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.

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Available from: Jesús De la Cruz, Oct 05, 2015
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    • "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). "
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    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
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    • "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. "
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    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
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    • "R-protein L8 is required for association of A3 factors with 66S pre-ribosomes and processing of 27SA3 pre-rRNA, but does not depend on the A3 factors to enter pre-rRNPs (31). The presence of A3 factors is necessary for assembly of r-proteins L17, L26, L35 and L37 with pre-ribosomes (18), which are required for efficient cleavage at the C2 site in 27SB pre-rRNA (25,32). All of these factors must be in place for association of Nsa2 and Nog2 with pre-ribosomes, which are required for cleavage of 27SB pre-rRNA (18,25,31,32). "
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    ABSTRACT: Ribosome biogenesis requires ∼200 assembly factors in Saccharomyces cerevisiae. The pre-ribosomal RNA (rRNA) processing defects associated with depletion of most of these factors have been characterized. However, how assembly factors drive the construction of ribonucleoprotein neighborhoods and how structural rearrangements are coupled to pre-rRNA processing are not understood. Here, we reveal ATP-independent and ATP-dependent roles of the Has1 DEAD-box RNA helicase in consecutive pre-rRNA processing and maturation steps for construction of 60S ribosomal subunits. Has1 associates with pre-60S ribosomes in an ATP-independent manner. Has1 binding triggers exonucleolytic trimming of 27SA3 pre-rRNA to generate the 5' end of 5.8S rRNA and drives incorporation of ribosomal protein L17 with domain I of 5.8S/25S rRNA. ATP-dependent activity of Has1 promotes stable association of additional domain I ribosomal proteins that surround the polypeptide exit tunnel, which are required for downstream processing of 27SB pre-rRNA. Furthermore, in the absence of Has1, aberrant 27S pre-rRNAs are targeted for irreversible turnover. Thus, our data support a model in which Has1 helps to establish domain I architecture to prevent pre-rRNA turnover and couples domain I folding with consecutive pre-rRNA processing steps.
    Nucleic Acids Research 06/2013; 41(16). DOI:10.1093/nar/gkt545 · 9.11 Impact Factor
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