Structure of the pre-60S ribosomal subunit with nuclear export factor Arx1 bound at the exit tunnel

1] Biochemistry Center, Universität Heidelberg, Heidelberg, Germany. [2].
Nature Structural & Molecular Biology (Impact Factor: 13.31). 11/2012; 19(12). DOI: 10.1038/nsmb.2438
Source: PubMed


Preribosomal particles evolve in the nucleus through transient interaction with biogenesis factors before export to the cytoplasm. Here, we report the architecture of the late pre-60S particle, purified from Saccharomyces cerevisiae, through Arx1, a nuclear export factor with structural homology to methionine aminopeptidases, or its binding partner Alb1. Cryo-EM reconstruction of the Arx1 particle at 11.9-Å resolution reveals regions of extra density on the pre-60S particle attributed to associated biogenesis factors, confirming the immature state of the nascent subunit. One of these densities could be unambiguously assigned to Arx1. Immunoelectron microscopy and UV cross-linking localize Arx1 close to the ribosomal exit tunnel, in direct contact with ES27, a highly dynamic eukaryotic rRNA expansion segment. The binding of Arx1 at the exit tunnel may position this export factor to prevent premature recruitment of ribosome-associated factors active during translation.

  • Source
    • "This might also be the reason why we failed to observe any significant methyltransferase activity of heterologous Bmt5 using mature 60S subunits. Proteomics and structure analysis (rRNA) of preribosomal particles have revealed that precursors of ribosomal subunits differ both in composition and conformation as compared with mature subunits (35,36). Also, as ribosome biogenesis is a highly dynamic process with several fleeting interaction of different trans-acting factors, identification of the in vitro substrates for the base methyltransferases is a major challenge. "
    [Show abstract] [Hide abstract]
    ABSTRACT: RNA contains various chemical modifications that expand its otherwise limited repertoire to mediate complex processes like translation and gene regulation. 25S rRNA of the large subunit of ribosome contains eight base methylations. Except for the methylation of uridine residues, methyltransferases for all other known base methylations have been recently identified. Here we report the identification of BMT5 (YIL096C) and BMT6 (YLR063W), two previously uncharacterized genes, to be responsible for m(3)U2634 and m(3)U2843 methylation of the 25S rRNA, respectively. These genes were identified by RP-HPLC screening of all deletion mutants of putative RNA methyltransferases and were confirmed by gene complementation and phenotypic characterization. Both proteins belong to Rossmann-fold-like methyltransferases and the point mutations in the S-adenosyl-l-methionine binding pocket abolish the methylation reaction. Bmt5 localizes in the nucleolus, whereas Bmt6 is localized predominantly in the cytoplasm. Furthermore, we showed that 25S rRNA of yeast does not contain any m(5)U residues as previously predicted. With Bmt5 and Bmt6, all base methyltransferases of the 25S rRNA have been identified. This will facilitate the analyses of the significance of these modifications in ribosome function and cellular physiology.
    Nucleic Acids Research 12/2013; 42(5). DOI:10.1093/nar/gkt1281 · 9.11 Impact Factor
  • Source
    • "However, why specific steps are blocked in the absence of individual assembly factors or how rRNA folding and r-protein binding are coupled to pre-rRNA processing is not known. Recently, crosslinking and cryo-EM assays have been adapted to locate the binding sites of assembly factors on preribosomes (Bohnsack et al. 2009; Granneman et al. 2010, 2011; Strunk et al. 2011; Bradatsch et al. 2012; Greber et al. 2012; Segerstolpe et al. 2013). Identification of binding sites of assembly factors is necessary to understand the specific functions of these proteins during ribosome assembly and to "
    [Show abstract] [Hide abstract]
    ABSTRACT: Eukaryotic ribosome assembly requires over 200 assembly factors that facilitate rRNA folding, ribosomal protein binding, and pre-rRNA processing. One such factor is Rlp7, an essential RNA binding protein required for consecutive pre-rRNA processing steps for assembly of yeast 60S ribosomal subunits: exonucleolytic processing of 27SA3 pre-rRNA to generate the 5' end of 5.8S rRNA and endonucleolytic cleavage of the 27SB pre-rRNA to initiate removal of internal transcribed spacer 2 (ITS2). To better understand the functions of Rlp7 in 27S pre-rRNA processing steps, we identified where it crosslinks to pre-rRNA. We found that Rlp7 binds at the junction of ITS2 and the ITS2-proximal stem, between the 3' end of 5.8S rRNA and the 5' end of 25S rRNA. Consistent with Rlp7 binding to this neighborhood during assembly, two-hybrid and affinity copurification assays showed that Rlp7 interacts with other assembly factors that bind to or near ITS2 and the proximal stem. We used in vivo RNA structure probing to demonstrate that the proximal stem forms prior to Rlp7 binding and that Rlp7 binding induces RNA conformational changes in ITS2 that may chaperone rRNA folding and regulate 27S pre-rRNA processing. Our findings contradict the hypothesis that Rlp7 functions as a placeholder for ribosomal protein L7, from which Rlp7 is thought to have evolved in yeast. The binding site of Rlp7 is within eukaryotic-specific RNA elements, which are not found in bacteria. Thus, we propose that Rlp7 coevolved with these RNA elements to facilitate eukaryotic-specific functions in ribosome assembly and pre-rRNA processing.
    RNA 10/2013; 19(12). DOI:10.1261/rna.041194.113 · 4.94 Impact Factor
  • Source
    • "The proposed exchangeability of eukaryotic rpL10 and phospho-stalk proteins rpP1/rpP2 furthermore suggests that the structural organization of the phospho-stalk and the central protuberance is subject to dynamic changes even in mature LSUs. In line with this, recent single-molecule cryo-electron microscopic analyses indicated that in LSU particles associated with Arx1, which binds to late LSUs precursors and free mature LSUs (Figure 2 and [61,78–80]), the definite organization of the central protuberance and the phospho-stalk, including correct assembly of rpL10 and rpP1/rpP2 proteins, is not accomplished [55]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: During the assembly process of ribosomal subunits, their structural components, the ribosomal RNAs (rRNAs) and the ribosomal proteins (r-proteins) have to join together in a highly dynamic and defined manner to enable the efficient formation of functional ribosomes. In this work, the assembly of large ribosomal subunit (LSU) r-proteins from the eukaryote S. cerevisiae was systematically investigated. Groups of LSU r-proteins with specific assembly characteristics were detected by comparing the protein composition of affinity purified early, middle, late or mature LSU (precursor) particles by semi-quantitative mass spectrometry. The impact of yeast LSU r-proteins rpL25, rpL2, rpL43, and rpL21 on the composition of intermediate to late nuclear LSU precursors was analyzed in more detail. Effects of these proteins on the assembly states of other r-proteins and on the transient LSU precursor association of several ribosome biogenesis factors, including Nog2, Rsa4 and Nop53, are discussed.
    PLoS ONE 07/2013; 8(7):e68412. DOI:10.1371/journal.pone.0068412 · 3.23 Impact Factor
Show more