The metabolism of a poly(A) minus mRNA fraction in HeLa cells (original) (raw)
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European Journal of Biochemistry, 1979
The mRNA species which exist in the HeLa cell polyribosomes in a form devoid of A sequences longer than 8 nucleotides constitute the poly(A)-free class of mRNA. The rapidly labelled component of this mRNA class shares no measurable sequence homology with poly(A)-containing RNA. If poly(A)-free mRNA larger than 12 S labelled for 2 h in vivo is hybridized with total cellular DNA, it hybridizes primarily with single-copy DNA. When a large excess of steady poly(A)-containing RNA is added before hybridization of labelled poly(A)-free RNA, no inhibition of hybridization occurs. This indicates the existence of a class of poly(A)-free mRNA with no poly(A)containing counterpart. Some mRNA species can exist solely as poly(A)-containing mRNAs. These mRNAs in HeLa cells are found almost exclusively in the mRNA species present only a few times per cell (scarce sequences). Some mRNA species can exist in two forms, poly(A)containing and lacking, as evidenced by the translation data in vitvo of Kaufmann et al. [Pvoc. Nut1 Acud. Sci. U.S.A. 74, 4801-4805 (1977)l. In addition, if cDNA to total poly(A)-containing mRNA is fractionated into abundant and scarce classes, 47 7; of the scarce class cDNA can be readily hybridized with poly(A)-free mRNA.
A major species of mammalian messenger RNA lacking a polyadenylate segment
Proceedings of the National Academy of Sciences, 1976
Translation of total polysomal RNA from sarcoma 180 ascites cells in a wheat germ cell-free system produces two major polypeptides, A and B, with molecular weights of 50,000 and 45,000, respectively. Fractionation on Millipore filters or on oligo(dT)-cellulose leads to retention of the mRNA specific for protein A in the poly(A)-containing fraction and to accumulation of the B mRNA in the unadsorbed poly(A)-deficient fraction. The mRNA for B sediments at approximately 18 S; it is released as a 50S ribonucleorprotein upon EDTA treatment of polysomes. Its translation is particularly sensitive to an inhibitor present in the polysomal RNA. The poly(A)-deficient mRNA for the 45,000 dalton polypeptide is also present in mouse myeloma MPC-11 cells, where it seems to be localized in membrane-bound polysomes.
1979
Cytoplasmic extracts of mouse Taper ascites cells were centrifuged on sucrose gradients to give O-80 S, monosome, and polysome fractions. CsCl equilibrium density centrifugation of formaldehyde-fixed material from the O-80 S fractiorr demonstrated th& the messenger RNA in the O-80 S fraction was in the form of free ribonucleoprotein. The size of the poly(A+)RNA and t,he size of the poly(A) segments of t,hese molecules were shown to be very similar in both the free rnRNPt and polysome fractions. The labeling kinetics of the free mRNP poly(A+)RNA was similar to that of the polysomal poly(A+)RNA. The free mRNP poly(A+)RNA efficiently stimulated protein synthesis in tire wheat germ cell-free system, supporting the view that it was mRNA. Twodimensional gel electrophoresis was used to analyze the proteins whose synt,hesis was directed by free mRNP and polysomal poly(A+)RNA. The free mRNP poly(A+)RNA directed the synthesis of a simpler set of abundant protein products than did the polysomal poly(A+)RNA. Most of the free mRNP abundant protein products were also present in the polysomal products, though obvious quantitative differences were evident, indicating that each individual mRNA had it,s own characteristic distribution between polysomes and the translationally innct,ive RNP form.
Role of poly(A) polymerase in the cleavage and polyadenylation of mRNA precursor
Molecular and cellular biology, 1989
To determine the role of poly(A) polymerase in 3'-end processing of mRNA, the effect of purified poly(A) polymerase antibodies on endonucleolytic cleavage and polyadenylation was studied in HeLa nuclear extracts, using adenovirus L3 pre-mRNA as the substrate. Both Mg2+- and Mn2+-dependent reactions catalyzing addition of 200 to 250 and 400 to 800 adenylic acid residues, respectively, were inhibited by the antibodies, which suggested that the two reactions were catalyzed by the same enzyme. Anti-poly(A) polymerase antibodies also inhibited the cleavage reaction when the reaction was coupled or chemically uncoupled with polyadenylation. These antibodies also prevented formation of specific complexes between the RNA substrate and components of nuclear extracts during cleavage or polyadenylation, with the concurrent appearance of another, antibody-specific complex. These studies demonstrate that (i) previously characterized poly(A) polymerase is the enzyme responsible for addition o...