GRS1, a Yeast tRNA Synthetase with a Role in mRNA 3′ End Formation

Abstract

Transcription termination and 3 end formation are essential processes necessary for gene expression. However, the specific mechanisms responsible for these events remain elusive. A screen designed to identify trans-acting factors involved in these mechanisms in Saccharomyces cerevisiae identified a temperature-sensitive mutant that displayed phenotypes consistent with a role in transcription termination. The complementing gene was identified as GRS1, which encodes the S. cerevisiae glycyl-tRNA synthetase. This result, although unusual, is not unprecedented given that the involvement of tRNA synthetases in a variety of cellular processes other than translation has been well established. A direct role for the synthetase in transcription termination was determined through several in vitro assays using purified wild type and mutant enzyme. First, binding to two well characterized yeast mRNA 3 ends was demonstrated by cross-linking studies. In addition, it was found that all three substrates compete with each other for binding to GlyRS enzyme. Next, the affinity of the synthetase for the two mRNA 3 ends was found to be similar to that of its "natural" substrate, glycine tRNA in a nitrocellulose filter binding assay. The effect of the grs1-1 mutation was also examined and found to significantly reduce the affinity of the enzyme for the three RNA substrates. Taken together, these data indicate that not only does this synthetase interact with several different RNA substrates, but also that these substrates bind to the same site. These results establish a direct role for GRS1 in mRNA 3 end formation.The formation of a eukaryotic mRNA 3Ј end is a complex process involving factors that recognize, cleave, and process the pre-mRNA as well as those that facilitate the dissociation of RNA polymerase II from the DNA template. Although many of these factors have been identified, a comprehensive model of how the termination event interfaces with the processing activities remains elusive. The favored model suggests that termination is dependent on the recognition of a polyadenylation signal that then triggers a change in the polymerase, resulting in dissociation of the enzyme, followed by the termination event(s) (1). The most likely alteration in the actively transcribing enzyme would be a result of the "offloading" of 3Ј end processing factors that associate with the carboxyl-terminal domain of the largest subunit of RNAP II.

Functional and Crystal Structure Analysis of Active Site Adaptations of a Potent Anti-Angiogenic Human tRNA Synthetase

“…Possibly, in lower eukaryotes the eukaryote-specific patch is associated with an unidentified, novel function of TrpRS. Expanded functions of tRNA synthetases in lower eukaryotes are known, such as the roles in RNA splicing of TyrRS in N. crassa and LeuRS in S. cerevisiae (Hsu et al, 2006;Myers et al, 2002), and in transcription termination of GlyRS in S. cerevisiae (Johanson et al, 2003). In vertebrates, TrpRSs have both the VSE and eukaryotespecific patch appended to the N-terminus.…”

“…In addition to the novel functions of proteins such as p43 (or AIMP1) (Han et al, 2006), p38 (or AIMP2) (Kim et al, 2003) and p18 (or AIMP3) (Park et al, 2005a) that are bound to tRNA synthetases in mammalian cells, human synthetases with well-documented expanded functions include Tyr- (Wakasugi and Schimmel, 1999), Trp- (Wakasugi et al, 2002), His- (Howard et al, 2002), Lys- (Park et al, 2005c), and Glu-Pro (Sampath et al, 2004) tRNA synthetases. In lower eukaryotes, TyrRS, LeuRS and GlyRS are well-studied examples (Hsu et al, 2006;Myers et al, 2002;Johanson et al, 2003).…”

The GARS gene is rarely mutated in Japanese patients with Charcot–Marie–Tooth neuropathy

“…11 In yeast, GARS plays a role in mRNA 3¢-end formation. 12 Peripheral neurons can be vulnerable because of their characteristically long structures, which require the transport of organelles and proteins over long distances to maintain their function. In fact, GARS is a homodimer, and, on the basis of results of in vitro expression experiments, the GARS mutations causing CMT or dSMA are predicted to alter the dimer interface and engender a neurite distribution defect.…”

Altered sensory neuron development in CMT2D mice is site-specific and linked to increased GlyRS levels

“…Furthermore, the increase was not observed in all lumbar sensory neurons, but preferentially in a portion of NF200 + neurons (Figure 6). This argues against GlyRS upregulation being a compensatory response to impaired charging, an alteration in a non-canonical function (Johanson et al, 2003;Park et al, 2012;Mo et al, 2016), or that mutant GlyRS protein stability is altered, because if any of those scenarios were true, then GlyRS increase would also likely occur in cervical DRG and across all sensory neurons equally. That is, unless there is a greater requirement for glycine charging in cell bodies of larger sensory neurons with the longest axons (i.e.…”