Functional separation of pre-rRNA processing steps revealed by truncation of the U3 small nucleolar ribonucleoprotein component, Mpp10

Abstract

The U3 small nucleolar ribonucleoprotein (snoRNP) is required for three cleavage events that generate the mature 18S rRNA from the pre-rRNA. In Saccharomyces cerevisiae, depletion of Mpp10, a U3 snoRNP-specific protein, halts 18S rRNA production and impairs cleavage at the three U3 snoRNP-dependent sites: A0, A1, and A2. We have identified truncation mutations of Mpp10 that affect 18S rRNA synthesis and confer cold-sensitivity and slow growth. However, distinct from yeast cells depleted of Mpp10, the mutants carrying these truncated Mpp10 proteins accumulate a novel precursor, resulting from cleavage at only A0. The Mpp10 truncations do not alter association of Mpp10 with the U3 snoRNA, nor do they affect snoRNA or protein stability. Thus, the role in processing of the U3 snoRNP can be separated into cleavage at the A0 site, which occurs in the presence of truncated Mpp10, and cleavage at the A1͞A2 sites, which occurs only with intact Mpp10. These results strongly argue for a role for Mpp10 in processing at the A1͞A2 sites.Four rRNAs and many proteins comprise the eukaryotic ribosome: the 60S subunit contains 25S (Saccharomyces cerevisiae), 5.8S, and 5S rRNAs, and the 40S subunit houses 18S rRNA. In the nucleolus RNA polymerase I transcribes long, precursor molecules that must be processed to yield the mature 25S, 5.8S, and 18S rRNAs. A schematic of pre-rRNA processing (Fig. 1) shows that the rRNA sequences are flanked by two external transcribed spacers (5Ј and 3Ј) and separated by two internal transcribed spacers (ITS1 and ITS2). Complexes of a small nucleolar RNA and proteins, called snoRNPs, and other nucleolar proteins process this original transcript into the mature rRNAs. The depletion or mutation of many of these processing molecules disrupts pre-rRNA processing (1, 2).Many small nucleolar RNAs (snoRNAs) have been identified but U3 is the most abundant in vertebrate cells and exists in a wide evolutionary range of species (from trypanosomes to humans), making it an attractive molecule for studies aimed at elucidation of pre-rRNA processing. In vivo and͞or in vitro studies in three different systems, mouse, Xenopus laevis oocytes, and S. cerevisiae, have demonstrated that it is as an essential component for production of 18S rRNA (3-9).In yeast, processing of the pre-18S rRNA occurs before the cleavage steps that release the mature 5.8S and 25S rRNAs (Fig.

An unexpected, conserved element of the U3 snoRNA is required for Mpp10p association

“…Our results place Mpp10p in the functional center of the U3 snoRNP, consistent with previous genetic analyses (Lee & Baserga, 1997)+ Mpp10p interacts with two other U3 snoRNP-specific proteins, Imp3p and Imp4p+ We do not yet know if Mpp10p or one of its interacting proteins contact the U3 snoRNA directly+ Future studies will aim to verify the organization of Mpp10p, Imp3p, and Imp4p in the U3 snoRNP and to elucidate their topology with respect to the U3 snoRNA+…”

Ribosomal RNA processing in Candida albicans

“…The identification of the 23S pre-rRNA as a normal feature of C. albicans growth was unexpected since depletion or protein truncation of SSU processome components also leads to a 23S or 22S rRNA with 59 ETS and A3 endpoints Dunbar et al 1997;Lee and Baserga 1997;Dragon et al 2002;Gallagher et al 2004;Osheim et al 2004;. However, Hughes and Ares determined that the 23S pre-rRNA can be detected at low levels in wild-type S. cerevisiae and appears to be a normal component of the processing pathway (Hughes and Ares 1991; also see Billy et al 2000;Wehner and Baserga 2002).…”

Slx9p facilitates efficient ITS1 processing of pre-rRNA in Saccharomyces cerevisiae

“…1B). In previously studied mutants an increase in 21S and/or 23S pre-rRNA resulted from a defect in cleavage at site A2 (as well as A0 and A1 in the case of 23S), and was accompanied by a decrease in the level of the 27SA2 precursor (Jansen et al 1993;Lee and Baserga 1997;Lafontaine and Tollervey 1999;Gallagher et al 2004). Inactivation of SLX9, thus, appears to have complex consequences for processing in ITS1, resulting in a phenotype that has not been observed before.…”