Cells depleted for RPS19, a protein associated with Diamond Blackfan Anemia, show defects in 18S ribosomal RNA synthesis and small ribosomal subunit production

RNA synthesis can be maintained in large oocytes of Xenopus laevis during periods of in vitro culture of at least 10 days. A simple salt medium, modified Barth's solution, is found to be as effective a culture medium for these oocytes as several other complex media. The newly synthesized RNA is characterized electrophoretically and shown to consist predominantly of ribosomal RNA precursor, 28S and 18S ribosomal RNA, and 4S RNA. The distribution of this RNA within the oocyte is detected autoradiographically, where it is found to be greatly concentrated over the nucleoli. No qualitative alterations in either of these parameters are found during culture, within the limits of sensitivity of the assay procedures.

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The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase activity in 40S ribosome assembly

10.1101/2021.09.28.462141 ◽

2021 ◽

Author(s):

Haina Huang ◽

Melissa Parker ◽

Katrin Karbstein

Keyword(s):

Quality Control ◽

Binding Site ◽

Ribosomal Rna ◽

Ribosomal Proteins ◽

Kinase Activity ◽

Ribosomal Subunit ◽

Ribosome Assembly ◽

Small Ribosomal Subunit ◽

Yeast Strains

AbstractRibosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AF) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate “handover” from one highly related AF to another remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the activities of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1. Inactive Tsr3 blocks Rio1, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

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The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase activity in 40S ribosome assembly

RNA ◽

10.1261/rna.078994.121 ◽

2022 ◽

pp. rna.078994.121

Author(s):

Haina Huang ◽

Melissa D Parker ◽

Katrin Karbstein

Keyword(s):

Quality Control ◽

Binding Site ◽

Ribosomal Rna ◽

Ribosomal Proteins ◽

Kinase Activity ◽

Ribosomal Subunit ◽

Ribosome Assembly ◽

Small Ribosomal Subunit ◽

Yeast Strains

Ribosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AF) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate “handover” from one highly related AF to another remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the activities of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1. Inactive Tsr3 blocks Rio1, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

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Mobility restriction in vivo of the heavy ribosomal subunit in a secretory cell.

The Journal of Cell Biology ◽

10.1083/jcb.70.3.573 ◽

1976 ◽

Vol 70 (3) ◽

pp. 573-580 ◽

Cited By ~ 14

Author(s):

U Lönn ◽

J E Edström

Keyword(s):

Endoplasmic Reticulum ◽

Ribosomal Rna ◽

Rna Synthesis ◽

Ribosomal Subunit ◽

Chironomus Tentans ◽

Salivary Gland Cells ◽

Unique Parameter ◽

Labeled Rna

Analysis in insect (Chironomus tentans) salivary gland cells of labeled RNA as a function of time after precursor injection and its distance to the nuclear membrane, cytoplasmic zone analysis, could previously be used to demonstrate the presence of short-lasting gradients in newly labeled ribosomal RNA. Since the gradients were sensitive to puromycin, they are likely to be a result of diffusion restriction due to an engagement of the subunits into polysomes. In the present paper the possibility was explored of recording gradients that were caused by labeled subunits in puromycin-resistant associations, which, in all probability, involve the endoplasmic reticulum. It was found that labeled 28 S and 5 S RNA but not 18 S RNA were present in radioactivity gradients lasting for at least 2 days but less than 6 days. The gradients also remained during inhibition of RNA synthesis by actinomycin, and they were completely resistant to puromycin whether given in vivo or in vitro. The semipermanent gradients formed fhere offer a unique parameter for the in vivo study of conditions for formation and maintenance of heavy subunits in puromycin-resistant bonds. An explanation for these and previous results is that the light subunit, although restricted in movement by engagement to polysomes, is nevertheless free to exchange and spread between rounds of translation, whereas at least part of the heavy subunit population is bound to the endoplasmic reticulum and less free to spread. These results offer a good in vivo correlate to previous results on in vitro exchangeability of subunits.

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Conformational switches control early maturation of the eukaryotic small ribosomal subunit

eLife ◽

10.7554/elife.45185 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 11

Author(s):

Mirjam Hunziker ◽

Jonas Barandun ◽

Olga Buzovetsky ◽

Caitlin Steckler ◽

Henrik Molina ◽

...

Keyword(s):

Ribosomal Rna ◽

Conformational Changes ◽

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Small Subunit ◽

Ribosome Assembly ◽

Small Ribosomal Subunit ◽

Early Maturation ◽

External Transcribed Spacer ◽

Assembly Factors

Eukaryotic ribosome biogenesis is initiated with the transcription of pre-ribosomal RNA at the 5’ external transcribed spacer, which directs the early association of assembly factors but is absent from the mature ribosome. The subsequent co-transcriptional association of ribosome assembly factors with pre-ribosomal RNA results in the formation of the small subunit processome. Here we show that stable rRNA domains of the small ribosomal subunit can independently recruit their own biogenesis factors in vivo. The final assembly and compaction of the small subunit processome requires the presence of the 5’ external transcribed spacer RNA and all ribosomal RNA domains. Additionally, our cryo-electron microscopy structure of the earliest nucleolar pre-ribosomal assembly - the 5’ external transcribed spacer ribonucleoprotein – provides a mechanism for how conformational changes in multi-protein complexes can be employed to regulate the accessibility of binding sites and therefore define the chronology of maturation events during early stages of ribosome assembly.

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