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Association of poly(adenylate)-deficient messenger ribonucleic acid with membranes in mouse kidney
Abstract:
To describe further the metabolism of messenger ribonucleic acid (mRNA) in mouse kidney, we examined newly synthesized mRNA deficient in poly(adenylate) [poly(A)]. Approximately 50% of renal polysomal mRNA that labeled selectively in the presence of the pyrimidine analogue 5-fluoroorotic acid lacks or is deficient in poly(A) as defined by its ability to bind to poly(A) affinity columns. Nearly one-half of this poly(A)-deficient mRNA is associated uniquely with a cellular membrane fraction detected by sedimentation of renal cytoplasm in sucrose density gradients containing EDTA and nonionic detergents. Poly(A+) mRNA and poly(A)-deficient mRNA [poly(A-) mRNA] have similar modal sedimentation coefficients (20-22 S) and similar cytoplasmic distribution. Although 95% of newly synthesized poly(A+) mRNA is released in 10 mM EDTA as 20-90 S ribonucleoproteins from polysomes greater than 80 S, only 55% of poly(A)-deficient mRNA is released under the same conditions. Poly(A)-deficient mRNA recovered from greater than 80 S ribonucleoproteins resistant to EDTA treatment lacks ribosomal RNA, is similar in size to poly(A+) mRNA, and is associated with membranous structures, since 70% of poly(A)-deficient mRNA in EDTA-resistant ribonucleoproteins is released into the 20-80 S region by solubilizing membranes with 1% Triton X-100. These membrane-associated renal poly(A-) mRNAs could have unique coding or regulatory functions.
Insights
Mouse kidney messenger RNA (mRNA) metabolism reveals that approximately half of newly synthesized mRNA lacks poly(adenylate) [poly(A)]. This poly(A)-deficient mRNA is uniquely associated with cellular membranes, suggesting novel functions.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Messenger ribonucleic acid (mRNA) metabolism is crucial for gene expression.
- The poly(adenylate) [poly(A)] tail plays a significant role in mRNA stability and translation.
- Understanding mRNA processing in specific tissues like the kidney is essential.
Purpose of the Study:
- To investigate the characteristics and cellular localization of newly synthesized poly(adenylate)-deficient mRNA in mouse kidney.
- To explore the potential functional significance of poly(A)-deficient mRNA.
Main Methods:
- Selective labeling of newly synthesized mRNA using 5-fluoroorotic acid.
- Poly(A) affinity chromatography to isolate poly(A)-deficient mRNA.
- Sucrose density gradient centrifugation of renal cytoplasm with EDTA and detergents to identify cellular fractions.
- Analysis of ribonucleoprotein complex dissociation and association with membranes.
Main Results:
- Approximately 50% of newly synthesized renal mRNA lacks or is deficient in poly(A).
- Nearly half of this poly(A)-deficient mRNA is uniquely associated with a cellular membrane fraction.
- Poly(A)-deficient mRNA exhibits differential release from polysomes under EDTA treatment compared to poly(A+) mRNA.
- Membrane-associated poly(A)-deficient mRNA is resistant to EDTA but can be released by membrane solubilization.
Conclusions:
- Mouse kidney contains a significant population of newly synthesized poly(A)-deficient mRNA.
- This poly(A)-deficient mRNA is preferentially associated with cellular membranes.
- Membrane-associated poly(A)-deficient mRNAs may possess distinct coding or regulatory roles in renal cells.