PRMT3 is a ribosomal protein methyltransferase that affects the cellular levels of 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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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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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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The eukaryote-specific N-terminal extension of ribosomal protein S31 contributes to the assembly and function of 40S ribosomal subunits

Nucleic Acids Research ◽

10.1093/nar/gkw641 ◽

2016 ◽

Vol 44 (16) ◽

pp. 7777-7791 ◽

Cited By ~ 8

Author(s):

Antonio Fernández-Pevida ◽

Sara Martín-Villanueva ◽

Guillaume Murat ◽

Thierry Lacombe ◽

Dieter Kressler ◽

...

Keyword(s):

Ribosomal Protein ◽

Ribosomal Subunits ◽

And Function

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A Drosophila ribosomal protein functions in mammalian cells.

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4524 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4524-4528 ◽

Cited By ~ 2

Author(s):

C G Maki ◽

D D Rhoads ◽

J J Diaz ◽

D J Roufa

Keyword(s):

Ribosomal Protein ◽

Chinese Hamster Ovary ◽

Cho Cells ◽

Mammalian Cells ◽

Expression Vector ◽

Resistance Mutation ◽

Chinese Hamster ◽

Ribosomal Subunits ◽

Cdna Expression ◽

Protein Functions

A cDNA expression vector encoding Drosophila ribosomal protein S14 was transfected into cultured Chinese hamster ovary (CHO) cells that harbor a recessive RPS14 emetine resistance mutation. Transformants synthesized the insect mRNA and polypeptide and consequently displayed an emetine-sensitive phenotype. These observations indicate that the insect protein was accurately expressed and correctly assembled into functional mammalian 40S ribosomal subunits.

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Effect of RP51 gene dosage alterations on ribosome synthesis in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.5.12.3429-3435.1985 ◽

1985 ◽

Vol 5 (12) ◽

pp. 3429-3435

Author(s):

N Abovich ◽

L Gritz ◽

L Tung ◽

M Rosbash

Keyword(s):

Saccharomyces Cerevisiae ◽

Growth Rate ◽

Ribosomal Protein ◽

Dosage Compensation ◽

Ribosomal Proteins ◽

Gene Dosage ◽

Translational Efficiency ◽

Phenotypic Analysis ◽

Ribosomal Subunits ◽

Single Deletion

The Saccharomyces cerevisiae ribosomal protein rp51 is encoded by two interchangeable genes, RP51A and RP51B. We altered the RP51 gene dose by creating deletions of the RP51A or RP51B genes or both. Deletions of both genes led to spore inviability, indicating that rp51 is an essential ribosomal protein. From single deletion studies in haploid cells, we concluded that there was no intergenic dosage compensation at the level of mRNA abundance or mRNA utilization (translational efficiency), although phenotypic analysis had previously indicated a small compensation effect on growth rate. Similarly, deletions in diploid strains indicated that no strong mechanisms exist for intragenic dosage compensation; in all cases, a decreased dose of RP51 genes was characterized by a slow growth phenotype. A decreased dose of RP51 genes also led to insufficient amounts of 40S ribosomal subunits, as evidenced by a dramatic accumulation of excess 60S ribosomal subunits. We conclude that inhibition of 40S synthesis had little or no effect on the synthesis of the 60S subunit components. Addition of extra copies of rp51 genes led to extra rp51 protein synthesis. The additional rp51 protein was rapidly degraded. We propose that rp51 and perhaps many ribosomal proteins are normally oversynthesized, but the unassembled excess is degraded, and that the apparent compensation seen in haploids, i.e., the fact that the growth rate of mutant strains is less depressed than the actual reduction in mRNA, is a consequence of this excess which is spared from proteolysis under this circumstance.

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A Drosophila ribosomal protein functions in mammalian cells

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4524-4528.1990 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4524-4528

Author(s):

C G Maki ◽

D D Rhoads ◽

J J Diaz ◽

D J Roufa

Keyword(s):

Ribosomal Protein ◽

Chinese Hamster Ovary ◽

Cho Cells ◽

Mammalian Cells ◽

Expression Vector ◽

Resistance Mutation ◽

Chinese Hamster ◽

Ribosomal Subunits ◽

Cdna Expression ◽

Protein Functions

A cDNA expression vector encoding Drosophila ribosomal protein S14 was transfected into cultured Chinese hamster ovary (CHO) cells that harbor a recessive RPS14 emetine resistance mutation. Transformants synthesized the insect mRNA and polypeptide and consequently displayed an emetine-sensitive phenotype. These observations indicate that the insect protein was accurately expressed and correctly assembled into functional mammalian 40S ribosomal subunits.

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Creation of a Ribosomal Protein Sa/LAMR1 Heterozygous Mouse.

Blood ◽

10.1182/blood.v106.11.3551.3551 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3551-3551

Author(s):

Steven R. Ellis ◽

Paula M. Logsdon ◽

Junying Han

Keyword(s):

Ribosomal Protein ◽

Ribosomal Proteins ◽

Small Subunit ◽

Fourth Generation ◽

Unknown Etiology ◽

Gene Encoding ◽

Mendelian Segregation ◽

Ribosomal Subunits ◽

Premature Deaths ◽

Ribosomal Protein S19

Abstract Diamond Blackfan anemia (DBA) is a severe hypoplastic anemia that generally presents early in infancy. Approximately 25% of DBA cases have been linked to mutations in the gene encoding ribosomal protein S19. The remaining cases are of unknown etiology. Our studies in yeast have identified a specific role for Rps19 in the maturation of 40S ribosomal subunits. While only one other small subunit ribosomal protein, Rps18, has a function virtually identical to Rps19 in subunit maturation, several others have functions closely related to Rps19. If the involvement of Rps19 in DBA is linked to its role in the synthesis of 40S ribosomal subunits, we would expect that one or more of these other ribosomal proteins may be responsible for DBA in patients with normal RPS19. To address a potential role for ribosomal proteins other than Rps19 in DBA we have created a transgenic mouse heterozygous at the LAMR1 locus. LAMR1 encodes ribosomal protein Sa, the mammalian homolog of the yeast ribosomal protein S0. Rps0, like Rps19, is required for the maturation of the 3′ end of 18S rRNA. We are currently in the fourth generation of out-crossing the original 129SvEv/C57BL6J chimeras to a C57BL6 background. The frequency of heterozygous pups is about that expected by Mendelian segregation suggesting that haploinsufficiency for ribosomal protein Sa does not lead to significant embryonic lethality. The heterozygous mice do, however, exhibit a higher frequency of craniofacial abnormalities and premature deaths relative to their wild-type littermates. The overall fitness of the heterozygous mice appears to be decreasing with each generation of outcrossing to the C57/BL6 background. Efforts are underway to understand the nature of the premature deaths and to obtain detailed hematological profiles on the LAMR1 heterozygous mice.

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