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Free cytoplasmic messenger ribonucleoprotein complexes from rabbit reticulocytes
Abstract:
Free cytoplasmic globin mRNA containing mRNP-particles were isolated from rabbit reticulocytes by zonal sucrose gradient centrifugation and their properties were compared with mRNP particles isolated in the same way from EDTA-dissociated reticulocyte polyribosomes. The average poly(A)-length of 9S mRNA from free cytoplasmic mRNP was 17-20 nucleotides being about two times shorter than the average poly(A)-length of polysomal 9S mRNA. The protein composition of the free cytoplasmic mRNP particles disclosed the absence of the 76,000 dalton protein which is associated with the 3'poly(A)-segment of polysomal globin mRNA. It was concluded that free cytoplasmic mRNP-particles from rabbit reticulocytes can be classified as "old" mRNP in a post-translational phase. Free cytoplasmic mRNPs were translated in heterologous cell-free systems as well as in Xenopus laevis oocytes. Addition of hemin stimulated the synthesis of alpha-globin in all systems, while the presence of the cap analogue m7G(5')p inhibited translation of free cytoplasmic mRNA completely. The latter finding suggested that free cytoplasmic mRNA has a 5' terminal "cap". Shortening of the poly(A)-segment with concomitant loss of the 76,000 dalton protein may lead to less efficient translation of free cytoplasmic mRNP.
Insights
Free cytoplasmic globin mRNA in rabbit reticulocytes has shorter poly(A) tails and lacks a specific protein, indicating an "older" stage. This mRNA can still be translated, but its efficiency may decrease with poly(A) shortening.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Messenger ribonucleoprotein particles (mRNPs) are essential for mRNA transport and translation.
- Globin mRNA in reticulocytes is a well-studied model for gene expression regulation.
Purpose of the Study:
- To characterize the properties of free cytoplasmic globin mRNA-containing mRNP particles.
- To compare these properties with mRNP particles from reticulocyte polyribosomes.
- To investigate the translational capacity of free cytoplasmic mRNPs.
Main Methods:
- Isolation of mRNP particles from rabbit reticulocytes using zonal sucrose gradient centrifugation.
- Analysis of poly(A) tail length and protein composition of mRNPs.
- In vitro translation assays using heterologous cell-free systems and Xenopus laevis oocytes.
Main Results:
- Free cytoplasmic mRNP exhibited shorter poly(A) tails (17-20 nucleotides) compared to polysomal mRNP.
- A 76,000 dalton protein, associated with the 3' poly(A) segment in polysomal mRNP, was absent in free cytoplasmic mRNP.
- Free cytoplasmic mRNPs were translatable, with hemin stimulating alpha-globin synthesis and m7G(5')p inhibiting translation, suggesting a functional 5' cap.
Conclusions:
- Free cytoplasmic mRNP particles represent an "older" post-translational stage of globin mRNA.
- Poly(A) tail shortening and loss of the 76,000 dalton protein may contribute to reduced translational efficiency.
- The presence of a 5' cap on free cytoplasmic mRNA is confirmed.