Interactions and activities of factors involved in the late stages of human 18S rRNA maturation

RIO proteins form a conserved family of atypical protein kinases. Humans possess three distinct RIO kinases—hRio1, hRio2, and hRio3, of which only hRio2 has been characterized with respect to its role in ribosomal biogenesis. Here we show that both hRio1 and hRio3, like hRio2, are associated with precursors of 40S ribosomal subunits in human cells. Furthermore, we demonstrate that depletion of hRio1 by RNA interference affects the last step of 18S rRNA maturation and causes defects in the recycling of several trans-acting factors (hEnp1, hRio2, hLtv1, hDim2/PNO1, and hNob1) from pre-40S subunits in the cytoplasm. Although the effects of hRio1 and hRio2 depletion are similar, we show that the two kinases are not fully interchangeable. Moreover, rescue experiments with a kinase-dead mutant of hRio1 revealed that the kinase activity of hRio1 is essential for the recycling of the endonuclease hNob1 and its binding partner hDim2 from cytoplasmic pre-40S. Kinase-dead hRio1 is trapped on pre-40S particles containing hDim2 and hNob1 but devoid of hEnp1, hLtv1, and hRio2. These data reveal a role of hRio1 in the final stages of cytoplasmic pre-40S maturation.

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In vitro processing at the 3'-terminal region of pre-18S rRNA by a nucleolar endoribonuclease.

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.3868 ◽

1990 ◽

Vol 10 (8) ◽

pp. 3868-3872 ◽

Cited By ~ 15

Author(s):

C M Shumard ◽

C Torres ◽

D C Eichler

Keyword(s):

18S Rrna ◽

Precise Determination ◽

In Vivo Studies ◽

Rrna Sequence ◽

S1 Nuclease ◽

In Vitro Processing ◽

Rrna Maturation ◽

A Site

In an investigation of the possible involvement of a highly purified nucleolar endoribonuclease in processing of pre-rRNA at the 3' end of the 18S rRNA sequence, an in vitro synthesized pre-18S rRNA transcript containing the 3' end region of 18S rRNA and the 5' region of the first internal transcribed spacer (ITS1) was used as a substrate for the enzyme. Cleavages generated by the nucleolar RNase were localized by S1 nuclease protection analysis and by the direct release of labeled rRNA products. Precise determination of the specificity of cleavage was achieved by RNA sequence analysis with end-labeled rRNA transcripts. These data demonstrated that the purified nucleolar RNase cleaved the pre-18S rRNA transcript at three specific sites relative to the 3' region of 18S rRNA. The first two sites included the mature 3'-end 18S rRNA sequence and a site approximately 55 nucleotides downstream of the 3'-end 18S rRNA sequence, both of which corresponded directly to recent results (Raziuddin, R. D. Little, T. Labella, and D. Schlessinger, Mol. Cell. Biol. 9:1667-1671, 1989) obtained with transfected mouse rDNA in hamster cells. The other cleavage occurred approximately 35 nucleotides upstream from the mature 3' end in the 18S rRNA sequence. The results from this study mimic the results obtained from in vivo studies for processing in the 3' region of pre-18S rRNA, supporting the proposed involvement of this nucleolar endoribonuclease in rRNA maturation.

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

Molecular and Cellular Biology ◽

10.1128/mcb.23.6.2083-2095.2003 ◽

2003 ◽

Vol 23 (6) ◽

pp. 2083-2095 ◽

Cited By ~ 89

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.

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Poly(A)-specific ribonuclease is a nuclear ribosome biogenesis factor involved in human 18S rRNA maturation

Nucleic Acids Research ◽

10.1093/nar/gkx253 ◽

2017 ◽

Vol 45 (11) ◽

pp. 6822-6836 ◽

Cited By ~ 24

Author(s):

Christian Montellese ◽

Nathalie Montel-Lehry ◽

Anthony K. Henras ◽

Ulrike Kutay ◽

Pierre-Emmanuel Gleizes ◽

...

Keyword(s):

Ribosome Biogenesis ◽

18S Rrna ◽

Rrna Maturation

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Synthetic lethal interactions with conditional poly(A) polymerase alleles identify LCP5, a gene involved in 18S rRNA maturation

RNA ◽

10.1017/s1355838298980955 ◽

1998 ◽

Vol 4 (11) ◽

pp. 1357-1372 ◽

Cited By ~ 33

Author(s):

THOMAS WIEDERKEHR ◽

RENÉ F. PRÉTÔT ◽

LIONEL MINVIELLE-SEBASTIA

Keyword(s):

18S Rrna ◽

Synthetic Lethal ◽

Synthetic Lethal Interactions ◽

Rrna Maturation

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The Putative NTPase Fap7 Mediates Cytoplasmic 20S Pre-rRNA Processing through a Direct Interaction with Rps14

Molecular and Cellular Biology ◽

10.1128/mcb.25.23.10352-10364.2005 ◽

2005 ◽

Vol 25 (23) ◽

pp. 10352-10364 ◽

Cited By ~ 67

Author(s):

Sander Granneman ◽

Madhusudan R. Nandineni ◽

Susan J. Baserga

Keyword(s):

High Throughput ◽

Structural Component ◽

Ribosome Biogenesis ◽

18S Rrna ◽

Direct Interaction ◽

Nucleoside Triphosphate ◽

Rrna Processing ◽

Conserved Residues ◽

Rrna Maturation

ABSTRACT One of the proteins identified as being involved in ribosome biogenesis by high-throughput studies, a putative P-loop-type kinase termed Fap7 (YDL166c), was shown to be required for the conversion of 20S pre-rRNA to 18S rRNA. However, the mechanism underlying this function has remained unclear. Here we demonstrate that Fap7 is strictly required for cleavage of the 20S pre-rRNA at site D in the cytoplasm. Genetic depletion of Fap7 causes accumulation of only the 20S pre-rRNA, which could be detected not only in 43S preribosomes but also in 80S-sized complexes. Fap7 is not a structural component of 43S preribosomes but likely transiently interacts with them by directly binding to Rps14, a ribosomal protein that is found near the 3′ end of the 18S rRNA. Consistent with an NTPase activity, conserved residues predicted to be required for nucleoside triphosphate (NTP) hydrolysis are essential for Fap7 function in vivo. We propose that Fap7 mediates cleavage of the 20S pre-rRNA at site D by directly interacting with Rps14 and speculate that it is an enzyme that functions as an NTP-dependent molecular switch in 18S rRNA maturation.

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The PINc domain protein Utp24, a putative nuclease, is required for the early cleavage steps in 18S rRNA maturation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0603673103 ◽

2006 ◽

Vol 103 (25) ◽

pp. 9464-9469 ◽

Cited By ~ 59

Author(s):

F. Bleichert ◽

S. Granneman ◽

Y. N. Osheim ◽

A. L. Beyer ◽

S. J. Baserga

Keyword(s):

18S Rrna ◽

Early Cleavage ◽

Rrna Maturation ◽

Domain Protein

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In vitro processing at the 3'-terminal region of pre-18S rRNA by a nucleolar endoribonuclease

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.3868-3872.1990 ◽

1990 ◽

Vol 10 (8) ◽

pp. 3868-3872

Author(s):

C M Shumard ◽

C Torres ◽

D C Eichler

Keyword(s):

18S Rrna ◽

Precise Determination ◽

In Vivo Studies ◽

Rrna Sequence ◽

S1 Nuclease ◽

In Vitro Processing ◽

Rrna Maturation ◽

A Site

In an investigation of the possible involvement of a highly purified nucleolar endoribonuclease in processing of pre-rRNA at the 3' end of the 18S rRNA sequence, an in vitro synthesized pre-18S rRNA transcript containing the 3' end region of 18S rRNA and the 5' region of the first internal transcribed spacer (ITS1) was used as a substrate for the enzyme. Cleavages generated by the nucleolar RNase were localized by S1 nuclease protection analysis and by the direct release of labeled rRNA products. Precise determination of the specificity of cleavage was achieved by RNA sequence analysis with end-labeled rRNA transcripts. These data demonstrated that the purified nucleolar RNase cleaved the pre-18S rRNA transcript at three specific sites relative to the 3' region of 18S rRNA. The first two sites included the mature 3'-end 18S rRNA sequence and a site approximately 55 nucleotides downstream of the 3'-end 18S rRNA sequence, both of which corresponded directly to recent results (Raziuddin, R. D. Little, T. Labella, and D. Schlessinger, Mol. Cell. Biol. 9:1667-1671, 1989) obtained with transfected mouse rDNA in hamster cells. The other cleavage occurred approximately 35 nucleotides upstream from the mature 3' end in the 18S rRNA sequence. The results from this study mimic the results obtained from in vivo studies for processing in the 3' region of pre-18S rRNA, supporting the proposed involvement of this nucleolar endoribonuclease in rRNA maturation.

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