The eukaryote-specific N-terminal extension of ribosomal protein S31 contributes to the assembly and function of 40S ribosomal subunits

Ribosomal protein L1 from Saccharomyces cerevisiae binds 5S rRNA and can be released from intact 60S ribosomal subunits as an L1-5S ribonucleoprotein (RNP) particle. To understand the nature of the interaction between L1 and 5S rRNA and to assess the role of L1 in ribosome assembly and function, we cloned the RPL1 gene encoding L1. We have shown that RPL1 is an essential single-copy gene. A conditional null mutant in which the only copy of RPL1 is under control of the repressible GAL1 promoter was constructed. Depletion of L1 causes instability of newly synthesized 5S rRNA in vivo. Cells depleted of L1 no longer assemble 60S ribosomal subunits, indicating that L1 is required for assembly of stable 60S ribosomal subunits but not 40S ribosomal subunits. An L1-5S RNP particle not associated with ribosomal particles was detected by coimmunoprecipitation of L1 and 5S rRNA. This pool of L1-5S RNP remained stable even upon cessation of 60S ribosomal subunit assembly by depletion of another ribosomal protein, L16. Preliminary results suggest that transcription of RPL1 is not autogenously regulated by L1.

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Yeast ribosomal protein L1 is required for the stability of newly synthesized 5S rRNA and the assembly of 60S ribosomal subunits.

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.2835 ◽

1993 ◽

Vol 13 (5) ◽

pp. 2835-2845 ◽

Cited By ~ 83

Author(s):

M Deshmukh ◽

Y F Tsay ◽

A G Paulovich ◽

J L Woolford

Keyword(s):

Ribosomal Protein ◽

Ribosomal Subunit ◽

Single Copy ◽

5S Rrna ◽

Gene Encoding ◽

Ribosomal Subunits ◽

Ribosomal Protein L1 ◽

The Stability ◽

And Function

Ribosomal protein L1 from Saccharomyces cerevisiae binds 5S rRNA and can be released from intact 60S ribosomal subunits as an L1-5S ribonucleoprotein (RNP) particle. To understand the nature of the interaction between L1 and 5S rRNA and to assess the role of L1 in ribosome assembly and function, we cloned the RPL1 gene encoding L1. We have shown that RPL1 is an essential single-copy gene. A conditional null mutant in which the only copy of RPL1 is under control of the repressible GAL1 promoter was constructed. Depletion of L1 causes instability of newly synthesized 5S rRNA in vivo. Cells depleted of L1 no longer assemble 60S ribosomal subunits, indicating that L1 is required for assembly of stable 60S ribosomal subunits but not 40S ribosomal subunits. An L1-5S RNP particle not associated with ribosomal particles was detected by coimmunoprecipitation of L1 and 5S rRNA. This pool of L1-5S RNP remained stable even upon cessation of 60S ribosomal subunit assembly by depletion of another ribosomal protein, L16. Preliminary results suggest that transcription of RPL1 is not autogenously regulated by L1.

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Structural Role of rRNAs on the Ribosomal Subunits from E. Coli by Scanning Transmission Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100098629 ◽

1981 ◽

Vol 39 ◽

pp. 424-425

Author(s):

M. Boublik ◽

N. Robakis ◽

J.S. Wall

Keyword(s):

Three Dimensional ◽

Uranyl Acetate ◽

Dimensional Structure ◽

Electron Microscopic ◽

Ribosomal Subunits ◽

E Coli ◽

Freeze Dried ◽

16S Rna ◽

Scanning Transmission ◽

And Function

The three-dimensional structure and function of biological supramolecular complexes are, in general, determined and stabilized by conformation and interactions of their macromolecular components. In the case of ribosomes, it has been suggested that one of the functions of ribosomal RNAs is to act as a scaffold maintaining the shape of the ribosomal subunits. In order to investigate this question, we have conducted a comparative TEM and STEM study of the structure of the small 30S subunit of E. coli and its 16S RNA.The conventional electron microscopic imaging of nucleic acids is performed by spreading them in the presence of protein or detergent; the particles are contrasted by electron dense solution (uranyl acetate) or by shadowing with metal (tungsten). By using the STEM on freeze-dried specimens we have avoided the shearing forces of the spreading, and minimized both the collapse of rRNA due to air drying and the loss of resolution due to staining or shadowing. Figure 1, is a conventional (TEM) electron micrograph of 30S E. coli subunits contrasted with uranyl acetate.

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Faculty Opinions recommendation of Introns within ribosomal protein genes regulate the production and function of yeast ribosomes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.13409007.14779129 ◽

2011 ◽

Author(s):

Simon Hubbard

Keyword(s):

Ribosomal Protein ◽

Ribosomal Protein Genes ◽

Yeast Ribosomes ◽

And Function

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Ribosomal protein S14 is altered by two-step emetine resistance mutations in Chinese hamster cells

Molecular and Cellular Biology ◽

10.1128/mcb.3.2.190-197.1983 ◽

1983 ◽

Vol 3 (2) ◽

pp. 190-197

Author(s):

J J Madjar ◽

M Frahm ◽

S McGill ◽

D J Roufa

Keyword(s):

Ribosomal Protein ◽

Ribosomal Proteins ◽

Ribosomal Subunit ◽

Chinese Hamster ◽

Complementation Group ◽

Resistance Mutations ◽

Cho Cell ◽

Ribosomal Subunits ◽

40S Ribosomal Subunit ◽

One Step

Four two-dimensional polyacrylamide gel electrophoresis systems were used to identify 78 Chinese hamster cell ribosomal proteins by the uniform nomenclature based on rat liver ribosomal proteins. The 40S ribosomal subunit protein affected by Chinese hamster ovary (CHO) cell one-step emetine resistance mutations is designated S14 in the standard nomenclature. To seek unambiguous genetic evidence for a cause and effect relationship between CHO cell emetine resistance and mutations in the S14 gene, we mutagenized a one-step CHO cell mutant and isolated second-step mutant clones resistant to 10-fold-higher concentrations of emetine. All of the highly resistant, two-step CHO cell mutants obtained displayed additional alterations in ribosomal protein S14. Hybridization complementation tests revealed that the two-step CHO cell emetine resistance mutants were members of the same complementation group defined by one-step CHO cell mutants, EmtB. Two-step mutants obtained from a Chinese hamster lung cell emetine-resistant clone belong to the EmtA complementation group. The two-step and EmtB mutants elaborated 40S ribosomal subunits, which dissociated to 32S and 40S core particles in buffers containing 0.5 M KCl at 4 degrees C. In contrast, 40S ribosomal subunits purified from all EmtA, one-step EmtB EmtC mutants, and wild-type CHO and lung cells were stable at this temperature in buffers containing substantially higher concentrations of salt. Thus, two-step emtB mutations affect the structure of S14 protein directly and the stability of the 40S ribosomal subunit indirectly.

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Molecular genetics of cryptopleurine resistance in Saccharomyces cerevisiae: expression of a ribosomal protein gene family.

Genetics ◽

10.1093/genetics/135.3.719 ◽

1993 ◽

Vol 135 (3) ◽

pp. 719-730

Author(s):

A G Paulovich ◽

J R Thompson ◽

J C Larkin ◽

Z Li ◽

J L Woolford

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Protein ◽

Gene Fusion ◽

Null Allele ◽

Ribosomal Subunit ◽

Null Alleles ◽

Protein Synthesis Inhibitor ◽

Lacz Gene ◽

Ribosomal Subunits ◽

Cry1 Gene

Abstract The Saccharomyces cerevisiae CRY1 gene encodes the 40S ribosomal subunit protein rp59 and confers sensitivity to the protein synthesis inhibitor cryptopleurine. A yeast strain containing the cry1-delta 1::URA3 null allele is viable, cryptopleurine sensitive (CryS), and expresses rp59 mRNA, suggesting that there is a second functional CRY gene. The CRY2 gene has been isolated from a yeast genomic library cloned in bacteriophage lambda, using a CRY1 DNA probe. The DNA sequence of the CRY2 gene contains an open reading frame encoding ribosomal protein 59 that differs at five residues from rp59 encoded by the CRY1 gene. The CRY2 gene was mapped to the left arm of chromosome X, centromere-proximal to cdc6 and immediately adjacent to ribosomal protein genes RPS24A and RPL46. Ribosomal protein 59 is an essential protein; upon sporulation of a diploid doubly heterozygous for cry1-delta 2::TRP1 cry2-delta 1::LEU2 null alleles, no spore clones containing both null alleles were recovered. Several results indicate that CRY2 is expressed, but at lower levels than CRY1: (1) Introduction of CRY2 on high copy plasmids into CryR yeast of genotype cry1 CRY2 confers a CryS phenotype. Transformation of these CryR yeast with CRY2 on a low copy CEN plasmid does not confer a CryS phenotype. (2) Haploids containing the cry1-delta 2::TRP1 null allele have a deficit of 40S ribosomal subunits, but cry2-delta 1::LEU2 strains have wild-type amounts of 40S ribosomal subunits. (3) CRY2 mRNA is present at lower levels than CRY1 mRNA. (4) Higher levels of beta-galactosidase are expressed from a CRY1-lacZ gene fusion than from a CRY2-lacZ gene fusion. Mutations that alter or eliminate the last amino acid of rp59 encoded by either CRY1 or CRY2 result in resistance to cryptopleurine. Because CRY2 (and cry2) is expressed at lower levels than CRY1 (and cry1), the CryR phenotype of cry2 mutants is only expressed in strains containing a cry1-delta null allele.

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Translation initiation in cancer at a glance

Journal of Cell Science ◽

10.1242/jcs.248476 ◽

2021 ◽

Vol 134 (1) ◽

pp. jcs248476

Author(s):

Rachael C. L. Smith ◽

Georgios Kanellos ◽

Nikola Vlahov ◽

Constantinos Alexandrou ◽

Anne E. Willis ◽

...

Keyword(s):

Translation Initiation ◽

Ribosomal Subunits ◽

Eukaryotic Translation ◽

Initiation Factors ◽

Eukaryotic Initiation Factors ◽

Tumour Formation ◽

Multistep Process ◽

Proliferation And Metastasis ◽

Regulated Gene Expression ◽

And Function

ABSTRACTCell division, differentiation and function are largely dependent on accurate proteome composition and regulated gene expression. To control this, protein synthesis is an intricate process governed by upstream signalling pathways. Eukaryotic translation is a multistep process and can be separated into four distinct phases: initiation, elongation, termination and recycling of ribosomal subunits. Translation initiation, the focus of this article, is highly regulated to control the activity and/or function of eukaryotic initiation factors (eIFs) and permit recruitment of mRNAs to the ribosomes. In this Cell Science at a Glance and accompanying poster, we outline the mechanisms by which tumour cells alter the process of translation initiation and discuss how this benefits tumour formation, proliferation and metastasis.

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Ribosomal protein L30 is dispensable in the yeast Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5235 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5235-5243 ◽

Cited By ~ 44

Author(s):

D M Baronas-Lowell ◽

J R Warner

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Protein ◽

Ribosomal Proteins ◽

Wild Type ◽

Diploid Strain ◽

Chain Reaction ◽

Yeast Saccharomyces Cerevisiae ◽

Ribosomal Subunits ◽

Strong Homology ◽

Polymerase Chain

In the yeast Saccharomyces cerevisiae, L30 is one of many ribosomal proteins that is encoded by two functional genes. We have cloned and sequenced RPL30B, which shows strong homology to RPL30A. Use of mRNA as a template for a polymerase chain reaction demonstrated that RPL30B contains an intron in its 5' untranslated region. This intron has an unusual 5' splice site, C/GUAUGU. The genomic copies of RPL30A and RPL30B were disrupted by homologous recombination. Growth rates, primer extension, and two-dimensional ribosomal protein analyses of these disruption mutants suggested that RPL30A is responsible for the majority of L30 production. Surprisingly, meiosis of a diploid strain carrying one disrupted RPL30A and one disrupted RPL30B yielded four viable spores. Ribosomes from haploid cells carrying both disrupted genes had no detectable L30, yet such cells grew with a doubling time only 30% longer than that of wild-type cells. Furthermore, depletion of L30 did not alter the ratio of 60S to 40S ribosomal subunits, suggesting that there is no serious effect on the assembly of 60S subunits. Polysome profiles, however, suggest that the absence of L30 leads to the formation of stalled translation initiation complexes.

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Ribosomal protein L30 is dispensable in the yeast Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5235-5243.1990 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5235-5243

Author(s):

D M Baronas-Lowell ◽

J R Warner

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Protein ◽

Ribosomal Proteins ◽

Wild Type ◽

Diploid Strain ◽

Chain Reaction ◽

Yeast Saccharomyces Cerevisiae ◽

Ribosomal Subunits ◽

Strong Homology ◽

Polymerase Chain

In the yeast Saccharomyces cerevisiae, L30 is one of many ribosomal proteins that is encoded by two functional genes. We have cloned and sequenced RPL30B, which shows strong homology to RPL30A. Use of mRNA as a template for a polymerase chain reaction demonstrated that RPL30B contains an intron in its 5' untranslated region. This intron has an unusual 5' splice site, C/GUAUGU. The genomic copies of RPL30A and RPL30B were disrupted by homologous recombination. Growth rates, primer extension, and two-dimensional ribosomal protein analyses of these disruption mutants suggested that RPL30A is responsible for the majority of L30 production. Surprisingly, meiosis of a diploid strain carrying one disrupted RPL30A and one disrupted RPL30B yielded four viable spores. Ribosomes from haploid cells carrying both disrupted genes had no detectable L30, yet such cells grew with a doubling time only 30% longer than that of wild-type cells. Furthermore, depletion of L30 did not alter the ratio of 60S to 40S ribosomal subunits, suggesting that there is no serious effect on the assembly of 60S subunits. Polysome profiles, however, suggest that the absence of L30 leads to the formation of stalled translation initiation complexes.

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60S acidic ribosomal protein P1 (RPLP1) is elevated in human endometriotic tissue and in a murine model of endometriosis and is essential for endometriotic epithelial cell survival in vitro

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaz065 ◽

2020 ◽

Vol 26 (1) ◽

pp. 53-64

Author(s):

Zahraa Alali ◽

Amanda Graham ◽

Kimberly Swan ◽

Rebecca Flyckt ◽

Tommaso Falcone ◽

...

Keyword(s):

Cell Proliferation ◽

Ribosomal Protein ◽

Cell Survival ◽

Murine Model ◽

Eutopic Endometrium ◽

Ectopic Tissue ◽

And Function ◽

Matched Samples ◽

Proliferation And Invasion

Abstract Endometriosis is a female disease which is defined as the presence of ectopic endometrial tissue and is dependent on estrogen for its survival in these ectopic locations. Expression of the ribosomal protein large P1 (RPLP1) is associated with cell proliferation and invasion in several pathologies, but a role in the pathophysiology of endometriosis has not been explored. In this study, we aimed to evaluate the expression and function of RPLP1 with respect to endometriosis pathophysiology. RPLP1 protein was localised by immunohistochemistry (IHC) in eutopic and ectopic tissue from 28 subjects with confirmed endometriosis and from 20 women without signs or symptoms of the disease, while transcript levels were evaluated by qRT-PCR in 77 endometriotic lesions and 55 matched eutopic endometrial biopsies, and protein expression was evaluated using western blotting in 20 of these matched samples. To evaluate the mechanism for enhanced lesion expression of RPLP1, an experimental murine model of endometriosis was used and RPLP1 expression was localized using IHC. In vitro studies using an endometriosis cell line coupled with shRNA knockdown was used to demonstrate its role in cell survival. Expression of RPLP1 mRNA and protein were significantly higher in ectopic lesion tissue compared to paired eutopic endometrium and immunohistochemical localisation revealed predominant localisation to epithelial cells. This pattern of lesion RPLP1 was recapitulated in mice with experimentally induced endometriosis. Stable knockdown of RPLP1 protein resulted in a significant decrease in cell survival in vitro. These studies reveal that RPLP1 is associated with cell proliferation and/or survival and may play a role in the pathophysiology of endometriosis.

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