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
    • "Arx1 has been suggested to support the nuclear export of the pre-60S particle in yeast by interacting with FG-repeat nucleoporins in a cavity corresponding to the catalytic pocket in the homologous MAP enzymes (Bradatsch et al., 2007). However, the cryo-EM structures showed that access to this cavity is restricted in 60S-bound Arx1 because it faces the ribosomal surface (Bradatsch et al., 2012; Greber et al., 2012). The solventexposed side of Arx1 interacts with expansion segment (ES) 27 and thereby stabilizes this long eukaryotic-specific rRNA helix (Greber et al., 2012). "
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    ABSTRACT: Eukaryotic ribosome biogenesis depends on several hundred assembly factors to produce functional 40S and 60S ribosomal subunits. The final phase of 60S subunit biogenesis is cytoplasmic maturation, which includes the proofreading of functional centers of the 60S subunit and the release of several ribosome biogenesis factors. We report the cryo-electron microscopy (cryo-EM) structure of the yeast 60S subunit in complex with the biogenesis factors Rei1, Arx1, and Alb1 at 3.4 Å resolution. In addition to the network of interactions formed by Alb1, the structure reveals a mechanism for ensuring the integrity of the ribosomal polypeptide exit tunnel. Arx1 probes the entire set of inner-ring proteins surrounding the tunnel exit, and the C terminus of Rei1 is deeply inserted into the ribosomal tunnel, where it forms specific contacts along almost its entire length. We provide genetic and biochemical evidence that failure to insert the C terminus of Rei1 precludes subsequent steps of 60S maturation.
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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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    • "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 "
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