Faithful cell-cycle regulation of a recombinant mouse histone H4 gene is controlled by sequences in the 3'-terminal part of the gene.

Abstract

We have analyzed the expression of endogenous histone H4 genes and of a newly introduced H4 gene in 21-Tb cells, a mouse mastocytoma cell-cycle mutant. Endogenous H4 mRNAs were less abundant by a factor of 120-180 in Gl-arrested than in exponentially multiplying cells. However, H4 transcription rates were only decreased by a factor of 3 under these conditions, as determined by in vitro elongation of nascent transcripts. This indicates that post-transcriptional control of histone mRNA levels is important, in accord with published data. We introduced a mouse H4 gene, modified by a 12-base-pair (bp) insertion in its coding sequence, into 21-Tb cells by DNA-mediated gene transfer. The levels of transcripts from this gene were regulated in parallel with those of the endogenous genes. Moreover, fusion of the simian virus 40 (SV40) early promoter to a 463-bp fragment containing the 3'-terminal half of the mouse H4 gene, including 230 bp of spacer sequences, led to the regulated expression of SV40/H4 fusion RNA. However, a small proportion of SV40-initiated transcripts were not processed to histone-specific 3' ends, but extended farther through the downstream Escherichia coli galactokinase gene to a SV40 polyadenylylation site. In contrast to the short SV40/H4 RNA, the levels of these longer transcripts were not reduced in Gl-arrested cells. These resultsshow that sequences in the 3'-terminal part of the H4 gene can regulate gene expression in the cell cycle, presumably at the post-transcriptional level, as long as they are not positioned much more distant from the terminus than normal.Histone biosynthesis in most eukaryotic cells is tightly coupled to nuclear DNA synthesis such that translatable histone mRNAs are present in significant amounts only during the S phase ofthe cell cycle (1-3). Recent studies using synchronized murine or human cells and homologous histone genes as hybridization probes have shown that histone mRNA steady-state levels vary over a 10-to 50-fold range, and similar variations were also observed in cells treated with inhibitors of DNA synthesis (4-9). Where analyzed, histone gene transcription varied only 2-to 5-fold (6-9), suggesting a major contribution of some post-transcriptional mechanism(s) to regulation of histone gene expression during the cell cycle.We are interested in defining the genetic information responsible for cell-cycle regulation of histone gene expression. To this end, we have transformed a temperature-sensitive mouse mastocytoma cell-cycle mutant with a recombinant mouse H4 gene. We describe the faithful regulation of this gene and present evidence indicating that sequences in the 3' part of the H4 gene can effectively control RNA metabolism during the cell cycle. MATERIALS AND METHODSCell Growth and Transformation. The K21 line of P-815-X2 mouse mastocytoma and its heat-sensitive cell-cycle variant 21-Tb (10, 11) were cultured in suspension in medium 1 (12) supplemented with 10% horse serum (Amimed, Basel, Switzerland). Before transfection, =106 cells were immobiliz...

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.…”

Generation of histone mRNA 3′ ends by endonucleolytic cleavage of the pre-mRNA in a snRNP-dependent in vitro reaction.

“…So far, the information on these points is based on in vivo experiments with either frog oocytes (Birchmeier et al, 1982(Birchmeier et al, , 1983(Birchmeier et al, , 1984Stunnenberg and Birnstiel, 1982;Galli et al, 1983), or tissue culture cells (Luscher et al, 1985) as test sytems. The discovery of the U7 RNA as an essential component of the processing machinery derives from one such experimental design.…”

Human La Protein: a Stabilizer of Histone mRNA

“…1B). The stem-loop is essential for proper cell cycle regulation and is the site at which mRNA degradation begins (4,14,29,31,39,47,56,57,65,68,75). It is also a binding site for a unique stemloop binding protein (40,46,72,73,76).…”

“…Specificity is achieved primarily via the unique 3Ј-terminal structure of histone mRNA. The 3Ј terminus lacks poly(A) and contains a 6-bp stem and 4-base loop (29,31,45) (see Fig. 1B).…”