Ebp2 and Brx1 function cooperatively in 60S ribosomal subunit assembly in Saccharomyces cerevisiae

Abstract

The yeast protein Ebp2 is required for early steps in production of 60S ribosomal subunits. To search for cofactors with which Ebp2 functions, or substrates on which it acts, we screened for mutants that were synthetically lethal (sl) with the ebp2-14 mutation. Four different mutant alleles of the 60S ribosomal subunit assembly factor Brx1 were found. To investigate defects of the double mutant, we constructed strains conditional for the ebp2-14 brx1- synthetic lethal phenotype. These ebp2-14 brx1 mutants were defective in processing of 27S pre-rRNA and production of 60S subunits, under conditions where each single mutant was not. Ebp2 and Brx1 exhibit a strong two-hybrid interaction, which is eliminated by some combinations of brx1 and ebp2 mutations. In one such mutant, Ebp2 and Brx1 can still associate with pre-ribosomes, but subunit maturation is perturbed. Depletion of either Ebp2 or Brx1 revealed that Brx1 requires Ebp2 for its stable association with pre-ribosomes, but Ebp2 does not depend on the presence of Brx1 to enter pre-ribosomes. These results suggest that assembly of 60S ribosomal subunits requires cooperation of Ebp2 with Brx1, together with other molecules present in pre-ribosomes, potentially including several found in assembly subcomplexes with Brx1 and Ebp2.

Ribosome Biogenesis in the YeastSaccharomyces cerevisiae

“…Assembly of these A3 factors into preribosomes is hierarchical (Figure 9). Ebp2 and L8 are each required for assembly of Nop7, Erb1, Ytm1, Nop15, Rlp7, Cic1, and Has1 Shimoji et al 2012). Nop7, Erb1, Ytm1, Rlp7, Nop15, and Cic1 are mutually interdependent for assembly and are necessary for association of Drs1 and Has1, but not the Ebp2 complex, with pre-rRNPs (Sahasranaman et al 2011).…”

Identification of the binding site of Rlp7 on assembling 60S ribosomal subunits in Saccharomyces cerevisiae

“…We tested for potential interactions between the three A 3 factors that bind to ITS2 (Rlp7, Nop15, and Cic1) and other assembly factors and r-proteins implicated in 27SA 2 (Brx1, Ebp2), 27SA 3 (Pwp1, Nop7, Ytm1, Erb1, Rrp1, Has1, Rrp17, Rat1, Rai1, L7, L8), or 27SB (L17, L26, L35, L37, Nsa2, Rrs1, Rpf2, Nog2, Nop2, Nip7, L11, Spb4, Dbp10, Tif6, Rlp24, Mak11, Nog1) pre-rRNA processing, as well as other r-proteins that contact domain I (L15, L25) (Henry et al 1994;Emery et al 2004;Zhang et al 2004;Oeffinger et al 2009;Ben-Shem et al 2011;Granneman et al 2011;Sahasranaman et al 2011;Babiano et al 2012;Jakovljevic et al 2012;Shimoji et al 2012;Talkish et al 2012;Gamalinda et al 2013). Only 11 of the 210 potential pair-wise two-hybrid interactions tested were positive, confirming the specificity of the assay ( Fig.…”

Nα-Acetyltransferase NatA Is Involved in Ribosome Synthesis inSaccharomyces cerevisiae

“…Genetic analysis revealed that a point mutation in the genome resulted in a synthetic growth defect in all of the strains, except for m5, and that each of these exhibited a recessive mutation (data not shown). We have found that each of the five completely synthetic-lethal mutants (two of them had the same mutation allele) had a mutation in BRX1, 16) which encodes a trans-acting factor involved in 60S ribosomal subunit synthesis. Three strains (m1, m4, and m8) were conditionally synthetic lethal.…”

“…We examined whether N -acetylation of Ebp2 or related proteins is important in the background of ebp2-14. We focused on three proteins: Ebp2, Brx1, a functional partner of Ebp2 in 60S ribosomal subunit biogenesis, 16) and ribosomal protein L36a/b, overexpression of which suppresses a growth defect in ebp2-14. Mutants designed such that the second amino acid was substituted for proline in each of the proteins revealed that brx1-S2P was synthetically lethal with ebp2-14, whereas ebp2-A2P, rpl36a-T2P, and rpl36b-A2P had little effect on the growth of the ebp2-14 mutant.…”

“…28) Ebp2 and Brx1 are required for the processing of 27SA 2 to 27SA 3 pre-rRNAs, and processing of 27SA 3 to 27SB S pre-rRNAs. 16,29) Recently it was found that depletion of L8 affects the recruitment of proteins L13, L15, and L36 to 60S ribosomal subunits, and the processing of 27SA 2 and 27SA 3 pre-rRNAs. 30) This suggests that Ebp2 functions cooperatively with Brx1, L36, L15, L8, Pwp1, and Nop12 in the processing of 27S pre-rRNA.…”