Abstract
Tgs1 is the hypermethylase responsible for m(3)G cap formation of U small nuclear RNAs (U snRNAs) and small nucleolar RNAs (snoRNAs). In vertebrates, hypermethylation of snRNAs occurs in the cytoplasm, whereas this process takes place in the nucleus for snoRNAs. Accordingly, the hypermethylase is found in both compartments with a diffuse localization in the cytoplasm and a concentration in Cajal bodies in the nucleoplasm. In this study, we report that the Tgs1 hypermethylase exists as two species, a full-length cytoplasmic isoform and a shorter nuclear isoform of 65-70 kDa. The short isoform exhibits methyltransferase activity and associates with components of box C/D and H/ACA snoRNPs, pointing to a role of this isoform in hypermethylation of snoRNAs. We also show that production of the short Tgs1 isoform is inhibited by MG132, suggesting that it results from proteasomal limited processing of the full-length Tgs1 protein. Together, our results suggest that proteasome maturation constitutes a mechanism regulating Tgs1 function by generating Tgs1 species with different substrate specificities, subcellular localizations, and functions.
Highlights
Small ribonucleoproteins (RNPs)2 are complexes required for processing RNA precursors into mature RNA species
Two Tgs1 Isoforms Are Expressed in HeLa Cells—We previously reported that polyclonal rabbit antibodies raised against a mixture of two peptides located in the C-terminal domain of the human Tgs1 hypermethylase allow the detection of the endogenous Tgs1 protein by immunofluorescence and Western blot analysis [17, 18]
We describe the existence of two human Tgs1 hypermethylase isoforms that display different subcellular localization and substrate binding
Summary
Plasmid Constructions—The plasmid expressing the Tgs1TAP fusion protein was constructed using the pZOME-N vector placed into the Gateway system according to the manufacturer’s instructions (Invitrogen). To perform nuclear protein extraction, the pellet was solubilized in extraction buffer (0.1 M Tris-HCl, pH 9, 0.1 M NaCl, 5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 0.5% Nonidet P-40, and protease inhibitor mixture) for 20 min on ice. Samples were. Antibodies were coupled to 40 l of protein A-Sepharose (0.1 g/ml; Amersham Biosciences) in 500 l of buffer HNTG (20 mM HEPES, pH 7.9, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM MgCl2, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor mixture) for 1 h at 4 °C. Images were acquired with a Coolsnap camera (Photometrics) controlled by the Metamorph software (Universal Imaging)
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