Heat-sensitive mutant strain of Neurospora crassa, 4M(t), conditionally defective in 25S ribosomal ribonucleic acid production

1981 ◽  
Vol 1 (3) ◽  
pp. 199-207
Author(s):  
M W Loo ◽  
N S Schricker ◽  
P J Russell
Keyword(s):  
Neurospora Crassa ◽  
Mutant Strain ◽  
Ribosomal Rna ◽  
Ribonucleic Acid ◽  
Ribosomal Subunit ◽  
Macromolecular Synthesis ◽  
Sensitive Mutant ◽  
Ribosomal Subunits ◽  
Sequence Of Events ◽  
Ribosomal Rna Processing

A heat-sensitive mutant strain of Neurospora crassa, 4M(t), was studied in an attempt to define its molecular lesion. The mutant strain is inhibited in conidial germination and mycelial extension at the nonpermissive temperature (37 degrees C). Macromolecular synthesis studies showed that both ribonucleic acid (RNA) and protein syntheses are inhibited when 4-h cultures are shifted from 20 to 37 degrees C. Density gradient analysis of ribosomal subunits made at 37 degrees C indicated that strain 4M(t) is deficient in the accumulation of 60S ribosomal subunits in that the ratio of 60S/37S subunits was 0.29:1 compared with 1.6:1 for the parental strain. This phenotype was shown to be the result of a slow rate of processing of, and a deficiency in the amount of, the immediate precursor to 25S ribosomal RNA (the large RNA of the 60S subunit) in the sequence of events constituting the production of mature ribosomal RNAs from the primary transcript of the ribosomal deoxyribonucleic acid, the precursor ribosomal RNA molecule. Analysis of polysomes suggested that the heat-sensitive gene product might function in both the assembly and the function of the 60S ribosomal subunit, since there was a smaller proportion of newly made 60S subunits synthesized at 37 degrees C in the polysome region of the gradients than in the monosome-plus-subunit region. The ribosomal RNA processing defect is apparently responsible for the observed defects in germination and macromolecular synthesis at 37 degrees C, but the precise molecular lesion is not known. On the basis of these results, the heat-sensitive mutant allele in the 4M(t) strain is considered to define the rip1 (ribosome production) gene locus.

scholarly journals Heat-sensitive mutant strain of Neurospora crassa, 4M(t), conditionally defective in 25S ribosomal ribonucleic acid production.

1981 ◽  
Vol 1 (3) ◽  
pp. 199-207 ◽  
Author(s):  
M W Loo ◽  
N S Schricker ◽  
P J Russell
Keyword(s):  
Neurospora Crassa ◽  
Mutant Strain ◽  
Ribosomal Rna ◽  
Ribonucleic Acid ◽  
Ribosomal Subunit ◽  
Macromolecular Synthesis ◽  
Sensitive Mutant ◽  
Ribosomal Subunits ◽  
Sequence Of Events ◽  
Ribosomal Rna Processing

A heat-sensitive mutant strain of Neurospora crassa, 4M(t), was studied in an attempt to define its molecular lesion. The mutant strain is inhibited in conidial germination and mycelial extension at the nonpermissive temperature (37 degrees C). Macromolecular synthesis studies showed that both ribonucleic acid (RNA) and protein syntheses are inhibited when 4-h cultures are shifted from 20 to 37 degrees C. Density gradient analysis of ribosomal subunits made at 37 degrees C indicated that strain 4M(t) is deficient in the accumulation of 60S ribosomal subunits in that the ratio of 60S/37S subunits was 0.29:1 compared with 1.6:1 for the parental strain. This phenotype was shown to be the result of a slow rate of processing of, and a deficiency in the amount of, the immediate precursor to 25S ribosomal RNA (the large RNA of the 60S subunit) in the sequence of events constituting the production of mature ribosomal RNAs from the primary transcript of the ribosomal deoxyribonucleic acid, the precursor ribosomal RNA molecule. Analysis of polysomes suggested that the heat-sensitive gene product might function in both the assembly and the function of the 60S ribosomal subunit, since there was a smaller proportion of newly made 60S subunits synthesized at 37 degrees C in the polysome region of the gradients than in the monosome-plus-subunit region. The ribosomal RNA processing defect is apparently responsible for the observed defects in germination and macromolecular synthesis at 37 degrees C, but the precise molecular lesion is not known. On the basis of these results, the heat-sensitive mutant allele in the 4M(t) strain is considered to define the rip1 (ribosome production) gene locus.

scholarly journals Both endonucleolytic and exonucleolytic cleavage mediate ITS1 removal during human ribosomal RNA processing

2013 ◽  
Vol 200 (5) ◽  
pp. 577-588 ◽  
Author(s):  
Katherine E. Sloan ◽  
Sandy Mattijssen ◽  
Simon Lebaron ◽  
David Tollervey ◽  
Ger J.M. Pruijn ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Rna Processing ◽  
Large Subunit ◽  
Genetic Diseases ◽  
Evolutionary Conservation ◽  
Rrna Processing ◽  
Ribosomal Subunits ◽  
Important Pathway ◽  
Ribosomal Rna Processing ◽  
Insight Into

Human ribosome production is up-regulated during tumorogenesis and is defective in many genetic diseases (ribosomopathies). We have undertaken a detailed analysis of human precursor ribosomal RNA (pre-rRNA) processing because surprisingly little is known about this important pathway. Processing in internal transcribed spacer 1 (ITS1) is a key step that separates the rRNA components of the large and small ribosomal subunits. We report that this was initiated by endonuclease cleavage, which required large subunit biogenesis factors. This was followed by 3′ to 5′ exonucleolytic processing by RRP6 and the exosome, an enzyme complex not previously linked to ITS1 removal. In contrast, RNA interference–mediated knockdown of the endoribonuclease MRP did not result in a clear defect in ITS1 processing. Despite the apparently high evolutionary conservation of the pre-rRNA processing pathway and ribosome synthesis factors, each of these features of human ITS1 processing is distinct from those in budding yeast. These results also provide significant insight into the links between ribosomopathies and ribosome production in human cells.

scholarly journals Unusual pattern of ribonucleic acid components in the ribosome of Crithidia fasciculata, a trypanosomatid protozoan.

1981 ◽  
Vol 1 (4) ◽  
pp. 347-357 ◽  
Author(s):  
M W Gray
Keyword(s):  
Ribosomal Rna ◽  
Ribonucleic Acid ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Ribosomal Rnas ◽  
High Potassium ◽  
Intact Cells ◽  
Crithidia Fasciculata ◽  
Low Magnesium ◽  
Chain Lengths

In a previous study from this laboratory, presumptive ribosomal ribonucleic acid (RNA) species were identified in the total cellular RNA directly extracted from intact cells of the trypanosomatid protozoan Crithidia fasciculata (M. W. Gray, Can. J. Biochem. 57:914-926, 1979). The results suggested that the C. fasciculata ribosome might be unusual in containing three novel, low-molecular-weight ribosomal RNA components, designated e, f, and g (apparent chain lengths 240, 195, and 135 nucleotides, respectively), in addition to analogs of eucaryotic 5S (species h) and 5.8S (species i) ribosomal RNAs. In the present study, all of the presumptive ribosomal RNAs were indeed found to be associated with purified C. fasciculata ribosomes, and their localization was investigated in subunits produced under different conditions of ribosome dissociation. When ribosomes were dissociated in a high-potassium (880 mM K+, 12.5 mM Mg2+) medium, species e to i were all found in the large ribosomal subunit, which also contained an additional, transfer RNA-sized component (species j). However, when subunits were prepared in a low-magnesium (60 mM K+, 0.1 mM Mg2+) medium, two of the novel species (e and g) did not remain with the large subunit, but were released, apparently as free RNAs. Control experiments have eliminated the possibility that the small RNAs are generated by quantitative and highly specific (albeit artifactual) ribonuclease cleavage of large ribosomal RNAs during isolation. In terms of RNA composition and dissociation properties, therefore, the ribosome of C. fasciculata is the most "atypical" eucaryotic ribosome yet described. These observations raise interesting questions about the function and evolutionary origin of C. fasciculata ribosomes and about the organization and expression of ribosomal RNA genes in this organism.

scholarly journals A functional network involved in the recycling of nucleocytoplasmic pre-60S factors

2006 ◽  
Vol 173 (3) ◽  
pp. 349-360 ◽  
Author(s):  
Alice Lebreton ◽  
Cosmin Saveanu ◽  
Laurence Decourty ◽  
Jean-Christophe Rain ◽  
Alain Jacquier ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Rna Processing ◽  
Functional Network ◽  
Ribosomal Subunits ◽  
Small Complex ◽  
Key Factor ◽  
Ribosomal Rna Processing ◽  
Cytoplasmic Maturation ◽  
Nucleocytoplasmic Proteins ◽  
Late Stages

Eukaryotic pre-ribosomes go through cytoplasmic maturation steps before entering translation. The nucleocytoplasmic proteins participating in these late stages of maturation are reimported to the nucleus. In this study, we describe a functional network focused on Rei1/Ybr267w, a strictly cytoplasmic pre-60S factor indirectly involved in nuclear 27S pre-ribosomal RNA processing. In the absence of Rei1, the nuclear import of at least three other pre-60S factors is impaired. The accumulation in the cytoplasm of a small complex formed by the association of Arx1 with a novel factor, Alb1/Yjl122w, inhibits the release of the putative antiassociation factor Tif6 from the premature large ribosomal subunits and its recycling to the nucleus. We propose a model in which Rei1 is a key factor for the coordinated dissociation and recycling of the last pre-60S factors before newly synthesized large ribosomal subunits enter translation.

Electron Microscopy of EDTA-Treated Ribosomal Subunits from Escherichia coli

1971 ◽  
Vol 29 ◽  
pp. 432-433
Author(s):  
John H. Nisbet ◽  
Henry S. Slayter
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Rna ◽  
Gradient Analysis ◽  
Ribosomal Subunit ◽  
Nuclease Activity ◽  
Final Concentration ◽  
Ribosomal Subunits ◽  
Magnesium Removal ◽  
Low Ionic Strength ◽  
Endogenous Nuclease

Several studies have indicated that treatment of ribosomes and ribosomal subunits with EDTA at low ionic strength results in the formation of slower-sedimenting species which have not lost any significant amount of protein (see, for example, reference 1). Here we attempt to follow morphologically the process of magnesium removal from the 50S ribosomal subunit of Escherichia coli, using strain Q13 which exhibits a low RNase I activity. 50S particles, initially in 0.01M Tris, 0.0001M MgCl2, pH 7.4, were treated with EDTA either by dialysis against 0.002M Tris, 0.001M EDTA, pH 7.4, or by direct addition of a 0.1M solution of EDTA to a final concentration of 0.001M. On sucrose gradients made up in the Tris-EDTA buffer, the particles sedimented as a sharp peak in the 10-12S region, with no detectable slower - sedimenting species. Sucrose gradient analysis of the ribosomal RNA following EDTA treatment of 50S subunits showed that 23S RNA were largely converted to 16S, reflecting a low level of endogenous nuclease activity in the subunit preparation.

A Stable 44S Intermediate in the Autodigestion of 50S Ribosomal Subunits

1971 ◽  
Vol 29 ◽  
pp. 430-431
Author(s):  
John H. Nisbet ◽  
Henry S. Slayter
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Ribosomal Rna ◽  
Gel Electrophoresis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Ribosomal Subunit ◽  
Wild Type ◽  
Ribosomal Subunits ◽  
Type Strains

Wild - type strains of Escherichia coli are known to contain as many as four endogenous nucleases (Ref. 1). These are commonly found associated with the ribosomes after extraction from the cell, but may be removed, with the exception of RNase IV, by washing the ribosomes in NH4Cl (at 0.2 M and higher concentrations). We have examined the effect of these nucleases on the 50S ribosomal subunit of one wild-type strain, K12 (Hfr 3000), by incubating the unwashed particles at 37° in the presence of varying magnesium concentrations.At 10-4 molar magnesium (slower at 10-3 molar), the 50S particle is converted to a species sedimenting at about 44S. About 20% of the total O.D260 is liberated at the same time. Continued incubation leads to the release of more O.D260 material while the RNA remaining in the 44S (Fig. 1) particle is progressively cleaved, eventually to the point where it consists of one principal fragment of molecular weight 0.42 x 106 daltons and several lesser fragments. The ribosomal RNA and proteins have been characterized by acrylamide gel electrophoresis.

scholarly journals The size and conformation of Artemia (brine-shrimp) ribosomal RNA free in solution

1982 ◽  
Vol 205 (3) ◽  
pp. 495-501 ◽  
Author(s):  
K Donceel ◽  
P Nieuwenhuysen ◽  
J Clauwaert
Keyword(s):  
Ribosomal Rna ◽  
Diffusion Coefficients ◽  
Brine Shrimp ◽  
Ribosomal Proteins ◽  
Hydrodynamic Radius ◽  
Ribosomal Subunit ◽  
Ribosomal Subunits ◽  
Rrna Species ◽  
Reported Data ◽  
And Diffusion

The RNA was isolated from the large ribosomal subunits of the brine shrimp Artemia, and its conformation free in solution was studied by determining its sedimentation and diffusion coefficients. A comparison was made of the hydrodynamic radius of the ribosomal subunit and its isolated RNA in various buffers. The conformation of the rRNA free in solution is more extended than when it is incorporated in the ribosome. This is not only the case when the rRNA solution lacks bivalent and polyvalent cations, but even in the presence of Mg2+ and spermidine, which cause a tightening of RNA. Thus the ribosomal proteins should induce a further tightening of the rRNA during the assembly of the ribosome. In the discussion, the reported data on Escherichia coli rRNA species are presented in such a way that large discrepancies between various studied are revealed, and that they can be compared with the data reported here on the larger rRNA of an eukaryote.

Unusual pattern of ribonucleic acid components in the ribosome of Crithidia fasciculata, a trypanosomatid protozoan

1981 ◽  
Vol 1 (4) ◽  
pp. 347-357
Author(s):  
M W Gray
Keyword(s):  
Ribosomal Rna ◽  
Ribonucleic Acid ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Ribosomal Rnas ◽  
High Potassium ◽  
Intact Cells ◽  
Crithidia Fasciculata ◽  
Low Magnesium ◽  
Chain Lengths

In a previous study from this laboratory, presumptive ribosomal ribonucleic acid (RNA) species were identified in the total cellular RNA directly extracted from intact cells of the trypanosomatid protozoan Crithidia fasciculata (M. W. Gray, Can. J. Biochem. 57:914-926, 1979). The results suggested that the C. fasciculata ribosome might be unusual in containing three novel, low-molecular-weight ribosomal RNA components, designated e, f, and g (apparent chain lengths 240, 195, and 135 nucleotides, respectively), in addition to analogs of eucaryotic 5S (species h) and 5.8S (species i) ribosomal RNAs. In the present study, all of the presumptive ribosomal RNAs were indeed found to be associated with purified C. fasciculata ribosomes, and their localization was investigated in subunits produced under different conditions of ribosome dissociation. When ribosomes were dissociated in a high-potassium (880 mM K+, 12.5 mM Mg2+) medium, species e to i were all found in the large ribosomal subunit, which also contained an additional, transfer RNA-sized component (species j). However, when subunits were prepared in a low-magnesium (60 mM K+, 0.1 mM Mg2+) medium, two of the novel species (e and g) did not remain with the large subunit, but were released, apparently as free RNAs. Control experiments have eliminated the possibility that the small RNAs are generated by quantitative and highly specific (albeit artifactual) ribonuclease cleavage of large ribosomal RNAs during isolation. In terms of RNA composition and dissociation properties, therefore, the ribosome of C. fasciculata is the most "atypical" eucaryotic ribosome yet described. These observations raise interesting questions about the function and evolutionary origin of C. fasciculata ribosomes and about the organization and expression of ribosomal RNA genes in this organism.

scholarly journals Terminal-sequence studies of high-molecular-weight ribonucleic acid. The 3′-termini of rabbit reticulocyte ribosomal ribonucleic acid

1970 ◽  
Vol 120 (2) ◽  
pp. 353-363 ◽  
Author(s):  
John A. Hunt
Keyword(s):  
Ribonucleic Acid ◽  
Periodate Oxidation ◽  
Ribosomal Subunit ◽  
Large Ribosomal Subunit ◽  
Terminal Sequence ◽  
Ribosomal Subunits ◽  
Rna Molecules ◽  
Ribosomal Ribonucleic Acid ◽  
Rabbit Reticulocytes

Sequences of the polynucleotide chains of RNA found in the large and small ribosomal subunits of rabbit reticulocytes have been determined from the 3′-end by use of periodate oxidation and condensation with [3H]isoniazid and by stepwise degradation. By these methods the hexanucleotide sequences have been found as -pGpUpUpUpGpU for the 28S RNA and -pGpUpCpGpCpU for the 6S RNA of the large ribosomal subunit and the octanucleotide sequence -pGpApUpCpApUpUpA for the 18S rRNA of the small ribosomal subunit. These sequences are present in at least 70% of all the RNA molecules and are discussed in relation to the specific cleavage of rRNA from its precursors and the role of multiple cistrons for rRNA in the DNA of higher organisms. The feasibility of using the method for longer sequence determinations is discussed.

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