Studying nuclear functions of aminoacyl tRNA synthetases

Abstract

Aminoacyl tRNA synthetases (AARSs) are best known for their essential role in translation in the cytoplasm. The concept that AARSs also exist in the nucleus started to draw attention around the turn of the new millennium, when aminoacylated tRNAs were first found in the nuclei of Xenopus oocytes. It is now expected that all cytoplasmic AARSs are present in the nucleus. In addition to tRNA aminoacylation, nuclear AARSs were found to regulate a spectrum of biological processes and responses, with many AARSs functioning through regulation at the level of gene transcription. In this paper, we focus on describing methods that have been successfully implemented to study AARSs in transcriptional regulation. These include a cell fractionation assay to detect nuclear localization, an in vitro DNA-cellulose pull-down assay to determine DNA binding capacity, and a chromatin immunoprecipitation (ChIP)-DNA deep sequencing assay to identify DNA binding sites. Application of these methods would expand our understanding of AARS functions and reveal critical insights on the coordination of gene transcription and translation.

Regulation of ex-translational activities is the primary function of the multi-tRNA synthetase complex

“…We therefore investigated alternative functions of the MSC and asked whether the MSC determines cellular localization of its aaRSs. MSC-bound aaRSs have been previously found in the nucleus ( 62 ) where they contribute to tRNA quality control ( 63 ) as well as ex-translational functions ( 64 ). We probed the cellular localization of MSC aaRSs by comparing the levels of MSC components in the cell nucleus of wildtype (MSC) versus ΔLZ cells (MSCΔRQ).…”

GlyRS is a new mediator of amino acid‐induced milk synthesis in bovine mammary epithelial cells

“…In this study, we further show that GlyRS modulates mRNA expresssion of the mTOR‐S6K1/4EBP1 signaling; these results are in agreement with our previous reports that extracellular stimuli such as Met and estrogen trigger GlyRS nuclear localization in BMEC (Huang et al, ; Lu et al, ). In recent years, many aaRSs have been found to be localized in the nucleus and regulated gene expression beyond their aminoacylation roles (Fang & Guo, ; Shi, Wei, & Yang, ). For examples, a unique motif appended to SerRS in species with closed circulatory systems harbors a robust nuclear leading sequence (NLS) (Xu et al, ); under stress conditions, TyrRS discloses its NLS and translocates to the nucleus to induce survival genes (Fu, Xu, Shi, Wei, & Yang, ); LysRS phosphorylation creates steric clashes at the domain interface and disrupts its binding grooves for p38/AIMP2, releasing LysRS for nuclear translocation (Ofir‐Birin et al, ).…”

Transcriptional dysregulation by a nucleus-localized aminoacyl-tRNA synthetase associated with Charcot-Marie-Tooth neuropathy

“…Nuclear/cytoplasmic fractionation was performed with either the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Fisher Scientific) following manufacturer’s instructions or a similar custom protocol 46 . Briefly, Swelling Buffer (SB) [10 mM Tris-HCl (pH 7.4), 2 mM EDTA, proteinase inhibitor cocktail (added before use)], Plasma Membrane Lysis Buffer (PMLB) [10 mM Tris-HCl (pH 7.1), 2 mM MgCl2, 1% Triton X-100], and Nuclear Extraction Buffer (NEB) [20 mM HEPES (pH 7.6), 300 mM NaCl, 2 mM EDTA, 1 mM 1,4-Dithiothreitol (DTT), 10% glycerol, 1% Triton X-100, protease inhibitor cocktail (added before use)] were prepared.…”