Intersection of the Kap123p-Mediated Nuclear Import and Ribosome Export Pathways

ABSTRACT Kap123p is a yeast β-karyopherin that imports ribosomal proteins into the nucleus prior to their assembly into preribosomal particles. Surprisingly, Kap123p is not essential for growth, under normal conditions. To further explore the role of Kap123p in nucleocytoplasmic transport and ribosome biogenesis, we performed a synthetic fitness screen designed to identify genes that interact with KAP123. Through this analysis we have identified three other karyopherins, Pse1p/Kap121p, Sxm1p/Kap108p, and Nmd5p/Kap119p. We propose that, in the absence of Kap123p, these karyopherins are able to supplant Kap123p's role in import. In addition to the karyopherins, we identified Rai1p, a protein previously implicated in rRNA processing. Rai1p is also not essential, but deletion of the RAI1 gene is deleterious to cell growth and causes defects in rRNA processing, which leads to an imbalance of the 60S/40S ratio and the accumulation of halfmers, 40S subunits assembled on polysomes that are unable to form functional ribosomes. Rai1p localizes predominantly to the nucleus, where it physically interacts with Rat1p and pre-60S ribosomal subunits. Analysis of the rai1/kap123 double mutant strain suggests that the observed genetic interaction results from an inability to efficiently export pre-60S subunits from the nucleus, which arises from a combination of compromised Kap123p-mediated nuclear import of the essential 60S ribosomal subunit export factor, Nmd3p, and a ΔRAI1-induced decrease in the overall biogenesis efficiency.

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NSR1 is required for pre-rRNA processing and for the proper maintenance of steady-state levels of ribosomal subunits

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3865-3871.1992 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3865-3871

Author(s):

W C Lee ◽

D Zabetakis ◽

T Mélèse

Keyword(s):

Nuclear Localization ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Protein Translation ◽

Striking Similarity ◽

Growth Defect ◽

Nuclear Localization Sequence ◽

Rrna Processing ◽

Ribosomal Subunits ◽

Localization Sequence

NSR1 is a yeast nuclear localization sequence-binding protein showing striking similarity in its domain structure to nucleolin. Cells lacking NSR1 are viable but have a severe growth defect. We show here that NSR1, like nucleolin, is involved in ribosome biogenesis. The nsr1 mutant is deficient in pre-rRNA processing such that the initial 35S pre-rRNA processing is blocked and 20S pre-rRNA is nearly absent. The reduced amount of 20S pre-rRNA leads to a shortage of 18S rRNA and is reflected in a change in the distribution of 60S and 40S ribosomal subunits; there is no free pool of 40S subunits, and the free pool of 60S subunits is greatly increased in size. The lack of free 40S subunits or the improper assembly of these subunits causes the nsr1 mutant to show sensitivity to the antibiotic paromomycin, which affects protein translation, at concentrations that do not affect the growth of the wild-type strain. Our data support the idea that NSR1 is involved in the proper assembly of pre-rRNA particles, possibly by bringing rRNA and ribosomal proteins together by virtue of its nuclear localization sequence-binding domain and multiple RNA recognition motifs. Alternatively, NSR1 may also act to regulate the nuclear entry of ribosomal proteins required for proper assembly of pre-rRNA particles.

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Good Vibrations: Structural Remodeling of Maturing Yeast Pre-40S Ribosomal Particles Followed by Cryo-Electron Microscopy

Molecules ◽

10.3390/molecules25051125 ◽

2020 ◽

Vol 25 (5) ◽

pp. 1125 ◽

Cited By ~ 3

Author(s):

Ramtin Shayan ◽

Dana Rinaldi ◽

Natacha Larburu ◽

Laura Plassart ◽

Stéphanie Balor ◽

...

Keyword(s):

Electron Microscopy ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Particle Analysis ◽

Ribosomal Subunits ◽

Early Maturation ◽

Cryo Electron Microscopy ◽

Molecular Events ◽

Final Maturation

Assembly of eukaryotic ribosomal subunits is a very complex and sequential process that starts in the nucleolus and finishes in the cytoplasm with the formation of functional ribosomes. Over the past few years, characterization of the many molecular events underlying eukaryotic ribosome biogenesis has been drastically improved by the “resolution revolution” of cryo-electron microscopy (cryo-EM). However, if very early maturation events have been well characterized for both yeast ribosomal subunits, little is known regarding the final maturation steps occurring to the small (40S) ribosomal subunit. To try to bridge this gap, we have used proteomics together with cryo-EM and single particle analysis to characterize yeast pre-40S particles containing the ribosome biogenesis factor Tsr1. Our analyses lead us to refine the timing of the early pre-40S particle maturation steps. Furthermore, we suggest that after an early and structurally stable stage, the beak and platform domains of pre-40S particles enter a “vibrating” or “wriggling” stage, that might be involved in the final maturation of 18S rRNA as well as the fitting of late ribosomal proteins into their mature position.

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Nop53p is a novel nucleolar 60S ribosomal subunit biogenesis protein

Biochemical Journal ◽

10.1042/bj20041297 ◽

2005 ◽

Vol 388 (3) ◽

pp. 819-826 ◽

Cited By ~ 21

Author(s):

Yaroslav SYDORSKYY ◽

David J. DILWORTH ◽

Brendan HALLORAN ◽

Eugene C. YI ◽

Taras MAKHNEVYCH ◽

...

Keyword(s):

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Rrna Genes ◽

Ribosomal Subunits ◽

Nuclear Compartment ◽

Growth Defects ◽

Polysome Profiling ◽

Rrna Maturation ◽

Polymerase I

Ribosome biogenesis in Saccharomyces cerevisiae occurs primarily in a specialized nuclear compartment termed the nucleolus within which the rRNA genes are transcribed by RNA polymerase I into a large 35 S rRNA precursor. The ensuing association/dissociation and catalytic activity of numerous trans-acting protein factors, RNAs and ribosomal proteins ultimately leads to the maturation of the precursor rRNAs into 25, 5.8 and 18 S rRNAs and the formation of mature cytoplasmic 40 and 60 S ribosomal subunits. Although many components involved in ribosome biogenesis have been identified, our understanding of this essential cellular process remains limited. In the present study we demonstrate a crucial role for the previously uncharacterized nucleolar protein Nop53p (Ypl146p) in ribosome biogenesis. Specifically, Nop53p appears to be most important for biogenesis of the 60 S subunit. It physically interacts with rRNA processing factors, notably Cbf5p and Nop2p, and co-fractionates specifically with pre-60 S particles on sucrose gradients. Deletion or mutations within NOP53 cause significant growth defects and display significant 60 S subunit deficiencies, an imbalance in the 40 S:60 S ratio, as revealed by polysome profiling, and defects in progression beyond the 27 S stage of 25 S rRNA maturation during 60 S biogenesis.

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NSR1 is required for pre-rRNA processing and for the proper maintenance of steady-state levels of ribosomal subunits.

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3865 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3865-3871 ◽

Cited By ~ 76

Author(s):

W C Lee ◽

D Zabetakis ◽

T Mélèse

Keyword(s):

Nuclear Localization ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Protein Translation ◽

Striking Similarity ◽

Growth Defect ◽

Nuclear Localization Sequence ◽

Rrna Processing ◽

Ribosomal Subunits ◽

Localization Sequence

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The nucleolar DExD/H protein Hel66 is involved in ribosome biogenesis in Trypanosoma brucei

10.1101/2021.06.11.448066 ◽

2021 ◽

Author(s):

Majeed Bakari-Soale ◽

Nonso Josephat Ikenge ◽

Marion Scheibe ◽

Falk Butter ◽

Nicola Gail Jones ◽

...

Keyword(s):

Ribosomal Rna ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Growth Defect ◽

Rrna Processing ◽

Ribosomal Subunits ◽

Comprehensive Picture ◽

Severe Growth Defect ◽

H Protein ◽

Severe Growth

The biosynthesis of ribosomes is a complex cellular process involving ribosomal RNA, ribosomal proteins and several further trans-acting factors. DExD/H box proteins constitute the largest family of trans-acting protein factors involved in this process. Several members of this protein family have been directly implicated in ribosome biogenesis in yeast. In trypanosomes, ribosome biogenesis differs in several features from the process described in yeast. Here, we have identified the DExD/H box helicase Hel66 as being involved in ribosome biogenesis. The protein is unique to Kinetoplastida, localises to the nucleolus and its depletion via RNAi caused a severe growth defect. Loss of the protein resulted in a decrease of global translation and accumulation of rRNA processing intermediates for both the small and large ribosomal subunits. Only a few factors involved in trypanosome rRNA biogenesis have been described so far and our findings contribute to gaining a more comprehensive picture of this essential process.

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Impaired ribosome biogenesis in Diamond-Blackfan anemia

Blood ◽

10.1182/blood-2006-07-038372 ◽

2006 ◽

Vol 109 (3) ◽

pp. 1275-1283 ◽

Cited By ~ 148

Author(s):

Valérie Choesmel ◽

Daniel Bacqueville ◽

Jacques Rouquette ◽

Jacqueline Noaillac-Depeyre ◽

Sébastien Fribourg ◽

...

Keyword(s):

Ribosome Biogenesis ◽

18S Rrna ◽

Ribosomal Subunit ◽

Rrna Synthesis ◽

Rrna Processing ◽

Gene Encoding ◽

Ribosomal Subunits ◽

Diamond Blackfan Anemia ◽

Nucleolar Organization ◽

Ribosomal Protein S19

Abstract The gene encoding the ribosomal protein S19 (RPS19) is frequently mutated in Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia. The consequence of these mutations on the onset of the disease remains obscure. Here, we show that RPS19 plays an essential role in biogenesis of the 40S small ribosomal subunit in human cells. Knockdown of RPS19 expression by siRNAs impairs 18S rRNA synthesis and formation of 40S subunits and induces apoptosis in HeLa cells. Pre-rRNA processing is altered, which leads to an arrest in the maturation of precursors to the 18S rRNA. Under these conditions, pre-40S particles are not exported to the cytoplasm and accumulate in the nucleoplasm of the cells in perinuclear dots. Consistently, we find that ribosome biogenesis and nucleolar organization is altered in skin fibroblasts from DBA patients bearing mutations in the RPS19 gene. In addition, maturation of the 18S rRNA is also perturbed in cells from a patient bearing no RPS19-related mutation. These results support the hypothesis that DBA is directly related to a defect in ribosome biogenesis and indicate that yet to be discovered DBA-related genes may be involved in the synthesis of the ribosomal subunits.

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Prp43p Is a DEAH-Box Spliceosome Disassembly Factor Essential for Ribosome Biogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.26.2.523-534.2006 ◽

2006 ◽

Vol 26 (2) ◽

pp. 523-534 ◽

Cited By ~ 75

Author(s):

D. Joshua Combs ◽

Roland J. Nagel ◽

Manuel Ares ◽

Scott W. Stevens

Keyword(s):

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Mrna Splicing ◽

Splicing Factors ◽

Rrna Processing ◽

Ribosomal Subunits ◽

Splicing Defects ◽

Essential Function ◽

Polymerase I ◽

Kinetics Of

ABSTRACT The known function of the DEXH/D-box protein Prp43p is the removal of the U2, U5, and U6 snRNPs from the postsplicing lariat-intron ribonucleoprotein complex. We demonstrate that affinity-purified Prp43p-associated material includes the expected spliceosomal components; however, we also identify several preribosomal complexes that are specifically purified with Prp43p. Conditional prp43 mutant alleles confer a 35S pre-rRNA processing defect, with subsequent depletion of 27S and 20S precursors. Upon a shift to a nonpermissive temperature, both large and small-ribosomal-subunit proteins accumulate in the nucleolus of prp43 mutants. Pulse-chase analysis demonstrates delayed kinetics of 35S, 27S, and 20S pre-rRNA processing with turnover of these intermediates. Microarray analysis of pre-mRNA splicing defects in prp43 mutants shows a very mild effect, similar to that of nonessential pre-mRNA splicing factors. Prp43p is the first DEXH/D-box protein shown to function in both RNA polymerase I and polymerase II transcript metabolism. Its essential function is in its newly characterized role in ribosome biogenesis of both ribosomal subunits, positioning Prp43p to regulate both pre-mRNA splicing and ribosome biogenesis.

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The Nucleolar DExD/H Protein Hel66 is Involved in Ribosome Biogenesis in Trypanosoma Brucei

10.21203/rs.3.rs-612898/v1 ◽

2021 ◽

Author(s):

Majeed Bakari-Soale ◽

Nonso Josephat Ikenga ◽

Marion Scheibe ◽

Falk Butter ◽

Nicola Gail Jones ◽

...

Keyword(s):

Ribosomal Rna ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Growth Defect ◽

Rrna Processing ◽

Ribosomal Subunits ◽

Comprehensive Picture ◽

Severe Growth Defect ◽

H Protein ◽

Severe Growth

Abstract The biosynthesis of ribosomes is a complex cellular process involving ribosomal RNA, ribosomal proteins and several further trans-acting factors. DExD/H box proteins constitute the largest family of trans-acting protein factors involved in this process. Several members of this protein family have been directly implicated in ribosome biogenesis in yeast. In trypanosomes, ribosome biogenesis differs in several features from the process described in yeast. Here, we have identified the DExD/H box helicase Hel66 as being involved in ribosome biogenesis. The protein is unique to Kinetoplastida, localises to the nucleolus and its depletion via RNAi caused a severe growth defect. Loss of the protein resulted in a decrease of global translation and accumulation of rRNA processing intermediates for both the small and large ribosomal subunits. Only a few factors involved in trypanosome rRNA biogenesis have been described so far and our findings contribute to gaining a more comprehensive picture of this essential process.

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The nucleolar DExD/H protein Hel66 is involved in ribosome biogenesis in Trypanosoma brucei

Scientific Reports ◽

10.1038/s41598-021-97020-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Majeed Bakari-Soale ◽

Nonso Josephat Ikenga ◽

Marion Scheibe ◽

Falk Butter ◽

Nicola G. Jones ◽

...

Keyword(s):

Ribosomal Rna ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Growth Defect ◽

Rrna Processing ◽

Ribosomal Subunits ◽

Comprehensive Picture ◽

Severe Growth Defect ◽

H Protein ◽

Severe Growth

AbstractThe biosynthesis of ribosomes is a complex cellular process involving ribosomal RNA, ribosomal proteins and several further trans-acting factors. DExD/H box proteins constitute the largest family of trans-acting protein factors involved in this process. Several members of this protein family have been directly implicated in ribosome biogenesis in yeast. In trypanosomes, ribosome biogenesis differs in several features from the process described in yeast. Here, we have identified the DExD/H box helicase Hel66 as being involved in ribosome biogenesis. The protein is unique to Kinetoplastida, localises to the nucleolus and its depletion via RNAi caused a severe growth defect. Loss of the protein resulted in a decrease of global translation and accumulation of rRNA processing intermediates for both the small and large ribosomal subunits. Only a few factors involved in trypanosome rRNA biogenesis have been described so far and our findings contribute to gaining a more comprehensive picture of this essential process.

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The in vivo functions of ARPF2 and ARRS1 in ribosomal RNA processing and ribosome biogenesis in Arabidopsis

Journal of Experimental Botany ◽

10.1093/jxb/eraa019 ◽

2020 ◽

Vol 71 (9) ◽

pp. 2596-2611

Author(s):

Ilyeong Choi ◽

Young Jeon ◽

Youngki Yoo ◽

Hyun-Soo Cho ◽

Hyun-Sook Pai

Keyword(s):

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Critical Role ◽

Ribosomal Subunit ◽

Ribosome Profiling ◽

Production Factor ◽

Rrna Synthesis ◽

Binary Complex ◽

Rrna Processing ◽

Loss Of Function

Abstract Yeast Rpf2 plays a critical role in the incorporation of 5S rRNA into pre-ribosomes by forming a binary complex with Rrs1. The protein characteristics and overexpression phenotypes of Arabidopsis Ribosome Production Factor 2 (ARPF2) and Arabidopsis Regulator of Ribosome Synthesis 1 (ARRS1) have been previously studied. Here, we analyze loss-of-function phenotypes of ARPF2 and ARRS1 using virus-induced gene silencing to determine their functions in pre-rRNA processing and ribosome biogenesis. ARPF2 silencing in Arabidopsis led to pleiotropic developmental defects. RNA gel blot analysis and circular reverse transcription–PCR revealed that ARPF2 depletion delayed pre-rRNA processing, resulting in the accumulation of multiple processing intermediates. ARPF2 fractionated primarily with the 60S ribosomal subunit. Metabolic rRNA labeling and ribosome profiling suggested that ARPF2 deficiency mainly affected 25S rRNA synthesis and 60S ribosome biogenesis. ARPF2 and ARRS1 formed the complex that interacted with the 60S ribosomal proteins RPL5 and RPL11. ARRS1 silencing resulted in growth defects, accumulation of processing intermediates, and ribosome profiling similar to those of ARPF2-silenced plants. Moreover, depletion of ARPF2 and ARRS1 caused nucleolar stress. ARPF2-deficient plants excessively accumulated anthocyanin and reactive oxygen species. Collectively, these results suggest that the ARPF2–ARRS1 complex plays a crucial role in plant growth and development by modulating ribosome biogenesis.

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