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

Abstract

Bud23 is responsible for the conserved methylation of G1575 of 18S rRNA, in the P-site of the small subunit of the ribosome. bud23Δ mutants have severely reduced small subunit levels and show a general failure in cleavage at site A2 during rRNA processing. Site A2 is the primary cleavage site for separating the precursors of 18S and 25S rRNAs. Here, we have taken a genetic approach to identify the functional environment of BUD23. We found mutations in UTP2 and UTP14, encoding components of the SSU processome, as spontaneous suppressors of a bud23Δ mutant. The suppressors improved growth and subunit balance and restored cleavage at site A2. In a directed screen of 50 ribosomal trans-acting factors, we identified strong positive and negative genetic interactions with components of the SSU processome and strong negative interactions with components of RNase MRP. RNase MRP is responsible for cleavage at site A3 in pre-rRNA, an alternative cleavage site for separating the precursor rRNAs. The strong negative genetic interaction between RNase MRP mutants and bud23Δ is likely due to the combined defects in cleavage at A2 and A3. Our results suggest that Bud23 plays a role at the time of A2 cleavage, earlier than previously thought. The genetic interaction with the SSU processome suggests that Bud23 could be involved in triggering disassembly of the SSU processome, or of particular subcomplexes of the processome.

Utp14 Recruits and Activates the RNA Helicase Dhr1 To Undock U3 snoRNA from the Preribosome

“…We took advantage of the slowgrowth defect of bud23 mutants as a genetic entry point to dissect a constellation of functionally linked proteins. We found that mutations in DHR1, UTP2, or UTP14 partially suppressed the growth defect and 40S assembly defect of bud23⌬ mutants (18,21,23). We also showed that Bud23 binds to the N-terminal extension of Dhr1, making Bud23 a candidate for recruiting or regulating Dhr1 (24).…”

“…We found previously that an alanine-to-glycine change at position 758 in Utp14 suppressed the growth and rRNA processing defects of bud23⌬ (23). To better define the functional domain of Utp14 important for bud23⌬ suppression, we screened for additional suppressing mutations.…”

“…3A) and is the primary event that separates the RNAs of the pre-40S subunit from the pre-60S subunit. We previously showed that deletion of BUD23, loss of UTP14, or a catalytically inactive dhr1 mutant results in loss of the 27SA2 pre-rRNA intermediate (18,22,23), indicating either a failure in A2 cleavage or a delay in A2 cleavage such that cleavage at A3 precedes cleavage at A2. We compared pre-rRNA processing in WT and utp14 mutant cells.…”

“…The methyltransferase Bud23 modifies the guanosine base at position 1575 in 18S rRNA (21,22). Bud23 is recruited to the preribosome at a relatively late step in 40S biogenesis, before A2 cleavage, and remains associated with the pre-40S particle in the nucleus (21,23). We took advantage of the slowgrowth defect of bud23 mutants as a genetic entry point to dissect a constellation of functionally linked proteins.…”

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).…”