeRIP-seq) illustrated that myocyte enhancer factor 2c (MEF2C) mRNA contained m6A modification. Further experiments demonstrated that METTL3 enhanced the differentiation of MuSCs by upregulating m6A levels and expression of MEF2C. Moreover, the m6A reader YTH N6-methyladenosine RNA binding protein C1 (YTHDC1) was bound and stabilized to MEF2C mRNA. The present study reveals that METTL3 enhances myogenic differentiation in MuSCs by regulating MEF2C and provides evidence of a post-transcriptional mechanism in the development of goat skeletal muscle.

Hindered rotor benchmarks for the transition states of free radical additions to unsaturated hydrocarbons.

The hindered internal rotors of 32 transition states (TSs) formed through four free radicals, namely methyl, vinyl, ethyl, methoxy (CH3, C2H3, C2H5, CH3) additions to acetylene, ethylene, allene, propyne, and propene (C2H2/C2H4/C3H4-a/C3H4-p/C3H6) are studied. To validate the uncertainties of rate constants that stem from the use of different electronic structure methods to treat hindered rotors, the rotations of the newly formed C-C and/or C-O rotors in the transition states are calculated using commonly used DFT methods (B3LYP, M06-2X, ωB97X-D and B2PLYP-D3 with two Pople basis sets (6-31+G(d,p), 6-311++G(d,p)) and cc-pVTZ). The hindrance potential energies V(χ) calculated using the M06-2X/6-311++G(d,p) method are benchmarked at the CCSD(T), CCSD(T)-F12, DLPNO-CCSD(T) levels of theory with cc-pVTZ-F12 and cc-pVXZ (X = T, Q) basis sets and are extrapolated to the complete basis set (CBS) limit. The DLPNO-CCSD(T)/CBS method is proven to reproduce the CCSD(T)/CBS energies within 0.5 kJ mol-1 and this method is selected as the benchmark for all of the rotors in this study. Rotational constants B(χ) are computed for each method based on the optimized geometries for the hindrance potential via the I(2,3) approximation. Thereafter, the V(χ) and B(χ) values are used to compute hindered internal rotation partition functions, QHR, as a function of temperature. The uncertainties in the V(χ), B(χ) and QHR calculations stem from the use of different DFT methods for the internal rotor treatment are discussed for these newly formed rotors. For rotors formed by + C2 alkenes/alkynes, the V(χ) and QHR values calculated using DFT methods are compared with the DLPNO-CCSD(T)/CBS results and analysed according to reaction types. Based on comparisons of the DFT methods with the benchmarking method, reliable DFT methods are recommended for the treatment of internal rotors for different reaction types considering both accuracy and computational cost. This work, to the authors' knowledge, is the first to systematically benchmark hindrance potentials which can be used to estimate uncertainties in theoretically derived rate constants arising from the choice of different electronic structure methods.

An ab Initio/Transition State Theory Study of the Reactions of C5H9 Species of Relevance to 1,3-Pentadiene, Part II: Pressure Dependent Rate Constants and Implications for Combustion Modeling.

The temperature- and pressure-dependence of rate constants for several radicals and unsaturated hydrocarbons reactions (1,3-C5H8/1,4-C5H8/cyC5H8 + H, C2H4 + C3H5-a, C3H6 + C2H3) are analyzed in this paper. The abstraction reactions of these systems are also calculated and compared with available literature data. C5H9 radicals can be produced via H atom addition reactions to the pentadiene isomers and cyclopentene, and also by H-atom abstraction reactions from 1- and 2-pentene and cyclopentane. Comprehensive C5H9 potential energy surface (PES) analyses and high-pressure limiting rate constants for related reactions have been explored in part I of this work ( J. Phys. Chem. A 2019, 123 (22), 9019-9052). In this work, a chemical kinetic model is constructed based on the computed thermochemistry and high-pressure limiting rate constants from part I, to further understand the chemistry of different C5H8 molecules. The most important channels for these addition reactions are discussed in the present work based on reaction pathway analyses. The dominant reaction pathways for these five systems are combined together to generate a simplified C5H9 PES including nine reactants, 25 transition states (TSs), and nine products. Spin-restricted single point energies are calculated for radicals and TSs on the simplified PES at the ROCCSD(T)/aug-cc-pVTZ level of theory with basis set corrections from MP2/aug-cc-pVXZ (where X = T and Q). Temperature- and pressure-dependent rate constants are calculated using RRKM theory with a Master Equation analysis, with restricted energies used for minima on the simplified C5H9 PES and unrestricted energies for other species, over a temperature range of 300-2000 K and in the pressure range 0.01-100 atm. The rate constants calculated are in good agreement with those in the literature. The chemical kinetic model is updated with pressure-dependent rate constants and is used to simulate the species concentration profiles for H atom addition to cyclopentane and cyclopentene. Through detailed analyses and comparisons, this model can reproduce the experimental measurements of species qualitatively and quantitatively with reasonably good agreement.

Ab Initio/Transition-State Theory Study of the Reactions of C5H9 Species of Relevance to 1,3-Pentadiene, Part I: Potential Energy Surfaces, Thermochemistry, and High-Pressure Limiting Rate Constants.

In this study, the reactions of C5H9 radicals are theoretically investigated, with a particular emphasis on hydrogen atom addition reactions to 1,3-pentadiene (C5H8) to form C5H9 radicals, although the subsequent isomerization and decomposition reactions of the C5H9 radicals are also of direct relevance to the radicals formed from the pyrolysis and oxidation of species including pentene and cyclopentane. Moreover, H-atom abstraction reactions by hydrogen atoms from 1,3-pentadiene are also investigated. The geometries and frequencies of 63 potential energy surface (PES) minima and 88 transition states are optimized at the ωB97XD/aug-cc-pVTZ level of theory. Spin-unrestricted open-shell single-point energies for all the species are calculated at the CCSD(T)/aug-cc-pVTZ level of theory with basis set corrections from MP2/aug-cc-pVXZ (where X = T and Q). A one-dimensional hindered rotor treatment is employed for torsional modes, with the M06-2X/6-311++G(D,P) method used to compute the potential energy as a function of the dihedral angle. The high-pressure limiting rate constants and the thermochemical properties for C5 species are calculated using the Master Equation System Solver (MESS) with conventional transition-state theory and comparisons made with existing available literature data. A hydrogen atom can add to the terminal carbon atom of 1,3-pentadiene to form the 2,4-C5H9 radical and/or the internal carbon atoms to form 2,5-C5H9, 1,4-C5H9, and 1,3-C5H9 radicals. Among the four entrance channels for H atom addition reactions, the formation of 2,4-C5H9 and 1,3-C5H9 radicals is more exothermic in comparison to the other C5H9 isomers (2,5-C5H9, 1,4-C5H9) because of the resonantly stabilized allylic structure. Consequently, the formation of the former is generally dominant in terms of barrier heights. H atom addition reactions to 1,3-pentadiene are compared to available C3-C5 alkenes and dienes, with external addition calculated to be kinetically favored over internal addition. However, the correlation between heats of formation and energy barriers for H atom addition to 1,2-dienes is different from that for 1,3- and 1,4-dienes. Hydrogen atom addition and abstraction rate constants are also compared for 1,3-pentadiene, with addition found to be dominant. The subsequent unimolecular reactions on the C5H9 PES are found to be highly complex with reactions taking place on a multiple-well multiple-channel PES. For clarity, the reaction mechanism and kinetics of each C5H9 radical are discussed individually in terms of the computed enthalpy of the reaction and activation, the transition-state structure/reaction class, and also in terms of the combustion species for which the reactions are of potential importance. The reactions on the C5H9 PES are divided into three reaction classes (H-shift isomerization, cycloaddition, and ß-scission reactions), and the reactivity-structure-based estimation rules for energy barriers are derived for these three reaction classes and compared to literature results for alkyl radicals.

Chemical Kinetics of Hydrogen Atom Abstraction from Propargyl Sites by Hydrogen and Hydroxy Radicals.

Hydrogen atom abstraction from propargyl C-H sites of alkynes plays a critical role in determining the reactivity of alkyne molecules and understanding the formation of soot precursors. This work reports a systematic theoretical study on the reaction mechanisms and rate constants for hydrogen abstraction reactions by hydrogen and hydroxy radicals from a series of alkyne molecules with different structural propargyl C-H atoms. Geometry optimizations and frequency calculations for all species are performed at M06-2X/cc-pVTZ level of theory and the hindered internal rotations are also treated at this level. The high-level W1BD and CCSD(T)/CBS theoretical calculations are used as a benchmark for a series of DFT calculations toward the selection of accurate DFT functionals for large reaction systems in this work. Based on the quantum chemistry calculations, rate constants are computed using the canonical transition state theory with tunneling correction and the treatment of internal rotations. The effects of the structure and reaction site on the energy barriers and rate constants are examined systematically. To the best of our knowledge, this work provides the first systematic study for one of the key initiation abstraction reactions for compounds containing propargyl hydrogen atoms.