Yeast polypeptide exit tunnel ribosomal proteins L17, L35 and L37 are necessary to recruit late-assembling factors required for 27SB pre-rRNA processing

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA and Departamento de Genética, Universidad de Sevilla, Sevilla, E-41012, Spain.
Nucleic Acids Research (Impact Factor: 9.11). 12/2012; 41(3). DOI: 10.1093/nar/gks1272
Source: PubMed


Ribosome synthesis involves the coordinated folding and processing of pre-rRNAs with assembly of ribosomal proteins. In eukaryotes, these events are facilitated by trans-acting factors that propel ribosome maturation from the nucleolus to the cytoplasm. However, there is a gap in understanding how ribosomal proteins configure pre-ribosomes in vivo to enable processing to occur. Here, we have examined the role of adjacent yeast r-proteins L17, L35 and L37 in folding and processing of pre-rRNAs, and binding of other proteins within assembling ribosomes. These three essential ribosomal proteins, which surround the polypeptide exit tunnel, are required for 60S subunit formation as a consequence of their role in removal of the ITS2 spacer from 27SB pre-rRNA. L17-, L35- and L37-depleted cells exhibit turnover of aberrant pre-60S assembly intermediates. Although the structure of ITS2 does not appear to be grossly affected in their absence, these three ribosomal proteins are necessary for efficient recruitment of factors required for 27SB pre-rRNA processing, namely, Nsa2 and Nog2, which associate with pre-60S ribosomal particles containing 27SB pre-rRNAs. Altogether, these data support that L17, L35 and L37 are specifically required for a recruiting step immediately preceding removal of ITS2.

Download full-text


Available from: Jesús De la Cruz,
    • "We also found that depletion of mouse Rpl17 promoted the formation of " cropped " 32S C pre-rRNA. Similar 5 ′ -truncated 28SB forms were observed in yeast as an intermediate in Rat1-mediated turnover of pre-rRNA in stalled pre-60S subunits (Sahasranaman et al. 2011; Gamalinda et al. 2013). Surprisingly, mammalian 32S C pre-rRNA gives rise to functional ribosomes in a fraction of maturation events. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Processing of rRNA during ribosome assembly can proceed through alternative pathways but it is unclear whether this could affect the structure of the ribosome. Here, we demonstrate that shortage of a ribosomal protein can change pre-rRNA processing in a way that over time alters ribosome diversity in the cell. Reducing the amount of Rpl17 in mouse cells led to stalled 60S subunit maturation, causing degradation of most of the synthesized precursors. A fraction of pre-60S subunits, however, were able to complete maturation, but with a 5'-truncated 5.8S rRNA, which we named 5.8SC. The 5' exoribonuclease Xrn2 is involved in the generation of both 5.8SC and the canonical long form of 5.8S rRNA. Ribosomes containing 5.8SC rRNA are present in various mouse and human cells and engage in translation. These findings uncover a previously undescribed form of mammalian 5.8S rRNA and demonstrate that perturbations in ribosome assembly can be a source of heterogeneity in mature ribosomes. © 2015 Wang et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
    RNA 05/2015; 21(7). DOI:10.1261/rna.051169.115 · 4.94 Impact Factor
  • Source
    • "The A3 factors are also intriguing because their association with pre-ribosomal particles appears to be interdependent (6). Moreover, A3 factors are required for proper assembly of four r-proteins (L17, L26, L35 and L37) that predominantly bind to 5.8S/25S rRNA domain I, which in turn enable cleavage of ITS2 at site C2 (6,14,18,47). Consistently, our CRAC analyses show that Rlp7 binds to ITS2 at a position adjacent with that of the 3′ end of mature 5.8S rRNA (site E) and the 5′ end of mature 25S rRNA (site C1). The Rlp7 binding sites on pre-rRNA partially overlap with those previously reported for the A3 factor Nsa3 and are close to those of other A3 factors (Nop12, Nop15, Erb1 and Nop7) (7). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Ribosome biogenesis requires >300 assembly factors in Saccharomyces cerevisiae. Ribosome assembly factors Imp3, Mrt4, Rlp7 and Rlp24 have sequence similarity to ribosomal proteins S9, P0, L7 and L24, suggesting that these pre-ribosomal factors could be placeholders that prevent premature assembly of the corresponding ribosomal proteins to nascent ribosomes. However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles. Cross-linking and cDNA analysis experiments showed that Rlp7 binds to the ITS2 region of 27S pre-rRNAs, at two sites, in helix III and in a region adjacent to the pre-rRNA processing sites C1 and E. However, L7 binds to mature 25S and 5S rRNAs and cross-linked predominantly to helix ES7(L)b within 25S rRNA. Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles. Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation.
    Nucleic Acids Research 08/2013; 41(20). DOI:10.1093/nar/gkt726 · 9.11 Impact Factor
  • Source
    • "These studies indicated that the final incorporation of rpL10 [56] and rpL40 [57] into LSUs is established only after most of the processing steps of LSU rRNA precursors are accomplished. For rpL5, rpL11 [31] and rpL35 [58] association was detected already with early, and for rpL26 [59], rpL17 and rpL37 [30,32] association was seen mainly starting at the level of intermediate nuclear LSU precursor populations. "
    [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