Histone mRNA degradation in vivo: the first detectable step occurs at or near the 3' terminus.

The first detectable step in the degradation of human H4 histone mRNA occurs at the 3' terminus in a cell-free mRNA decay system (J. Ross and G. Kobs, J. Mol. Biol. 188:579-593, 1986). Most or all of the remainder of the mRNA is then degraded in a 3'-to-5' direction. The experiments described here were designed to determine whether a similar degradation pathway is followed in whole cells. Two sets of short-lived histone mRNA decay products were detected in logarithmically growing erythroleukemia (K562) cells. These products, designated the -5 and -12 RNAs, were generated by the loss of approximately 4 to 6 and 11 to 13 nucleotides, respectively, from the 3' terminus of histone mRNA. The same decay products were observed after a brief incubation in vitro. They were in low abundance or absent from cells that were not degrading histone mRNA. In contrast, they were readily detectable in cells that degraded the mRNA at an accelerated rate, i.e., in cells cultured with a DNA synthesis inhibitor, either cytosine arabinoside or hydroxyurea. During the initial stages of the decay process, as the 3' terminus of the mRNA was being degraded, the 5'-terminal region remained intact. These results indicate that the first detectable step in human H4 histone mRNA decay occurs at the 3' terminus and that degradation proceeds 3' to 5', both in cells and in cell-free reactions.

H4 histone messenger RNA decay in cell-free extracts initiates at or near the 3' terminus and proceeds 3' to 5'.

The relative decay of four human messenger RNAs, gamma globin, delta globin, c-myc and H4 histone, were compared in a cell-free system. Under appropriate conditions, they are degraded in vitro in approximately the same relative order as in vivo: histone faster than c-myc and delta globin faster than gamma globin. Degradation of polysome-associated H4 histone mRNA and of deproteinized histone mRNA begins at or near the 3' terminus. At least a portion of the mRNA then continues to be degraded in a 3' to 5' direction. Discrete 3'-terminal degradation hold-up points are observed, suggesting that 3' to 5' degradation occurs non-uniformly. Cycloheximide and puromycin inhibit protein synthesis but do not affect the rate or directionality of histone mRNA decay in vitro. We conclude that the rate-limiting step in H4 histone mRNA decay occurs at or near the 3' terminus and that at least a portion of the mRNA molecule is subsequently degraded 3' to 5', probably via a processive exonuclease.

Properties of the exonuclease activity that degrades H4 histone mRNA.

We have described a cell-free system for studying mRNA degradation (Ross, J., and Kobs, G. (1986) J. Mol. Biol. 188, 579-593). Using that system we found that human H4 histone mRNA was degraded in a 3' to 5' direction by an exonucleolytic activity. Here we investigate several properties of the crude system and of the exonuclease. A RNase inhibitor, such as that from placenta, was required to block nonspecific ribonucleases and thereby to permit different mRNAs to be degraded at different rates. The histone mRNA exonuclease required divalent cation (magnesium) but not exogenously added ATP or GTP. It functioned efficiently at monovalent cation concentrations ranging from 0.5 to 200 mM. It was bound to ribosomes isolated from cells lysed in low salt buffers. However, it was eluted in active form from the ribosomes by exposing them to 0.3 M KCl. The enzyme rapidly degraded deproteinized, 32P-labeled histone mRNA prepared enzymatically.

Substrate specificity of the exonuclease activity that degrades H4 histone mRNA.

We have investigated the substrate specificity of an exonuclease that degrades human H4 histone mRNA, using synthetic RNA templates incubated in a cell-free mRNA decay system (Ross, J., and Kobs, G. (1986) J. Mol. Biol. 188, 579-593). Five RNAs that lacked poly(A), including histone, were degraded rapidly in vitro. Polyadenylated histone mRNA was degraded at least 10-fold more slowly than unmodified histone mRNA. Double-stranded RNA and DNA were very stable. Single-stranded DNA was degraded approximately 20-fold more slowly than single-stranded, non-polyadenylated RNA, and RNA with a 3' phosphoryl group was degraded more slowly than RNA with a 3'-hydroxyl group. Uncapped RNAs were degraded rapidly in the unfractionated system but were stable in reactions containing a ribosomal high salt wash extract. Therefore, the exonuclease activity released from ribosomes by high salt extraction was separated from the enzyme(s) that degraded uncapped RNAs.