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.     

Electrostatic contribution of serine phosphorylation to the Drosophila SLBP--histone mRNA complex

Unlike all other metazoan mRNAs, mRNAs encoding the replication-dependent histones are not polyadenylated but end in a unique 26 nucleotide stem-loop structure. The protein that binds the 3' end of histone mRNA, the stem-loop binding protein (SLBP), is essential for histone pre-mRNA processing, mRNA translation, and mRNA degradation. Using biochemical, biophysical, and nuclear magnetic resonance (NMR) experiments, we report the first structural insight into the mechanism of SLBP-RNA recognition. In the absence of RNA, phosphorylated and unphosphorylated forms of the RNA binding and processing domain (RPD) of Drosophila SLBP (dSLBP) possess helical secondary structure but no well-defined tertiary fold. Drosophila SLBP is phosphorylated at four out of five potential serine or threonine sites in the sequence DTAKDSNSDSDSD at the extreme C-terminus, and phosphorylation at these sites is necessary for histone pre-mRNA processing. Here, we provide NMR evidence for serine phosphorylation of the C-terminus using (31)P direct-detect experiments and show that both serine phosphorylation and RNA binding are necessary for proper folding of the RPD. The electrostatic effect of protein phosphorylation can be partially mimicked by a mutant form of SLBP wherein four C-terminal serines are replaced with glutamic acids. Hence, both RNA binding and protein phosphorylation are necessary for stabilization of the SLBP RPD.     

Early evolution of histone mRNA 3' end processing

The replication-dependent histone mRNAs in metazoa are not polyadenylated, in contrast to the bulk of mRNA. Instead, they contain an RNA stem-loop (SL) structure close to the 3' end of the mature RNA, and this 3' end is generated by cleavage using a machinery involving the U7 snRNP and protein factors such as the stem-loop binding protein (SLBP). This machinery of 3' end processing is related to that of polyadenylation as protein components are shared between the systems. It is commonly believed that histone 3' end processing is restricted to metazoa and green algae. In contrast, polyadenylation is ubiquitous in Eukarya. However, using computational approaches, we have now identified components of histone 3' end processing in a number of protozoa. Thus, the histone mRNA stem-loop structure as well as the SLBP protein are present in many different protozoa, including Dictyostelium, alveolates, Trypanosoma, and Trichomonas. These results show that the histone 3' end processing machinery is more ancient than previously anticipated and can be traced to the root of the eukaryotic phylogenetic tree. We also identified histone mRNAs from both metazoa and protozoa that are polyadenylated but also contain the signals characteristic of histone 3' end processing. These results provide further evidence that some histone genes are regulated at the level of 3' end processing to produce either polyadenylated RNAs or RNAs with the 3' end characteristic of replication-dependent histone mRNAs.     

Nuclear export of metazoan replication-dependent histone mRNAs is dependent on RNA length and is mediated by TAP

Replication-dependent histone mRNAs are the only metazoan mRNAs that are not polyadenylated, ending instead in a conserved stem-loop sequence. Histone pre-mRNAs lack introns and are processed in the nucleus by a single cleavage step, which produces the mature 3' end of the mRNA. We have systematically examined the requirements for the nuclear export of a mouse histone mRNA using the Xenopus oocyte system. Histone mRNAs were efficiently exported when injected as mature mRNAs, demonstrating that the process of 3' end cleavage is not required for export factor binding. Export also does not depend on the stem-loop binding protein (SLBP) since mutations of the stem-loop that prevent SLBP binding and competition with a stem-loop RNA did not affect export. Only the length of the region upstream of the stem-loop, but not its sequence, was important for efficient export. Histone mRNA export was blocked by competition with constitutive transport element (CTE) RNA, indicating that the mRNA export receptor TAP is involved in histone mRNA export. Consistent with this observation, depletion of TAP from Drosophila cells by RNAi resulted in the restriction of mature histone mRNAs to the nucleus.     

ZFP100, a component of the active U7 snRNP limiting for histone pre-mRNA processing, is required for entry into S phase

Metazoan replication-dependent histone mRNAs are the only eukaryotic mRNAs that are not polyadenylated. The cleavage of histone pre-mRNA to form the unique 3' end requires the U7 snRNP and the stem-loop binding protein (SLBP) that binds the 3' end of histone mRNA. U7 snRNP contains three novel proteins, Lsm10 and Lsm11, which are part of the core U7 Sm complex, and ZFP100, a Zn finger protein that helps stabilize binding of the U7 snRNP to the histone pre-mRNA by interacting with the SLBP/pre-mRNA complex. Using a reporter gene that encodes a green fluorescent protein mRNA ending in a histone 3' end and mimics histone gene expression, we demonstrate that ZFP100 is the limiting factor for histone pre-mRNA processing in vivo. The overexpression of Lsm10 and Lsm11 increases the cellular levels of U7 snRNP but has no effect on histone pre-mRNA processing, while increasing the amount of ZFP100 increases histone pre-mRNA processing but has no effect on U7 snRNP levels. We also show that knocking down the known components of U7 snRNP by RNA interference results in a reduction in cell growth and an unsuspected cell cycle arrest in early G(1), suggesting that active U7 snRNP is necessary to allow progression through G(1) phase to S phase.     

A human histone H2B.1 variant gene, located on chromosome 1, utilizes alternative 3' end processing.

A variant human H2B histone gene (GL105), previously shown to encode a 2300 nt replication independent mRNA, has been cloned. We demonstrate this gene expresses alternative mRNAs regulated differentially during the HeLa S3 cell cycle. The H2B-Gl105 gene encodes both a 500 nt cell cycle dependent mRNA and a 2300 nt constitutively expressed mRNA. The 3' end of the cell cycle regulated mRNA terminates immediately following the region of hyphenated dyad symmetry typical of most histone mRNAs, whereas the constitutively expressed mRNA has a 1798 nt non-translated trailer that contains the same region of hyphenated dyad symmetry but is polyadenylated. The cap site for the H2B-GL105 mRNAs is located 42 nt upstream of the protein coding region. The H2B-GL105 histone gene was localized to chromosome region 1q21-1q23 by chromosomal in situ hybridization and by analysis of rodent-human somatic cell hybrids using an H2B-GL105 specific probe. The H2B-GL105 gene is paired with a functional H2A histone gene and this H2A/H2B gene pair is separated by a bidirectionally transcribed intergenic promoter region containing consensus TATA and CCAAT boxes and an OTF-1 element. These results demonstrate that cell cycle regulated and constitutively expressed histone mRNAs can be encoded by the same gene, and indicate that alternative 3' end processing may be an important mechanism for regulation of histone mRNA. Such control further increases the versatility by which cells can modulate the synthesis of replication-dependent as well as variant histone proteins during the cell cycle and at the onset of differentiation.