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Translation and M1 double-stranded RNA propagation: MAK18 = RPL41B and cycloheximide curing
1Section on Genetics of Simple Eukaryotes, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0830, USA.
Abstract:
MAK18 is one of nearly 30 chromosomal genes of Saccharomyces cerevisiae necessary for propagation of the killer toxin-encoding M1 double-stranded RNA satellite of the L-A double-stranded RNA virus. We have cloned and sequenced MAK18 and find that it is identical to RPL41B, one of the two genes encoding large ribosomal subunit protein L41. The mak18-1 mutant is deficient in 60S subunits, which we suggest results in a preferential decrease in translation of viral poly(A)-deficient mRNA. We have reexamined the curing of M1 by low concentrations of cycloheximide (G. R. Fink and C. A. Styles, Proc. Natl. Acad. Sci. USA 69:2846-2849, 1972), which is known to act on ribosomal large subunit protein L29. We find that when M1 is supported by L-A proteins made from the poly(A)+ mRNA of a cDNA clone of L-A, cycloheximide does not decrease the M1 copy number, consistent with our hypothesis.
Insights
The MAK18 gene in Saccharomyces cerevisiae is identified as RPL41B, a ribosomal protein. A mak18-1 mutant shows reduced 60S subunits, impacting viral mRNA translation and M1 satellite propagation.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Virology
Background:
- MAK18 is essential for the propagation of the M1 double-stranded RNA satellite in Saccharomyces cerevisiae.
- The M1 satellite depends on the L-A double-stranded RNA virus for its replication.
- Previous studies indicated that cycloheximide can cure M1, suggesting a link to ribosomal function.
Purpose of the Study:
- To clone and sequence the MAK18 gene.
- To investigate the molecular mechanism by which MAK18 influences M1 propagation.
- To elucidate the role of ribosomal subunits in viral RNA replication.
Main Methods:
- Gene cloning and sequencing of MAK18.
- Construction and analysis of the mak18-1 mutant.
- Investigation of ribosomal subunit composition.
- Analysis of viral mRNA translation efficiency.
- Cycloheximide sensitivity assays.
Main Results:
- MAK18 was found to be identical to RPL41B, a gene encoding ribosomal protein L41.
- The mak18-1 mutant exhibits a deficiency in 60S ribosomal subunits.
- This deficiency leads to a preferential decrease in the translation of viral poly(A)-deficient mRNA.
- Cycloheximide did not reduce M1 copy number when L-A proteins were produced from poly(A)+ mRNA, supporting the hypothesis.
Conclusions:
- MAK18 (RPL41B) is crucial for yeast 60S ribosomal subunit biogenesis.
- Ribosomal subunit composition directly impacts the translation of specific viral mRNAs.
- This study provides a molecular explanation for M1 satellite propagation and its sensitivity to translation inhibitors.