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Size distribution of the urokinase mRNA decay intermediates in different tissues and cell lines
A V Timofeeva1, N A Skrypina, L P Savochkina
1Russian Cardiology Research and Development Center, 3rd Cherepkovskaya str., 15a, Moscow 121552, Russia. angelica_t@cardio.ru
Abstract:
Many genes, particularly those encoding the products participating in the regulation of transcription, replication and tissue remodeling, produce short-lived mRNA. It has been commonly accepted that once mRNA is disintegrated, the degradation process is so rapid that the decay intermediates cannot be detected. In the present study we verified this postulate and focused our attention on the quantification of the decay products of the urokinase-type plasminogen activator (uPA) mRNA that belongs to short-lived mRNAs. Using a previously described modified quantitative RT-PCR method, we have shown that intact uPA mRNA coexists in normal human tissues, Jurkat and 5637 cells with a great abundance of its degradation products. The uPA mRNA decay products were not detected in T24P cells. The content of intact uPA mRNA in normal tissues was as low as 5% of the total amount of its poly(A)(+) fraction. The size distribution of the mRNA decay products suggests that the mRNA is digested by exonucleases or/and non-specific endonuclease with cut sites evenly distributed along the mRNA chain. Different decay degrees were demonstrated for subpopulation of the uPA mRNA molecules with intact 3' and 5' ends.
Insights
Short-lived mRNA decay products are detectable and abundant, challenging previous assumptions. This study quantifies urokinase-type plasminogen activator (uPA) mRNA degradation products in human tissues and cells.
Area of Science:
- Molecular Biology
- Gene Expression Regulation
Background:
- Many genes involved in crucial cellular processes produce short-lived messenger RNA (mRNA).
- It was previously believed that mRNA degradation intermediates are too transient to be detected.
Purpose of the Study:
- To investigate the existence and abundance of urokinase-type plasminogen activator (uPA) mRNA decay products.
- To challenge the postulate that mRNA decay intermediates are undetectable.
Main Methods:
- Utilized a modified quantitative reverse transcription polymerase chain reaction (RT-PCR) method.
- Quantified intact uPA mRNA and its degradation products in various human tissues and cell lines (Jurkat, 5637, T24P).
Main Results:
- Intact uPA mRNA coexists with abundant degradation products in normal human tissues, Jurkat, and 5637 cells.
- uPA mRNA decay products were notably absent in T24P cells.
- Intact uPA mRNA constituted only about 5% of the total poly(A)(+) fraction in normal tissues.
- Analysis of decay product size distribution suggests digestion by exonucleases and/or non-specific endonucleases.
- Varied degrees of decay were observed among uPA mRNA subpopulations.
Conclusions:
- Short-lived mRNA decay intermediates, specifically for uPA mRNA, are detectable and abundant.
- The findings necessitate a revision of the understanding of mRNA degradation kinetics.
- The study highlights the presence of distinct mRNA decay pathways and subpopulations.