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.

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Available from: Sander Granneman, Oct 07, 2015
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    • "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. "
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    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.
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    • "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]. "
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    PLoS ONE 07/2013; 8(7):e68412. DOI:10.1371/journal.pone.0068412 · 3.23 Impact Factor
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