Abstract
Many mRNAs in mammalian cells decay via a sequential pathway involving rapid conversion of polyadenylated molecules to a poly(A)-deficient state followed by rapid degradation of the poly(A)-deficient molecules. However, the rapidity of this latter step(s) has precluded further analyses of the decay pathways involved. Decay intermediates derived from degradation of poly(A)-deficient molecules could offer clues regarding decay pathways, but these intermediates have not been readily detected. Cell-free mRNA decay systems have proven useful in analyses of decay pathways because decay intermediates are rather stable in vitro. Cell-free systems indicate that many mRNAs decay by a sequential 3'-5' pathway because 3'-terminal decay intermediates form following deadenylation. However, if 3'-terminal, in vitro decay intermediates reflect a biologically significant aspect of mRNA turnover, then similar intermediates should be present in cells. Here, I have compared the in vivo and in vitro decay of mRNA encoded by the c-myc proto-oncogene. Its decay both in vivo and in vitro occurs by rapid removal of the poly(A) tract and generation of a 3'-terminal decay intermediate. These data strongly suggest that a 3'-5' pathway contributes to turnover of c-myc mRNA in cells. It is likely that 3'-5' decay represents a major turnover pathway in mammalian cells.
Highlights
The steady-state levels of mRNAs depend upon their combined rates of synthesis and processing in the nucleus, transport from the nucleus to cytoplasm, and decay in the cytoplasm
Degradation is likely because of an exoribonuclease that pauses within the 3Ј-terminal stem-loop structure, generating progressively shorter decay intermediates that lack 5 nt, 12 nt, from the 3Ј end [13]. (iii) In vitro mRNA decay extracts prepared from mammalian cells degrade labile mRNAs 3Ј-5Ј by rapid deadenylation followed by generation of 3Ј-terminal decay intermediates
Rapid deadenylation of c-myc mRNA was specific because the poly(A) tract of ␥-globin was not rapidly shortened, and the mRNA was stable over the 3-h time course (Fig. 1, lower panel)
Summary
Restriction enzymes and RNasin were obtained from Promega Corp. (Madison, WI). RNase H, oligo(dT), and oligo(dT)-cellulose were from Amersham Pharmacia Biotech. Radiolabeling of Probes—The same c-myc probe was used for both RNase protection assays and for the RNA blot (described above). It was prepared by in vitro transcription of SspI-digested plasmid pSP65myc(CLARI) [17] using SP6 RNA polymerase and [␣-32P]UTP (Ͼ800 Ci/mmol). A radiolabeled probe for detection of human H4 histone mRNA by nuclease S1 mapping was prepared by 3Ј-end labeling of plasmid pHh4A digested with NcoI as described [12]. A radiolabeled probe for detection of human ␥-globin mRNA by nuclease S1 mapping was prepared by 3Ј-end labeling of plasmid pDCY2 digested with EcoRI as described [17]. RNA was purified for each time point, and c-myc mRNA was analyzed by an RNase P1ϩT1 protection assay as described above
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