scholarly journals The Final Step in 5.8S rRNA Processing Is Cytoplasmic in Saccharomyces cerevisiae

2009 ◽  
Vol 30 (4) ◽  
pp. 976-984 ◽  
Author(s):  
Emma Thomson ◽  
David Tollervey
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Export ◽  
18S Rrna ◽  
Rrna Processing ◽  
Leptomycin B ◽  
5.8S Rrna ◽  
Final Maturation ◽  
Rrna Degradation

ABSTRACT The 18S rRNA component of yeast (Saccharomyces cerevisiae) 40S ribosomes undergoes cytoplasmic 3′ cleavage following nuclear export, whereas exported pre-60S subunits were believed to contain only mature 5.8S and 25S rRNAs. However, in situ hybridization detected 3′-extended forms of 5.8S rRNA in the cytoplasm, which were lost when Crm1-dependent preribosome export was blocked by treatment with leptomycin B (LMB). LMB treatment rapidly blocked processing of 6S pre-rRNA to 5.8S rRNA, leading to TRAMP-dependent pre-rRNA degradation. The 6S pre-rRNA was coprecipitated with the 60S export adapter Nmd3 and cytoplasmic 60S synthesis factor Lsg1. The longer 5.8S+30 pre-rRNA (a form of 5.8S rRNA 3′ extended by ∼30 nucleotides) is processed to 6S by the nuclear exonuclease Rrp6, and nuclear pre-rRNA accumulated in the absence of Rrp6. In contrast, 6S to 5.8S processing requires the cytoplasmic exonuclease Ngl2, and cytoplasmic pre-rRNA accumulated in strains lacking Ngl2. We conclude that nuclear pre-60S particles containing the 6S pre-rRNA bind Nmd3 and Crm1 and are exported to the cytoplasm prior to final maturation by Ngl2.

scholarly journals Exportin 1 Mediates Nuclear Export of the Kinetoplastid Spliced Leader RNA

2003 ◽  
Vol 2 (2) ◽  
pp. 222-230 ◽  
Author(s):  
Gusti M. Zeiner ◽  
Nancy R. Sturm ◽  
David A. Campbell
Keyword(s):  
Nuclear Export ◽  
Ectopic Expression ◽  
Specific Inhibitor ◽  
Leptomycin B ◽  
Spliced Leader ◽  
Small Nuclear Rnas ◽  
Rna Biogenesis ◽  
Common Substrate ◽  
The Common

ABSTRACT The kinetoplastid protozoan spliced leader (SL) RNA is the common substrate pre-mRNA utilized in all trans-splicing reactions. Here we show by fluorescence in situ hybridization that the SL RNA is present in the cytoplasm of Leishmania tarentolae and Trypanosoma brucei. Treatment with the karyopherin-specific inhibitor leptomycin B was toxic to T. brucei and eliminated the cytoplasmic SL RNA, suggesting that cytoplasmic SL RNA was dependent on the nuclear exporter exportin 1 (XPO1). Ectopic expression of xpo1 with a C506S mutation in T. brucei conferred resistance to leptomycin B. A reduction in SL RNA 3′ extension removal and 5′ methylation of nucleotide U4 was observed in wild-type T. brucei treated with leptomycin B, suggesting that the cytoplasmic stage is necessary for SL RNA biogenesis. This study demonstrates spatial and mechanistic similarities between the posttranscriptional trafficking of the kinetoplastid protozoan SL RNA and the metazoan cis-spliceosomal small nuclear RNAs.

scholarly journals Late Cytoplasmic Maturation of the Small Ribosomal Subunit Requires RIO Proteins in Saccharomyces cerevisiae

2003 ◽  
Vol 23 (6) ◽  
pp. 2083-2095 ◽  
Author(s):  
Emmanuel Vanrobays ◽  
Jean-Paul Gelugne ◽  
Pierre-Emmanuel Gleizes ◽  
Michele Caizergues-Ferrer
Keyword(s):  
18S Rrna ◽  
Nuclear Protein ◽  
Essential Gene ◽  
Sequence Similarity ◽  
Ribosomal Subunit ◽  
Rrna Processing ◽  
Ribosomal Subunits ◽  
Rrna Maturation ◽  
Cytoplasmic Maturation

ABSTRACT Numerous nonribosomal trans-acting factors involved in pre-rRNA processing have been characterized, but few of them are specifically required for the last cytoplasmic steps of 18S rRNA maturation. We have recently demonstrated that Rrp10p/Rio1p is such a factor. By BLAST analysis, we identified the product of a previously uncharacterized essential gene, YNL207W/RIO2, called Rio2p, that shares 43% sequence similarity with Rrp10p/Rio1p. Rio2p homologues were identified throughout the Archaea and metazoan species. We show that Rio2p is a cytoplasmic-nuclear protein and that its depletion blocks 18S rRNA production, leading to 20S pre-rRNA accumulation. In situ hybridization reveals that in Rio2p-depleted cells, 20S pre-rRNA localizes in the cytoplasm, demonstrating that its accumulation is not due to an export defect. We also show that both Rio1p and Rio2p accumulate in the nucleus of crm1-1 cells at the nonpermissive temperature. Nuclear as well as cytoplasmic Rio2p and Rio1p cosediment with pre-40S particles. These results strongly suggest that Rio2p and Rrp10p/Rio1p are shuttling proteins which associate with pre-40S particles in the nucleus and they are not necessary for export of the pre-40S complexes but are absolutely required for the cytoplasmic maturation of 20S pre-rRNA at site D, leading to mature 40S ribosomal subunits.

scholarly journals Nuclear RNase MRP is required for correct processing of pre-5.8S rRNA in Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (12) ◽  
pp. 7935-7941 ◽  
Author(s):  
M E Schmitt ◽  
D A Clayton
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Yeast Cells ◽  
Rrna Processing ◽  
Rnase Mrp ◽  
Gal1 Promoter ◽  
5.8S Rrna ◽  
Late Step ◽  
Conditional Mutations ◽  
A Site

RNase MRP is a site-specific ribonucleoprotein endoribonuclease that cleaves RNA from the mitochondrial origin of replication in a manner consistent with a role in priming leading-strand DNA synthesis. Despite the fact that the only known RNA substrate for this enzyme is complementary to mitochondrial DNA, the majority of the RNase MRP activity in a cell is found in the nucleus. The recent characterization of this activity in Saccharomyces cerevisiae and subsequent cloning of the gene coding for the RNA subunit of the yeast enzyme have enabled a genetic approach to the identification of a nuclear role for this ribonuclease. Since the gene for the RNA component of RNase MRP, NME1, is essential in yeast cells and RNase MRP in mammalian cells appears to be localized to nucleoli within the nucleus, we utilized both regulated expression and temperature-conditional mutations of NME1 to assay for a possible effect on rRNA processing. Depletion of the RNA component of the enzyme was accomplished by using the glucose-repressed GAL1 promoter. Shortly after the shift to glucose, the RNA component of the enzyme was found to be depleted severely, and rRNA processing was found to be normal at all sites except the B1 processing site. The B1 site, at the 5' end of the mature 5.8S rRNA, is actually composed of two cleavage sites 7 nucleotides apart. This cleavage normally generates two species of 5.8S rRNA at a ratio of 10:1 (small to large) in most eukaryotes. After RNase MRP depletion, yeast cells were found to have almost exclusively the larger species of 5.8S rRNA. In addition, an aberrant 309-nucleotide precursor that stretched from the A2 to E processing sites of rRNA accumulated in these cells. Temperature-conditional mutations in the RNase MRP RNA gene gave an identical phenotype.Translation in yeast cells depleted of the smaller 5.8S rRNA was found to remain robust, suggesting a possible function for two 5.8S rRNAs in the regulated translation of select messages. These results are consistent with RNase MRP playing a role in a late step of rRNA processing. The data also indicate a requirement for having the smaller form of 5.8S rRNA, and they argue for processing at the B1 position being composed of two separate cleavage events catalyzed by two different activities.

scholarly journals Ultrastructural localization of rRNA shows defective nuclear export of preribosomes in mutants of the Nup82p complex

2001 ◽  
Vol 155 (6) ◽  
pp. 923-936 ◽  
Author(s):  
Pierre-Emmanuel Gleizes ◽  
Jacqueline Noaillac-Depeyre ◽  
Isabelle Léger-Silvestre ◽  
Frédéric Teulières ◽  
Jean-Yves Dauxois ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Ultrastructural Localization ◽  
Small Subunit ◽  
Nuclear Accumulation ◽  
Rrna Processing ◽  
Wild Type ◽  
Tight Control ◽  
Yeast Saccharomyces Cerevisiae ◽  
In Cells

To study the nuclear export of preribosomes, ribosomal RNAs were detected by in situ hybridization using fluorescence and EM, in the yeast Saccharomyces cerevisiae. In wild-type cells, semiquantitative analysis shows that the distributions of pre-40S and pre-60S particles in the nucleolus and the nucleoplasm are distinct, indicating uncoordinated transport of the two subunits within the nucleus. In cells defective for the activity of the GTPase Gsp1p/Ran, ribosomal precursors accumulate in the whole nucleus. This phenotype is reproduced with pre-60S particles in cells defective in pre-rRNA processing, whereas pre-40S particles only accumulate in the nucleolus, suggesting a tight control of the exit of the small subunit from the nucleolus. Examination of nucleoporin mutants reveals that preribosome nuclear export requires the Nup82p–Nup159p–Nsp1p complex. In contrast, mutations in the nucleoporins forming the Nup84p complex yield very mild or no nuclear accumulation of preribosome. Interestingly, domains of Nup159p required for mRNP trafficking are not necessary for preribosome export. Furthermore, the RNA helicase Dbp5p and the protein Gle1p, which interact with Nup159p and are involved in mRNP trafficking, are dispensable for ribosomal transport. Thus, the Nup82p–Nup159p–Nsp1p nucleoporin complex is part of the nuclear export pathways of preribosomes and mRNPs, but with distinct functions in these two processes.

scholarly journals Unique Interactions of the Nuclear Export Receptors TbMex67 and TbMtr2 with Components of the 5S Ribonuclear Particle in Trypanosoma brucei

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Constance Rink ◽  
Noreen Williams
Keyword(s):  
Trypanosoma Brucei ◽  
Nuclear Export ◽  
Ribosome Biogenesis ◽  
5S Rrna ◽  
Ribosome Assembly ◽  
Rrna Processing ◽  
Ribosomal Subunits ◽  
Content Type ◽  
Final Maturation ◽  
Specific Proteins

ABSTRACT Eukaryotic ribosome biogenesis is a complicated and highly conserved biological process. A critical step in ribosome biogenesis is the translocation of the immature ribosomal subunits from the nucleoplasm, across the nucleopore complex, to the cytoplasm where they undergo final maturation. Many nonribosomal proteins are needed to facilitate export of the ribosomal subunits, and one complex participating in export of the pre-60S in Saccharomyces cerevisiae is the heterodimer Mex67-Mtr2. In Trypanomsoma brucei, the process of ribosome biogenesis differs from the yeast process in key steps and is not yet fully characterized. However, our laboratory has previously identified the trypanosome-specific proteins P34/P37 and has shown that P34/P37 are necessary for the formation of the 5S ribonuclear particle (RNP) and for the nuclear export of the pre-60S subunit. We have also shown that loss of TbMex67 or TbMtr2 leads to aberrant ribosome formation, rRNA processing, and polysome formation in T. brucei. In this study, we characterize the interaction of TbMex67 and TbMtr2 with the components of the 5S RNP (P34/P37, L5 and 5S rRNA) of the 60S subunit. We demonstrate that TbMex67 directly interacts with P34 and L5 proteins as well as 5S rRNA, while TbMtr2 does not. Using protein sequence alignments and structure prediction modeling, we show that TbMex67 lacks the amino acids previously shown to be essential for binding to 5S rRNA in yeast and in general aligns more closely with the human orthologue (NXF1 or TAP). This work suggests that the T. brucei Mex67-Mtr2 binds ribosomal cargo differently from the yeast system. IMPORTANCE Trypanosoma brucei is the causative agent for both African sleeping sickness in humans and nagana in cattle. Ribosome biogenesis in these pathogens requires both conserved and trypanosome-specific proteins to coordinate in a complex pathway. We have previously shown that the trypanosome-specific proteins P34/P37 are essential to the interaction of the TbNmd3-TbXpoI export complex with the 60S ribosomal subunits, allowing their translocation across the nuclear envelope. Our recent studies show that the trypanosome orthologues of the auxiliary export proteins TbMex67-TbMtr2 are required for ribosome assembly, proper rRNA processing, and polysome formation. Here we show that TbMex67-TbMtr2 interact with members of the 60S ribosomal subunit 5S RNP. Although TbMex67 has a unique structure among the Mex67 orthologues and forms unique interactions with the 5S RNP, particularly with trypanosome-specific P34/P37, it performs a conserved function in ribosome assembly. These unique structures and parasite-specific interactions may provide new therapeutic targets against this important parasite.

Nuclear RNase MRP is required for correct processing of pre-5.8S rRNA in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (12) ◽  
pp. 7935-7941
Author(s):  
M E Schmitt ◽  
D A Clayton
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Yeast Cells ◽  
Rrna Processing ◽  
Rnase Mrp ◽  
Gal1 Promoter ◽  
5.8S Rrna ◽  
Late Step ◽  
Conditional Mutations ◽  
A Site

RNase MRP is a site-specific ribonucleoprotein endoribonuclease that cleaves RNA from the mitochondrial origin of replication in a manner consistent with a role in priming leading-strand DNA synthesis. Despite the fact that the only known RNA substrate for this enzyme is complementary to mitochondrial DNA, the majority of the RNase MRP activity in a cell is found in the nucleus. The recent characterization of this activity in Saccharomyces cerevisiae and subsequent cloning of the gene coding for the RNA subunit of the yeast enzyme have enabled a genetic approach to the identification of a nuclear role for this ribonuclease. Since the gene for the RNA component of RNase MRP, NME1, is essential in yeast cells and RNase MRP in mammalian cells appears to be localized to nucleoli within the nucleus, we utilized both regulated expression and temperature-conditional mutations of NME1 to assay for a possible effect on rRNA processing. Depletion of the RNA component of the enzyme was accomplished by using the glucose-repressed GAL1 promoter. Shortly after the shift to glucose, the RNA component of the enzyme was found to be depleted severely, and rRNA processing was found to be normal at all sites except the B1 processing site. The B1 site, at the 5' end of the mature 5.8S rRNA, is actually composed of two cleavage sites 7 nucleotides apart. This cleavage normally generates two species of 5.8S rRNA at a ratio of 10:1 (small to large) in most eukaryotes. After RNase MRP depletion, yeast cells were found to have almost exclusively the larger species of 5.8S rRNA. In addition, an aberrant 309-nucleotide precursor that stretched from the A2 to E processing sites of rRNA accumulated in these cells. Temperature-conditional mutations in the RNase MRP RNA gene gave an identical phenotype.Translation in yeast cells depleted of the smaller 5.8S rRNA was found to remain robust, suggesting a possible function for two 5.8S rRNAs in the regulated translation of select messages. These results are consistent with RNase MRP playing a role in a late step of rRNA processing. The data also indicate a requirement for having the smaller form of 5.8S rRNA, and they argue for processing at the B1 position being composed of two separate cleavage events catalyzed by two different activities.

scholarly journals USP16 counteracts mono-ubiquitination of RPS27a and promotes maturation of the 40S ribosomal subunit

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Christian Montellese ◽  
Jasmin van den Heuvel ◽  
Caroline Ashiono ◽  
Kerstin Dörner ◽  
André Melnik ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Synthesis ◽  
Ribosome Biogenesis ◽  
18S Rrna ◽  
Ribosomal Subunit ◽  
Rrna Processing ◽  
Ribosomal Subunits ◽  
Novel Proteins ◽  
40S Ribosomal Subunit ◽  
Final Maturation

Establishment of translational competence represents a decisive cytoplasmic step in the biogenesis of 40S ribosomal subunits. This involves final 18S rRNA processing and release of residual biogenesis factors, including the protein kinase RIOK1. To identify novel proteins promoting the final maturation of human 40S subunits, we characterized pre-ribosomal subunits trapped on RIOK1 by mass spectrometry, and identified the deubiquitinase USP16 among the captured factors. We demonstrate that USP16 constitutes a component of late cytoplasmic pre-40S subunits that promotes the removal of ubiquitin from an internal lysine of ribosomal protein RPS27a/eS31. USP16 deletion leads to late 40S subunit maturation defects, manifesting in incomplete processing of 18S rRNA and retarded recycling of late-acting ribosome biogenesis factors, revealing an unexpected contribution of USP16 to the ultimate step of 40S synthesis. Finally, ubiquitination of RPS27a appears to depend on active translation, pointing at a potential connection between 40S maturation and protein synthesis.

scholarly journals Nop2p is required for pre-rRNA processing and 60S ribosome subunit synthesis in yeast.

1997 ◽  
Vol 17 (1) ◽  
pp. 378-388 ◽  
Author(s):  
B Hong ◽  
J S Brockenbrough ◽  
P Wu ◽  
J P Aris
Keyword(s):  
Steady State ◽  
Doubling Time ◽  
18S Rrna ◽  
Rrna Processing ◽  
Sequence Comparisons ◽  
Ribosomal Subunits ◽  
Rna Methylation ◽  
5.8S Rrna ◽  
Processing Step ◽  
Steady State Levels

To investigate the function of the nucleolar protein Nop2p in Saccharomyces cerevisiae, we constructed a strain in which NOP2 is under the control of a repressible promoter. Repression of NOP2 expression lengthens the doubling time of this strain about fivefold and reduces steady-state levels of 60S ribosomal subunits, 80S ribosomes, and polysomes. Levels of 40S subunits increase as the free pool of 60S subunits is reduced. Nop2p depletion impairs processing of the 35S pre-rRNA and inhibits processing of 27S pre-rRNA, which results in lower steady-state levels of 25S rRNA and 5.8S rRNA. Processing of 20S pre-rRNA to 18S rRNA is not significantly affected. Processing at sites A2, A3, B1L, and B1S and the generation of 5' termini of different pre-rRNA intermediates appear to be normal after Nop2p depletion. Sequence comparisons suggest that Nop2p may function as a methyltransferase. 2'-O-ribose methylation of the conserved site UmGm psi UC2922 is known to take place during processing of 27S pre-rRNA. Although Nop2p depletion lengthens the half-life of 27S pre-RNA, methylation of UmGm psi UC2922 in 27S pre-rRNA is low during Nop2p depletion. However, methylation of UmGm psi UC2922 in mature 25S rRNA appears normal. These findings provide evidence for a close interconnection between methylation at this conserved site and the processing step that yields the 25S rRNA.

The Saccharomyces cerevisiae Small GTPase, Gsp1p/Ran, Is Involved in 3′ Processing of 7S-to-5.8S rRNA and in Degradation of the Excised 5′-A0 Fragment of 35S Pre-rRNA, Both of Which Are Carried Out by the Exosome

Genetics ◽  
2001 ◽  
Vol 158 (2) ◽  
pp. 613-625
Author(s):  
Nobuhiro Suzuki ◽  
Eishi Noguchi ◽  
Nobutaka Nakashima ◽  
Masaya Oki ◽  
Tomoyuki Ohba ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleocytoplasmic Transport ◽  
Small Gtpase ◽  
Temperature Sensitive ◽  
Rrna Processing ◽  
5.8S Rrna ◽  
Allele Specific ◽  
A Subunit ◽  
Rrna Maturation ◽  
Nuclear Exosome

Abstract Dis3p, a subunit of the exosome, interacts directly with Ran. To clarify the relationship between the exosome and the RanGTPase cycle, a series of temperature-sensitive Saccharomyces cerevisiae dis3 mutants were isolated and their 5.8S rRNA processing was compared with processing in strains with mutations in a S. cerevisiae Ran homologue, Gsp1p. In both dis3 and gsp1 mutants, 3′ processing of 7S-to-5.8S rRNA was blocked at three identical sites in an allele-specific manner. In contrast, the 5′ end of 5.8S rRNA was terminated normally in gsp1 and in dis3. Inhibition of 5.8S rRNA maturation in gsp1 was rescued by overexpression of nuclear exosome components Dis3p, Rrp4p, and Mtr4p, but not by a cytoplasmic exosome component, Ski2p. Furthermore, gsp1 and dis3 accumulated the 5′-A0 fragment of 35S pre-rRNA, which is also degraded by the exosome, and the level of 27S rRNA was reduced. Neither 5.8S rRNA intermediates nor 5′-A0 fragments were observed in mutants defective in the nucleocytoplasmic transport, indicating that Gsp1p regulates rRNA processing through Dis3p, independent of nucleocytoplasmic transport.

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