Abstract
XendoU is the endoribonuclease involved in the biosynthesis of a specific subclass of Xenopus laevis intron-encoded small nucleolar RNAs. XendoU has no homology to any known cellular RNase, although it has sequence similarity with proteins tentatively annotated as serine proteases. It has been recently shown that XendoU represents the cellular counterpart of a nidovirus replicative endoribonuclease (NendoU), which plays a critical role in viral replication and transcription. In this paper, we combined prediction and experimental data to define the amino acid residues directly involved in XendoU catalysis. Specifically, we find that XendoU residues Glu-161, Glu-167, His-162, His-178, and Lys-224 are essential for RNA cleavage, which occurs in the presence of manganese ions. Furthermore, we identified the RNA sequence required for XendoU binding and showed that the formation of XendoU-RNA complex is Mn2+-independent.
Highlights
XendoU is an RNA processing enzyme that participates in the production of small nucleolar RNAs (snoRNA(s)),1 a large family of non-coding RNAs with essential roles in ribosome biogenesis
We showed that the box C/D U16 snoRNA, encoded by an intron of the L4 ribosomal protein gene of Xenopus laevis, originated from site-specific processing of the intron within the pre-mRNA [3]
Data base searches with nidovirus proteins led to the recent identification of XendoU as the cellular homolog of the nidovirus nidovirus replicative endoribonuclease (NendoU) [12]
Summary
Sequence Analysis—The non-redundant protein data base was searched with PSI-BLAST [14] for 10 iterations or until convergence; typical inclusion threshold E-value was 0.001, we experimented with E ϭ 0.01. In a typical processing reaction, 3 ϫ 104 cpm (corresponding to ϳ2 fmol of 32P-labeled RNA precursors) were incubated with recombinant proteins or oocyte nuclear extracts as described [5]. Analysis of 3Ј Terminus—Gel-purified 5Ј-terminal cleavage product (molecule I-1 derived by cleavage at the b site) was treated as described [5] After extraction and labeling in the presence of 5Ј-[32P]cytidine 3Ј,5Ј-bisphosphate, the RNA was analyzed by denaturing PAGE and autoradiography. Binding Assays—Binding assays were performed by incubating 2 fmol of [␣-32P]UTP in vitro transcribed mini-003 RNA with increasing amounts of recombinant proteins in a final volume of 10 l of binding buffer (10 mM Hepes, pH 7.5, 75 mM NaCl, 20 mM EGTA, 1 mM dithiothreitol, and 20% glycerol). In competition experiments, binding assays were carried out by incubating [␣-32P]UTP in vitro transcribed mini-003 RNA with His-XendoU, in the presence of increasing amounts of different unlabeled RNAs as competitors
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