Trm112 Is Required for Bud23-Mediated Methylation of the 18S rRNA at Position G1575

Abstract

Posttranscriptional and posttranslational modification of macromolecules is known to fine-tune their functions. Trm112 is unique, acting as an activator of both tRNA and protein methyltransferases. Here we report that in Saccharomyces cerevisiae, Trm112 is required for efficient ribosome synthesis and progression through mitosis. Trm112 copurifies with pre-rRNAs and with multiple ribosome synthesis trans-acting factors, including the 18S rRNA methyltransferase Bud23. Consistent with the known mechanisms of activation of methyltransferases by Trm112, we found that Trm112 interacts directly with Bud23 in vitro and that it is required for its stability in vivo. Consequently, trm112⌬ cells are deficient for Bud23-mediated 18S rRNA methylation at position G1575 and for small ribosome subunit formation. Bud23 failure to bind nascent preribosomes activates a nucleolar surveillance pathway involving the TRAMP complexes, leading to preribosome degradation. Trm112 is thus active in rRNA, tRNA, and translation factor modification, ideally placing it at the interface between ribosome synthesis and function.A ctively growing budding yeast cells produce an average of 33 ribosomes per second, which is considerable (61). Besides the synthesis of its constituents, i.e., 79 ribosomal proteins and 4 rRNAs, ribogenesis involves a large number of so-called transacting factors, including proteins and small nucleolar RNAs (13, 56). These interact only transiently with preribosomes and are required for pre-rRNA processing (i.e., cleavages), pre-rRNA modification, and preribosome assembly and transport.Ribosome synthesis is initiated in the nucleolus, a highly dynamic specialized subcompartment of the nucleus organized around clusters of actively transcribed rRNA genes (60). There, RNA polymerase I produces a 35S/47S (in yeasts and humans, respectively) primary transcript which is processed through a complex succession of endo-and exoribonucleolytic cleavages into three of the four mature rRNAs, the 18S, 5.8S, and 25S/28S rRNAs. The fourth rRNA, 5S, is independently transcribed by RNA polymerase III. Ribosome maturation starts cotranscriptionally in the nucleolus, progresses in the nucleoplasm until preribosomes reach the nuclear pore complex, and is finalized in the cytoplasm. Cytoplasmic maturation steps consisting of pre-rRNA processing and structural reorganization result in the assembly of prominent ribosomal structures, such as the "beak" of the small subunit (SSU) and the "stalk" of the large subunit, and are a prerequisite to the acquisition of functionality (43).It is not clear how the various facets of ribosome synthesis are integrated with ribosome function. However, there is growing evidence that such coordination exists. For the small subunit, recent cryoelectron microscopy (cryo-EM) maps of late pre-40S subunits indicate that the binding of trans-acting factors literally mask functional sites, preventing premature translation initiation (57). For the large subunit, trans-acting factors that resemble ribosomal proteins (suc...

WBSCR22/Merm1 is required for late nuclear pre-ribosomal RNA processing and mediates N7-methylation of G1639 in human 18S rRNA

“…In the case of WBSCR22, it will be interesting to investigate its interaction network and its essential role in SSU biogenesis. Bud23 was shown to interact with components of the SSU processome and the general methyltransferase cofactor Trm112 (Figaro et al 2012;Sardana et al 2013). We observed that WBSCR22 directly interacts with several human TRM112 isoforms (data not shown).…”

The rRNA methyltransferase Bud23 shows functional interaction with components of the SSU processome and RNase MRP

“…Despite the position of this base in a critical location of the subunit, it is the presence of Bud23 protein but not its methyl transferase activity that is important for 40S biogenesis (White et al 2008). More recently, work from our and the Lafontaine group identified Trm112 as a cofactor required for the stability of Bud23 in vivo (Figaro et al 2012;Sardana and Johnson 2012). In this study, we have further addressed the role of Bud23 in 40S subunit biogenesis.…”

Transfer RNA Post-Transcriptional Processing, Turnover, and Subcellular Dynamics in the YeastSaccharomyces cerevisiae

“…For example, the Trm7 Trm732 heterodimer is responsible for 29O methylation of C 32 (Cm 32 ), whereas the Trm7 Trm734 heterodimer is responsible for 29O methylation at position 34 (Cm 34 /Gm 34 and ncm 5 Um 34 ), indicating that Trm734 and Trm732 direct correct nucleoside modification sites (Guy et al 2012). Moreover, Trm112 is the activating subunit for both Trm9 and Trm11, required for mcm 5 U 34 / mcm 5 s 2 U 34 and m 2 G 10 , respectively, as well as for Mtq2, required for methylation of protein termination factor Sup45 and Bud23, required for rRNA G1575 methylation (Phizicky and Hopper 2010;Liger et al 2011;Figaro et al 2012). Analyses of the Mtq2 Trm112 complex structure provide insights into how Trm112 might alter Trm9 structure and its activity (Liger et al 2011).…”