Structure and regulation of histone H2B mRNAs from Leishmania enriettii.

We have studied the structure and expression of histone H2B mRNA and genes in the parasitic protozoan Leishmania enrietti. A genomic clone containing three tandemly repeated genes has been sequenced and shown to encode three identical histone proteins and two types of closely related mRNA sequence. We have also sequenced three independent cDNA clones and demonstrated that the Leishmania H2B mRNAs are polyadenylated, similar to the basal histone mRNAs of higher eucaryotes and the histone mRNAs of yeast. In addition, the Leishmania mRNAs contain inverted repeats near the poly(A) tail which could form stem-loops similar in secondary structure, but not in sequence, to the 3' stem-loops of nonpolyadenylated replication-dependent histones of higher eucaryotes. Unlike the replication-dependent histones, the Leishmania histone H2B mRNAs do not decrease in abundance following treatment with inhibitors of DNA synthesis. The histone mRNAs are differentially expressed during the parasite life cycle and accumulate to a higher level in the extracellular promastigotes (the form which in nature lives within the gut of the insect vector) than in the intracellular amastigotes (the form that lives within the mammalian host macrophages).     

Individual Xenopus histone genes are replication-independent in oocytes and replication-dependent in Xenopus or mouse somatic cells.

We have assessed the response of many histone H3 mRNAs and an H1C mRNA in Xenopus tissue culture cells after treatment with the DNA synthesis inhibitor hydroxyurea. The amount of the histone mRNAs falls rapidly in response to the inhibitor. This response is prevented by cycloheximide. Cloned Xenopus histone genes were transfected into mouse cells and a cell line was obtained in which the Xenopus genes were actively expressed giving rise to mRNA with correct 5'-termini. The Xenopus genes were correctly regulated at the level of mRNA amounts in the mouse cell line. Nuclear microinjection experiments with Xenopus oocytes and S1 nuclease analysis of normal ovary RNA showed that the H1C gene, and probably also two H3 genes, which are replication-dependent in somatic cells are expressed in oocytes and are therefore replication-independent in this cell type. The same promoters are used in both replication-dependent and independent expression.     

Isolation and characterization of a mouse fully replication-dependent H1 gene within a genomic cluster of core histone genes

We have used an oligonucleotide complementary to a sequence coding for the conserved central globular domain of H1s to screen a mouse genomic library for H1 genes. We then used a series of universal histone oligonucleotides to identify five different H1 genes which were linked to core histone genes. We characterized one of the H1 genes which was linked to an H2a, an H2b, an H3, and an H4 histone gene. This characterization involved: 1) sequencing of the coding region of the gene and several hundred base pairs of flanking region. 2) Comparison of this sequence to other H1 sequences from other organisms. This sequence analysis clearly showed that the gene coded for an H1 and identified H1 consensus sequences in the 5'- and 3'-flanking region. 3) Mapping of the 5'- and 3'-ends of the mRNA complementary to this gene by S1 nuclease analysis. 4) Identifying this gene and an adjacent H3 gene as being of the fully replication-dependent expression class, by measuring changes in the steady state levels of their mRNAs in the presence of hydroxyurea and during differentiation of murine erythroleukemia cells.     

Translation termination is involved in histone mRNA degradation when DNA replication is inhibited

The levels of replication-dependent histone mRNAs are coordinately regulated with DNA synthesis. A major regulatory step in histone mRNA metabolism is regulation of the half-life of histone mRNAs. Replication-dependent histone mRNAs are the only metazoan mRNAs that are not polyadenylated. Instead, they end with a conserved stem-loop structure, which is recognized by the stem-loop binding protein (SLBP). SLBP is required for histone mRNA processing, as well as translation. We show here, using histone mRNAs whose translation can be regulated by the iron response element, that histone mRNAs need to be actively translated for their rapid degradation following the inhibition of DNA synthesis. We also demonstrate the requirement for translation using a mutant SLBP which is inactive in translation. Histone mRNAs are not rapidly degraded when DNA synthesis is inhibited or at the end of S phase in cells expressing this mutant SLBP. Replication-dependent histone mRNAs have very short 3' untranslated regions, with the stem-loop located 30 to 70 nucleotides downstream of the translation termination codon. We show here that the stability of histone mRNAs can be modified by altering the position of the stem-loop, thereby changing the distance from the translation termination codon.     

Translation is required for regulation of histone mRNA degradation

When DNA synthesis is inhibited, the mRNAs coding for the replication-dependent histone proteins are selectively destabilized. The histone genes have been altered and reintroduced into tk- mouse L cells by cotransfection with the herpesvirus thymidine kinase gene. Two features of the mRNA are necessary for regulation of degradation: first, the hairpin loop must be present at the 3' end of the histone mRNA; and second, the histone mRNA must be capable of being translated to within 300 nucleotides of the 3' end of the RNA. Polyadenylated histone mRNAs are stable, as are histone mRNAs that contain in-frame termination codons early in the coding region or 500 nucleotide 3' untranslated regions with a normal hairpin loop at the 3' end.     

Histone gene expression and histone mRNA 3' end structure in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Background  

Histone protein synthesis is essential for cell proliferation and required for the packaging of DNA into chromatin. In animals, histone proteins are provided by the expression of multicopy replication-dependent histone genes. Histone mRNAs that are processed by a histone-specific mechanism to end after a highly conserved RNA hairpin element, and lack a poly(A) tail. In vertebrates and Drosophila, their expression is dependent on HBP/SLBP that binds to the RNA hairpin element. We showed previously that these cis and trans acting regulators of histone gene expression are conserved in C. elegans. Here we report the results of an investigation of the histone mRNA 3' end structure and of histone gene expression during C. elegans development.     

Metazoan replication-dependent histone mRNAs: a distinct set of RNA polymerase II transcripts

Metazoan replication-dependent histone mRNAs are the only eukaryotic mRNAs that lack polyA tails. The genes for the five histone proteins have remained physically linked during evolution. Expression of histone mRNAs and histone proteins requires a unique set of factors, and may be coordinated by association of the histone genes with Cajal bodies. Recently several novel factors, including components of the U7 snRNP, as well as proteins involved in regulation of histone gene expression, have been described.