Involvement of the 5'-leader sequence in coupling the stability of a human H3 histone mRNA with DNA replication.

Abstract

Two lines of evidence derived from fusion gene constructs indicate that sequences residing in the 5'-nontranslated region of a cell cycle-dependent human H3 histone mRNA are involved in the selective destabilization that occurs when DNA synthesis is terminated. The experimental approach was to construct chimeric genes in which fragments of the mRNA coding regions of the H3 histone gene were fused with fragments of genes not expressed in a cell cycle-dependent manner. After transfection in HeLa S3 cells with the recombinant plasmids, levels of fusion mRNAs were determined by S1 nuclease analysis prior to and following DNA synthesis inhibition. When the first 20 nucleotides of an H3 histone mRNA leader were replaced with 89 nucleotides of the leader from a Drosophila heat-shock (hsp70) mRNA, the fusion transcript remained stable during inhibition of DNA synthesis, in contrast to the rapid destabilization of the endogenous histone mRNA in these cells. In a reciprocal experiment, a histone-globin fusion gene was constructed that produced a transcript with the initial 20 nucleotides of the H3 histone mRNA substituted for the human f3-globin mRNA leader. In HeLa cells treated with inhibitors of DNA synthesis and/or protein synthesis, cellular levels of this histone-globin fusion mRNA appeared to be regulated in a manner similar to endogenous histone mRNA levels. These results suggest that the first 20 nucleotides of the leader are sufficient to couple histone mRNA stability with DNA replication.The human histone genes are a moderately repeated gene family that encode the major structural proteins ofchromatin. It has been well established that histone gene expression and DNA replication are temporally and functionally coupled. The synthesis of most histone proteins (1-6) and the steadystate levels of histone mRNAs (7-12) are closely correlated with DNA synthesis in the S phase of the cell cycle. At the natural end of S phase or following inhibitor-induced termination of DNA synthesis, there is a coordinate and stoichiometric decrease in histone mRNA levels and histone protein synthesis (8)(9)(10)(11)(12)(13). The rapid loss of histone mRNA under these conditions is in contrast to minimal changes in nonhistone mRNA levels. The selective destabilization of histone mRNA during DNA synthesis inhibition is posttranscriptionally mediated; destabilization is not dependent on transcription (11) but requires protein synthesis (11-15). The cellular and molecular basis for histone mRNA turnover, however, remains unresolved.To address molecular mechanisms operative in the selective destabilization of histone mRNAs, we are attempting to identify regions of a cloned, cell cycle-dependent human H3 histone gene (16,17) that are involved in the destabilization of its transcripts. Our approach is to construct fusion genes, in which fragments ofthe mRNA coding regions ofthe cloned human H3 histone gene are fused with fragments of other genes not expressed in a cell cycle-dependent manner. After transfection into HeLa S3 cells, le...

Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates.

“…This sequence is particularly noteworthy in light of the extremely high A+T content of noncoding regions in D. discoideum mRNAs (67). More definitive evidence for the importance of 5'-and 3'-UT regions has come from recent experiments in which these regions have been deleted, replaced, or transferred, resulting in substantial changes in the decay rates of individual mRNAs (22,42,53,64,74,76). The most well-characterized of the UT elements are the 3' AU-rich sequences associated with the GM-CSF mRNA (and other lymphokine and protooncogene mRNAs [12,50,74]) and the 3' stem-loop structure associated with the replication-dependent histone mRNAs (21,68 Fig.…”

“…It is likely that these complex decay kinetics reflect the sum of the decay rates of individual mRNAs (e.g., in D. discoideum, the most stable mRNAs have half-lives of approximately 10 h and the least stable mRNAs have half-lives which are approximately 10-fold shorter). It has been suggested that such large differences in the stability of individual mRNAs could be accounted for by differences in their respective sizes (39,52,58,63,78,81), poly(A) tail lengths (8,28,56,91), ribosome loading (1,26,33,41,45), or 5'-and 3'-untranslated (UT) sequences (42,53,64,69,74,76,89). To test the validity of these hypotheses, we have identified cloned cDNAs which encode mRNAs that are representative of the stability extremes in D. discoideum and have initiated a characterization of the properties of the respective mRNAs.…”

Transition from maternal to embryonic control in early mammalian development: A comparison of several species

“…In human histone H3 mRNA, however, the 5' noncoding region is important in linking the stability of the histone mRNA to DNA synthesis (Morris et al, 1986).…”

Autogenous regulation of histone mRNA decay by histone proteins in a cell-free system.

“…Although we know that puromycin and cycloheximide have no effect on the acceleration of histone mRNA degradation induced by S130 and histone proteins (S.W.P., unpublished observations), we do not know how translation rates and polysome structure affect autoregulation. Since histone mRNA metabolism tends to be affected by most mutations that alter its 5' or 3' sequences or its intracellular location (4,7,13,26,39,43,59,60,66,75), we suggest that normal regulation of histone mRNA turnover is strictly dependent not only on the stem and loop but also on additional histone-specific sequences that specify how and where it is translated. The constraints placed on normal…”