The Bipartite Structure of the tRNA m\u3csup\u3e1\u3c/sup\u3eA58 Methyltransferase from \u3cem\u3eS. cerevisiae\u3c/em\u3e is Conserved in Humans

Among all types of RNA, tRNA is unique given that it possesses the largest assortment and abundance of modified nucleosides. The methylation at N1 of adenosine 58 is a conserved modification, occurring in bacterial, archaeal, and eukaryotic tRNAs. In the yeast Saccharomyces cerevisiae, the tRNA 1-methyladenosine 58 (m1A58) methyltransferase (Mtase) is a two-subunit enzyme encoded by the essential genes TRM6 (GCD10) and TRM61 (GCD14). While the significance of many tRNA modifications is poorly understood, methylation of A58 is known to be critical for maintaining the stability of initiator tRNAMet in yeast. Furthermore, all retroviruses utilize m1A58-containing tRNAs to prime reverse transcription, and it has been shown that the presence of m1A58 in human tRNA3 Lys is needed for accurate termination of plus-strand strong-stop DNA synthesis during HIV-1 replication. In this study we have identified the human homologs of the yeast m1A Mtase through amino acid sequence identity and complementation of trm6 and trm61 mutant phenotypes. When coexpressed in yeast, human Trm6p and Trm61p restored the formation of m1A in tRNA, modifying both yeast initiator tRNAMet and human tRNA3 Lys. Stable hTrm6p/hTrm61p complexes purified from yeast maintained tRNA m1A Mtase activity in vitro. The human m1A Mtase complex also exhibited substrate specificity—modifying wild-type yeast tRNAi Met but not an A58U mutant. Therefore, the human tRNA m1A Mtase shares both functional and structural homology with the yeast tRNA m1A Mtase, possessing similar enzymatic activity as well as a conserved binary composition

A Functional Screen Provides Evidence for a Conserved, Regulatory, Juxtamembrane Phosphorylation Site in Guanylyl Cyclase A and B

Kinase homology domain (KHD) phosphorylation is required for activation of guanylyl cyclase (GC)-A and -B. Phosphopeptide mapping identified multiple phosphorylation sites in GC-A and GC-B, but these approaches have difficulty identifying sites in poorly detected peptides. Here, a functional screen was conducted to identify novel sites. Conserved serines or threonines in the KHDs of phosphorylated receptor GCs were mutated to alanine and tested for reduced hormone to detergent activity ratios. Mutation of Ser-489 in GC-B to alanine but not glutamate reduced the activity ratio to 60% of wild type (WT) levels. Similar results were observed with Ser-473, the homologous site in GC-A. Receptors containing glutamates for previously identified phosphorylation sites (GC-A-6E and GC-B-6E) were activated to ∼20% of WT levels but the additional glutamate substitution for S473 or S489 increased activity to near WT levels. Substrate-velocity assays indicated that GC-B-WT-S489E and GC-B-6E-S489E had lower Km values and that WT-GC-B-S489A, GC-B-6E and GC-B-6E-S489A had higher Km values than WT-GC-B. Homologous desensitization was enhanced when GC-A contained the S473E substitution, and GC-B-6E-S489E was resistant to inhibition by a calcium elevating treatment or protein kinase C activation – processes that dephosphorylate GC-B. Mass spectrometric detection of a synthetic phospho-Ser-473 containing peptide was 200–1300-fold less sensitive than other phosphorylated peptides and neither mass spectrometric nor 32PO4 co-migration studies detected phospho-Ser-473 or phospho-Ser-489 in cells. We conclude that Ser-473 and Ser-489 are Km-regulating phosphorylation sites that are difficult to detect using current methods

The bipartite structure of the tRNA m(1)A58 methyltransferase from S. cerevisiae is conserved in humans

Among all types of RNA, tRNA is unique given that it possesses the largest assortment and abundance of modified nucleosides. The methylation at N(1) of adenosine 58 is a conserved modification, occurring in bacterial, archaeal, and eukaryotic tRNAs. In the yeast Saccharomyces cerevisiae, the tRNA 1-methyladenosine 58 (m(1)A58) methyltransferase (Mtase) is a two-subunit enzyme encoded by the essential genes TRM6 (GCD10) and TRM61 (GCD14). While the significance of many tRNA modifications is poorly understood, methylation of A58 is known to be critical for maintaining the stability of initiator tRNA(Met) in yeast. Furthermore, all retroviruses utilize m(1)A58-containing tRNAs to prime reverse transcription, and it has been shown that the presence of m(1)A58 in human tRNA(3) (Lys) is needed for accurate termination of plus-strand strong-stop DNA synthesis during HIV-1 replication. In this study we have identified the human homologs of the yeast m(1)A Mtase through amino acid sequence identity and complementation of trm6 and trm61 mutant phenotypes. When coexpressed in yeast, human Trm6p and Trm61p restored the formation of m(1)A in tRNA, modifying both yeast initiator tRNA(Met) and human tRNA(3) (Lys). Stable hTrm6p/hTrm61p complexes purified from yeast maintained tRNA m(1)A Mtase activity in vitro. The human m(1)A Mtase complex also exhibited substrate specificity—modifying wild-type yeast tRNA(i) (Met) but not an A58U mutant. Therefore, the human tRNA m(1)A Mtase shares both functional and structural homology with the yeast tRNA m(1)A Mtase, possessing similar enzymatic activity as well as a conserved binary composition

Mutation of Val-472 in GC-A to glutamate or alanine had no effect on ANP-stimulated guanylyl cyclase activity.

<p>GC assays were conducted on membrane preparations from 293 cells transiently expressing WT or mutants of rat GC-A as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036747#pone-0036747-g002" target="_blank">figure 2</a>. The results are expressed as the mean ± SEM, where n = 12.</p

Alanine substitutions at Ser-489 in GC-B increase the Michaelis constant.

<p>293 cells were transiently transfected with the indicated form of GC-B and assayed for GC activity in the presence of 1 µM CNP, 1 mM ATP, and increasing concentrations of GTP. (A) Comparison between WT-GC-B, WT-GC-B-S489E, GC-B-6E and GC-B-6E-S489E. (B) Comparison between WT-GC-B-S489E and WT-GC-B-S489A. (C) Comparison between GC-B-6E and GC-B-6E-S489A. Vmax and Km values were calculated using nonlinear regression. * Indicates significantly different from WT-GC-B (A) or WT-GC-B-S489E (B) at p<0.02.</p

GC-B-6E-489E is resistant to inhibition by hyperosmotic medium and protein kinase C.

<p>293 cells transiently expressing WT-GC-B or GCB-6E-S489E were incubated ±0.1 M NaCl, 0.2 M NaCl or 1 µM PMA for 30 min at 37°C. Membranes were prepared and assayed for GC activity in the presence of CNP, ATP and magnesium-GTP or 1% Triton X-100 and manganese-GTP. The results were expressed as a ratio of hormone-stimulated/detergent-stimulated activity and were normalized to the activity ratio determined in membranes from control cells not exposed to any inhibitory agent. Data are presented as means ± SEM, where n = 4. ** Indicates a p value of <0.01, *** indicates a p value of <0.0001.</p

Glutamate substitution for serine 473 enhances homologous desensitization of GC-A.

<p>293 cells transiently expressing WT-GC-A or the indicated mutant forms of GC-A were incubated ±1 µM ANP for 1 hour at 37°C. Membranes were prepared and assayed for GC activity in the presence of ANP, ATP and magnesium-GTP or 1% Triton X-100 and manganese-GTP. The results are expressed as a ratio of hormone-stimulated/detergent-stimulated activity and were normalized to the percent of the activity ratio determined in membranes from cells not exposed to ANP. Data are means determined from multiple experiments ± SEM, where n≥6. * Indicates significantly different from control (GC-A or GC-A-6E) at p<0.01.</p

Mutation of Ser-489 in GC-B to alanine but not glutamate decreases CNP-stimulated guanylyl cyclase activity.

<p>293T cells were transiently transfected with WT-GC-B containing alanine or glutamate substitutions for Ser-489 (left side) or with the same substitutions engineered into GC-B-6E (right side). GC assays were conducted for 5 min in the presence of 1 µM CNP, 1 mM ATP and 1 mM magnesium-GTP or 1% Triton X-100 and 5 mM manganese-GTP. GC activity was expressed as CNP-dependent activity/Triton X-100-dependent activity ×100. The results are the mean ± SEM, where n = 8.</p

Mutation of Ser-473 in GC-A to alanine but not glutamate decreases ANP-stimulated guanylyl cyclase activity.

<p>293T cells were transiently transfected with WT-GC-A containing alanine or glutamate substitutions for Ser-473 (left side) or with the same substitutions engineered into GC-A-6E (right side). GC assays were conducted in the presence of 1 µM ANP, 1 mM ATP and 5 mM magnesium-GTP or 1% Triton X-100 and 5 mM manganese-GTP. GC activity was expressed as the ANP-dependent activity/Triton X-100-dependent activity ×100. The results are the mean ± SEM, where n = 14.</p

Comparison of the N-terminal region of the kinase homology domains of transmembrane guanylyl cyclase receptors.

<p>Purple RK indicates the beginning of intracellular domains. Red residues are confirmed phosphorylation sites. The green residue is a putative conserved phosphorylation site, which is Ser-489 in GC-B and Ser-473 in GC-A. Sequences were aligned with CLUSTAL version 2.0.1. Abbreviations are: rGC-A, rat GC-A; hGC-A, human GC-A; rGC-B, rat GC-B; hGC-B, human GC-B; hGC-C, human GC-C; A. punGC, GC from sea urchin species A. punctalata; S. purGC, GC from sea urchin species S. purpuratus.</p