Purification, Cloning, and Characterization of XendoU, a Novel Endoribonuclease Involved in Processing of Intron-encoded Small Nucleolar RNAs in Xenopus laevis

Abstract

Here we report the purification, from Xenopus laevis oocyte nuclear extracts, of a new endoribonuclease, XendoU, that is involved in the processing of the intronencoded box C/D U16 small nucleolar RNA (snoRNA) from its host pre-mRNA. Such an activity has never been reported before and has several uncommon features that make it quite a novel enzyme: it is poly(U)-specific, it requires Mn 2؉ ions, and it produces molecules with 2-3-cyclic phosphate termini. Even if XendoU cleaves U-stretches, it displays some preferential cleavage on snoRNA precursor molecules. XendoU also participates in the biosynthesis of another intron-encoded snoRNA, U86, which is contained in the NOP56 gene of Xenopus laevis. A common feature of these snoRNAs is that their production is alternative to that of the mRNA, suggesting an important regulatory role for all the factors involved in the processing reaction.Endoribonucleases play essential roles in RNA metabolism participating both in degradative pathways, such as mRNA decay, and in maturative pathways, to generate functional RNA molecules (1, 2). Despite the plethora of functions played by processing enzymes in RNA metabolism, in eukaryotes only a few endoribonucleases have been isolated to date. Most of these activities are involved in the biosynthesis of translation components. In particular, RNase P and RNase mitochondrial RNA processing are ribonucleoprotein enzymes, functioning as site-specific endoribonucleases (3, 4). Other well characterized endonucleolytic activities, such as the 3Ј-tRNase, the tRNA splicing endonuclease, and members of the RNase III-like family are protein-only enzymes (5-7). Although the majority of these activities participate in the biosynthesis of a specific class of RNA molecules, RNase III was shown to be required for a large number of different maturative pathways. Saccharomyces cerevisiae RNase III (Rnt1p) was shown to be involved in pre-rRNA, small nuclear RNA (snRNA), 1 and small nucleolar RNA (snoRNA) processing (8 -12). Recently, Rnt1p was also shown to participate in processing the intron-encoded snoRNAs U18 and snR38 from their host pre-mRNA (13). Furthermore, a new member of the metazoan RNase III family has been identified to be involved in the RNA interference process (14).Another process in which the participation of endoribonucleases was expected to play an important role is the biosynthesis of snoRNAs. These RNAs are part of a complex class of molecules that are localized in the nucleolus where they participate, as small ribonucleoprotein particles (snoRNPs), in different rRNA maturative events such as processing and nucleotide modifications (15,16). Most snoRNAs in vertebrates are encoded in introns of protein-coding genes and are released from the host primary transcript either by debranching and exotrimming of the spliced lariat (splicing-dependent pathway) or by endonucleolytic cleavage of the pre-mRNA (splicing-independent pathway) (15, 16). There are only a few cases of intronencoded snoRNAs in vertebrates, which are released throug...

The structure of the endoribonuclease XendoU: From small nucleolar RNA processing to severe acute respiratory syndrome coronavirus replication

“…After transcription, the RNA was gel purified, phenol extracted, and precipitated with ethanol. For the binding assay, Ϸ2 fmoles of 32 P-labeled precursor were incubated with the WT or the mutant recombinant proteins, in the absence of Mn 2ϩ (10); for the processing reaction, the same amount of RNA was incubated with proteins in the presence of 5 mM Mn 2ϩ (5). The reaction products were analyzed on native or denaturing PAGE respectively, and visualized by autoradiography.…”

“…In yeast, Rnt1p was shown to be the endonuclease responsible for the excision of intron-encoded snoRNAs and to be activated by the interaction with snoRNP factors assembled on the nascent transcript (3). Among higher eukaryotes, the endoribonuclease from Xenopus laevis, called XendoU, was shown to be responsible for processing the intronencoded U16 and U86 snoRNAs (2,(4)(5)(6). XendoU cuts the RNA substrate at the level of short single-stranded uridine stretches.…”

Origin of Informational Polymers

“…This RNA has the sequence 5Ј-GGAAACGUAUCCUUUGGGAG-3Ј, was purchased from RNATEC, and was kindly provided by E. Caffarelli (10,11).…”

“…Temperatures and incubation times are indicated. 10 Half-lives of the Bonds in the Ribooligonucleotide-For oligonucleotides, the half-lives of the phosphoester bonds were determined from the rate of disappearance of the band representing the intact 20-mer molecule. Cleavage of RNA normally requires participation of the 2Ј-OH group as an internal nucleophile (12) by two "nucleophilic cleavage" events: the trans-esterification and hydrolysis reactions (Fig.…”

Identification of novel components of NAD-utilizing metabolic pathways and prediction of their biochemical functions

“…the EndoU RNase), which can generate 2 0 -phosphate-containing cleavage products. 70,74,75 In all these cases, RNA repair via a KptA-dependent mechanism is likely to generate ADP-ribose derivatives, which when further processed by the NADAR domain might result in improvement of the overall efficiency of repair. Alternatively, the DNA viral gp33.3 like proteins could function in conjunction with the ADP ribosyltransferases encoded by several of these viruses, such as the T4 enzyme which modifies the host RNA polymerase a-subunit.…”