Processing of 20S pre-rRNA to 18S ribosomal RNA in yeast requires Rrp10p, an essential non-ribosomal cytoplasmic protein

Vanrobays, Emmanuel

doi:10.1093/emboj/20.15.4204

Cited by 113 publications

(149 citation statements)

References 50 publications

(90 reference statements)

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“…To examine this possibility, we imaged Rio1, labeled with green fluorescent protein (Rio1-GFP), in exponentially growing S. cerevisiae cells. We identified the protein both in the cytoplasm (consistent with its documented involvement in 20S pre-rRNA maturation 15 and pre-40S ribosome trans-factor recycling 12,17,18 ), and in the nucleus (Fig. 1a).…”

Section: Resultssupporting

confidence: 73%

“…Noteworthy, the RIO kinases may act especially as ATPases as they exhibit o0.1% kinase activity in vitro [12][13][14] . Cytoplasmic Rio1 contributes to pre-40S ribosome biogenesis by promoting 20S pre-rRNA maturation and by stimulating the recycling of trans-acting factors at the pre-40S subunit, both in yeast 12,[15][16][17][18] and human cells 19,20 . Roles in the nucleus are unknown for any RIO member, either in yeast or eukaryotes beyond.…”

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confidence: 99%

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Rio1 promotes rDNA stability and downregulates RNA polymerase I to ensure rDNA segregation

et al. 2015

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The conserved protein kinase Rio1 localizes to the cytoplasm and nucleus of eukaryotic cells. While the roles of Rio1 in the cytoplasm are well characterized, its nuclear function remains unknown. Here we show that nuclear Rio1 promotes rDNA array stability and segregation in Saccharomyces cerevisiae. During rDNA replication in S phase, Rio1 downregulates RNA polymerase I (PolI) and recruits the histone deacetylase Sir2. Both interventions ensure rDNA copy-number homeostasis and prevent the formation of extrachromosomal rDNA circles, which are linked to accelerated ageing in yeast. During anaphase, Rio1 downregulates PolI by targeting its subunit Rpa43, causing PolI to dissociate from the rDNA. By stimulating the processing of PolI-generated transcripts at the rDNA, Rio1 allows for rDNA condensation and segregation in late anaphase. These events finalize the genome transmission process. We identify Rio1 as an essential nucleolar housekeeper that integrates rDNA replication and segregation with ribosome biogenesis. A t anaphase onset, the replicated chromosomes separate and then segregate along the mitotic spindle into the daughter cells. In the budding yeast Saccharomyces cerevisiae, the locus containing the genes that encode the ribosomal RNAs (rDNA) segregates after the rest of the genome, in late anaphase [1][2][3][4] . The rDNA locus exists as a tandem-repeat array comprising B150 rDNA units containing the 35S and 5S genes, which are transcribed by RNA polymerase I (PolI) and PolIII, respectively. Processing of the 35S pre-rRNA generates 5.8S, 18S and 25S rRNA that, together with the 5S rRNA, become the catalytic backbones of each ribosome 5,6 . Only in anaphase does yeast repress rDNA transcription 4 , which allows the sister rDNA loci to condensate and segregate. PolI downregulation in anaphase is mediated by the Cdc14 phosphatase acting on PolI subunit Rpa43 (ref. 4), resulting in PolI dissociating from the 35S rDNA. The removal of PolI and the local resolution of its transcripts allow the condensin complex to bind. The latter compacts the rDNA array and recruits the DNA decatenating enzyme topoisomerase II (refs 1,3,4,7) resulting in the physical separation and subsequent segregation of the sister rDNA loci.S. cerevisiae Rio1 belongs to the atypical RIO protein kinase family whose members lack the activation loop and substrate recognition domain present in canonical eukaryotic protein kinases [8][9][10][11] . Noteworthy, the RIO kinases may act especially as ATPases as they exhibit o0.1% kinase activity in vitro [12][13][14] . Cytoplasmic Rio1 contributes to pre-40S ribosome biogenesis by promoting 20S pre-rRNA maturation and by stimulating the recycling of trans-acting factors at the pre-40S subunit, both in yeast 12,[15][16][17][18] and human cells 19,20 . Roles in the nucleus are unknown for any RIO member, either in yeast or eukaryotes beyond. Using S. cerevisiae, we now describe the first activities of Rio1 in the nucleus. Foremost, Rio1 downregulates PolI transcription through the cell cycle. In G...

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Section: Resultssupporting

confidence: 73%

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confidence: 99%

Rio1 promotes rDNA stability and downregulates RNA polymerase I to ensure rDNA segregation

et al. 2015

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“…A large number of factors are required for this step, most of which must be involved in regulating the cleavage as only two, Nob1 and Fap7, are actually implicated in the cleavage step itself. Depletion of Rio1, Rio2, Tsr1, or Tsr2 all lead to 20S accumulation (Gelperin et al 2001;Vanrobays et al 2001Vanrobays et al , 2003Geerlings et al 2003;Peng et al 2003;Leger-Silvestre et al 2004). In the case of Rio2 and Tsr1, depletion results in the cytoplasmic accumulation of 20S as measured by FISH (Vanrobays et al 2003;Leger-Silvestre et al 2004), indicating that yeast Rio2 and Tsr1 function not in pre-40S export but in the cytoplasmic maturation of the 20SrRNA.…”

Section: Resultsmentioning

confidence: 99%

Dominant Mutations in the Late 40S Biogenesis Factor Ltv1 Affect Cytoplasmic Maturation of the Small Ribosomal Subunit in Saccharomyces cerevisiae

et al. 2010

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In eukaryotes, 40S and 60S ribosomal subunits are assembled in the nucleus from rRNAs and ribosomal proteins, exported as premature complexes, and processed in final maturation steps in the cytoplasm. Ltv1 is a conserved 40S ribosome biogenesis factor that interacts with pre-40S complexes in vivo and is proposed to function in yeast in nuclear export. Cells lacking LTV1 grow slowly and are significantly impaired in mature 40S subunit production. Here we show that mutation or deletion of a putative nuclear export sequence in LTV1 is strongly dominant negative, but the protein does not accumulate in the nucleus, as expected for a mutation affecting export. In fact, most of the mutant protein is cytoplasmic and associated with pre-40S subunits. Cells expressing mutant Ltv1 have a 40S biogenesis defect, accumulate 20S rRNA in the cytoplasm as detected by FISH, and retain the late-acting biogenesis factor Tsr1 in the cytoplasm. Finally, overexpression of mutant Ltv1 is associated with nuclear retention of 40S subunit marker proteins, RpS2-GFP and RpS3-GFP. We suggest that the proximal consequence of these LTV1 mutations is inhibition of the cytoplasmic maturation of 40S subunits and that nuclear retention of pre-40S subunits is a downstream consequence of the failure to release and recycle critical factors back to the nucleus. R IBOSOME biogenesis is a major biosynthetic activity of eukaryotic cells and a significant ratelimiting factor in cell growth and proliferation. The pathway appears to be conserved in most respects in eukaryotes from yeast to humans and has been extensively analyzed in the model organism, Saccharomyces cerevisiae. Ribosome biogenesis begins cotranscriptionally in the nucleolus, continues in the nucleoplasm, and is completed in the cytoplasm where final maturation events must occur (for reviews, see More than 150 trans-acting factors have been identified by genetic and proteomic studies with roles in ribosome biogenesis. Generally, these factors are well conserved through eukaryotic evolution, but the specific function of many of the proteins remains largely unknown.In yeast, the 18S, 5.8S, and 25S ribosomal RNAs (rRNAs) are transcribed as a single 35S precursor rRNA. About 40 ribosome biogenesis factors, U3 snoRNA, and ribosomal proteins assemble cotranscriptionally on the nascent 35S pre-rRNA to form the 90S pre-ribosome (Dragon et al. 2002;Grandi et al. 2002;Schafer et al. 2003). Cleavage of the 35S pre-rRNA at site A2 releases a pre-40S particle containing 20S pre-rRNA, which then sheds most of the processing factors associated with it (Schafer et al. 2003). Pre-60S biogenesis factors and ribosomal proteins then assemble on the remaining transcript. The pre-60S subunit undergoes extensive remodeling and processing in the nucleolus and nucleoplasm, acquiring new processing factors and incorporating the independently transcribed 5S pre-rRNA before being exported through the nuclear pore to the cytoplasm (reviewed in Fromont-Racine et al. 2003;Tschochner and Hurt 2003). The pre-40S an...

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“…In the lsm1-⌬ strain, but not the lsm6-⌬ or lsm7-⌬ strains, the 20S pre-rRNA was depleted. It is notable that processing of the 20S pre-rRNA to the mature 18S rRNA takes place in the cytoplasm (48,49), and Lsm1p also localizes mainly to the cytoplasm (32).…”

Section: Depletion Of Any Essential Lsm Protein Leads To the Appearanmentioning

confidence: 99%

Lsm Proteins Are Required for Normal Processing and Stability of Ribosomal RNAs

Kufel

Allmang²,

Petfalski

et al. 2003

Journal of Biological Chemistry

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Depletion of any of the essential Lsm proteins, Lsm2-5p or Lsm8p, delayed pre-rRNA processing and led to the accumulation of many aberrant processing intermediates, indicating that an Lsm complex is required to maintain the normally strict order of processing events. In addition, high levels of degradation products derived from both precursors and mature rRNAs accumulated in Lsm-depleted strains. Depletion of the essential Lsm proteins reduced the apparent processivity of both 5 and 3 exonuclease activities involved in 5.8S rRNA processing, and the degradation intermediates that accumulated were consistent with inefficient 5 and 3 degradation. Many, but not all, pre-rRNA species could be coprecipitated with tagged Lsm3p, but not with tagged Lsm1p or non-tagged control strains, suggesting their direct interaction with an Lsm2-8p complex. We propose that Lsm proteins facilitate RNA protein interactions and structural changes required during ribosomal subunit assembly.The yeast 18S, 5.8S, and 25S rRNAs are transcribed by RNA polymerase I as a single precursor, the 35S pre-rRNA, which undergoes complex post-transcriptional processing to remove the external transcribed spacers (5Ј-ETS and 3Ј-ETS) 1 and internal transcribed spacers (ITS1 and ITS2) to release mature rRNAs (see Fig. 1A). This process involves multiple endonucleolytic and exonucleolytic steps (see Fig. 1B) and is largely carried out in the nucleolus. In Saccharomyces cerevisiae, enzymes directly involved in these reactions include the endonucleases RNase MRP and Rnt1p, the 5Ј 3 3Ј exonuclease Rat1p, and 3Ј 3 5Ј exonucleases, including the exosome complex, Rex1p, and Rex2p (1, 2; reviewed in Refs. 3 and 4). In addition to the RNA processing enzymes, around 110 other factors are known to be required for normal pre-rRNA processing in yeast. These include several small nucleolar ribonucleoprotein (snoRNP) particles, putative RNA helicases, GTPases, and many other assembly factors (5). It is very likely that these act to promote correct folding of the pre-rRNA, assembly of the ϳ80 ribosomal proteins, and assembly/disassembly of the processing complexes, with processing inhibition arising as a secondary consequence of defects in the structure of the preribosomal particles (see Refs. 3 and 4).All of the enzymes known to process the pre-rRNA also process other RNA species. Rnt1p, the exosome, and Rex proteins generate the 3Ј-ends of small nuclear and small nucleolar RNAs (snRNAs and snoRNAs), whereas Xrn1p and Rat1p produce 5Ј-ends of intron-encoded and polycistronic snoRNAs (2, 6 -13). Likewise, degradation of many RNAs, including cytoplasmic messenger RNAs (mRNAs) and nuclear pre-mRNAs, involves pre-rRNA processing exonucleases: the exosome, Xrn1p, and Rat1p (14 -18).Sm-like (Lsm) proteins have been identified in all kingdoms of life and participate in numerous RNA processing and degradation pathways. The Sm and Lsm complexes are all likely to form similar structures with seven-membered rings (or six in the case of Escherichia coli Hfq) with a central hole, th...

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Processing of 20S pre-rRNA to 18S ribosomal RNA in yeast requires Rrp10p, an essential non-ribosomal cytoplasmic protein

Cited by 113 publications

References 50 publications

Rio1 promotes rDNA stability and downregulates RNA polymerase I to ensure rDNA segregation

Rio1 promotes rDNA stability and downregulates RNA polymerase I to ensure rDNA segregation

Dominant Mutations in the Late 40S Biogenesis Factor Ltv1 Affect Cytoplasmic Maturation of the Small Ribosomal Subunit in Saccharomyces cerevisiae

Lsm Proteins Are Required for Normal Processing and Stability of Ribosomal RNAs

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