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.     

Structure of the histone mRNA hairpin required for cell cycle regulation of histone gene expression

Expression of replication-dependent histone genes requires a conserved hairpin RNA element in the 3' untranslated regions of poly(A)-less histone mRNAs. The 3' hairpin element is recognized by the hairpin-binding protein or stem-loop-binding protein (HBP/SLBP). This protein-RNA interaction is important for the endonucleolytic cleavage generating the mature mRNA 3' end. The 3' hairpin and presumably HBP/SLBP are also required for nucleocytoplasmic transport, translation, and stability of histone mRNAs. RNA 3' processing and mRNA stability are both regulated during the cell cycle. Here, we have determined the three-dimensional structure of a 24-mer RNA comprising a mammalian histone RNA hairpin using heteronuclear multidimensional NMR spectroscopy. The hairpin adopts a novel UUUC tetraloop conformation that is stabilized by base stacking involving the first and third loop uridines and a closing U-A base pair, and by hydrogen bonding between the first and third uridines in the tetraloop. The HBP interaction of hairpin RNA variants was analyzed in band shift experiments. Particularly important interactions for HBP recognition are mediated by the closing U-A base pair and the first and third loop uridines, whose Watson-Crick functional groups are exposed towards the major groove of the RNA hairpin. The results obtained provide novel structural insight into the interaction of the histone 3' hairpin with HBP, and thus the regulation of histone mRNA metabolism.     

The stem-loop structure at the 3'' end of histone mRNA is necessary and sufficient for regulation of histone mRNA stability.

Chimeric genes were made by fusing mouse histone genes with a human alpha-globin gene. The genes were introduced into mouse L cells and the stability of the chimeric mRNAs was measured when DNA synthesis was inhibited. An mRNA containing all the globin coding sequences and the last 30 nucleotides of the histone mRNA was degraded at the same rate as histone mRNA.     

Arabidopsis histone methyltransferase SET DOMAIN GROUP2 is required for regulation of various hormone responsive genes

Histone modifications are known to play important roles in plant development through epigenetic regulation of gene expression. How these modifications regulate downstream targets in response to various environmental cues and developmental stimuli is still largely unknown. Here, we provide evidence that Arabidopsis histone H3K4 methyltransferase SET DOMAIN GROUP2 (SDG2) is required for full activation of hormone responsive genes upon hormone treatment. The pleiotropic phenotypes of sdg2 were closely related to those of auxin deficient mutants and RNA analysis revealed that expression of early hormone responsive genes was significantly reduced in sdg2-5. By ChIP analyses we found that H3K4 tri-methylations on chromatin region of hormone responsive genes such as SAUR27, KIN1 and GASA6 were enriched in WT upon hormone treatments whereas these enrichments were largely abolished in sdg2-5. After hormone treatment, chromatin regions of responsive genes that accumulated H3K4me3 in WT overlapped with those displaying decreased H3K4me3 levels in sdg2-5. Histone H3K4 di-methylation levels on tested genes were increased rather than decreased in sdg2-5, suggesting that SDG2 mediates transition of H3K4me2 to H3K4me3. Taken together, we conclude that the SDG2 activity is required to regulate the expression of hormone responsive genes via histone H3K4 tri-methylation.     

Drosophila stem-loop binding protein intracellular localization is mediated by phosphorylation and is required for cell cycle-regulated histone mRNA expression

Stem-loop binding protein (SLBP) is an essential component of the histone pre-mRNA processing machinery. SLBP protein expression was examined during Drosophila development by using transgenes expressing hemagglutinin (HA) epitope-tagged proteins expressed from the endogenous Slbp promoter. Full-length HA-dSLBP complemented a Slbp null mutation, demonstrating that it was fully functional. dSLBP protein accumulates throughout the cell cycle, in contrast to the observed restriction of mammalian SLBP to S phase. dSLBP is located in both nucleus and cytoplasm in replicating cells, but it becomes predominantly nuclear during G2. dSLBP is present in mitotic cells and is down-regulated in G1 when cells exit the cell cycle. We determined whether mutation at previously identified phosphorylation sites, T120 and T230, affected the ability of the protein to restore viability and histone mRNA processing to dSLBP null mutants. The T120A SLBP restored viability and histone pre-mRNA processing. However, the T230A mutant, located in a conserved TPNK sequence in the RNA binding domain, did not restore viability and histone mRNA processing in vivo, although it had full activity in histone mRNA processing in vitro. The T230A protein is concentrated in the cytoplasm, suggesting that it is defective in nuclear targeting, and accounting for its failure to function in histone pre-mRNA processing in vivo.     

The stem-loop binding protein is required for efficient translation of histone mRNA in vivo and in vitro

Metazoan replication-dependent histone mRNAs end in a conserved stem-loop rather than in the poly(A) tail found on all other mRNAs. The 3' end of histone mRNA binds a single class of proteins, the stem-loop binding proteins (SLBP). In Xenopus, there are two SLBPs: xSLBP1, the homologue of the mammalian SLBP, which is required for processing of histone pre-mRNA, and xSLBP2, which is expressed only during oogenesis and is bound to the stored histone mRNA in Xenopus oocytes. The stem-loop is required for efficient translation of histone mRNAs and substitutes for the poly(A) tail, which is required for efficient translation of other eucaryotic mRNAs. When a rabbit reticulocyte lysate is programmed with uncapped luciferase mRNA ending in the histone stem-loop, there is a three- to sixfold increase in translation in the presence of xSLBP1 while xSLBP2 has no effect on translation. Neither SLBP affected the translation of a luciferase mRNA ending in a mutant stem-loop that does not bind SLBP. Capped luciferase mRNAs ending in the stem-loop were injected into Xenopus oocytes after overexpression of either xSLBP1 or xSLBP2. Overexpression of xSLBP1 in the oocytes stimulated translation, while overexpression of xSLBP2 reduced translation of the luciferase mRNA ending in the histone stem-loop. A small region in the N-terminal portion of xSLBP1 is required to stimulate translation both in vivo and in vitro. An MS2-human SLBP1 fusion protein can activate translation of a reporter mRNA ending in an MS2 binding site, indicating that xSLBP1 only needs to be recruited to the 3' end of the mRNA but does not need to be directly bound to the histone stem-loop to activate translation.     

Dexamethasone-induced selenoprotein S degradation is required for adipogenesis

Although adipogenesis is associated with induction of endoplasmic reticulum (ER) stress, the role of selenoprotein S (SEPS1), an ER resident selenoprotein known to regulate ER stress and ER-associated protein degradation, is unknown. We found an inverse relationship between SEPS1 level in adipose tissue and adiposity in mice. While SEPS1 expression was increased during adipogenesis, a markedly reduced SEPS1 protein level was found in the early phase of adipogenesis due to dexamethasone (DEX)-induced proteosomal degradation of SEPS1. Overexpression of SEPS1 in the early phase of cell differentiation resulted in impairment of adipogenesis with reduced levels of CCAAT/enhancer binding protein α and other adipocyte marker genes during the course of adipogenesis. Conversely, knockdown of SEPS1 resulted in the promotion of adipogenesis. Additionally, altered SEPS1 expression was associated with changes in expression of ER stress marker genes in the early phase of adipogenesis, and ubiquitin-proteasome system (UPS)-related ubiquitination and proteasome function. Our study reveals that SEPS1 is a novel anti-adipogenic selenoprotein that modulates ER stress- and UPS-dependent adipogenesis. Our results also identifies a novel function of DEX in the regulation of adipogenesis through induction of SEPS1 degradation. Taken together, DEX-dependent degradation of SEPS1 in the early phase of adipogenesis is necessary for initiating ER stress- and UPS-dependent maturation of adipocytes.     

The histone methyltransferase SET8 is required for S-phase progression

Chromatin structure and function is influenced by histone posttranslational modifications. SET8 (also known as PR-Set7 and SETD8) is a histone methyltransferase that monomethylates histonfe H4-K20. However, a function for SET8 in mammalian cell proliferation has not been determined. We show that small interfering RNA inhibition of SET8 expression leads to decreased cell proliferation and accumulation of cells in S phase. This is accompanied by DNA double-strand break (DSB) induction and recruitment of the DNA repair proteins replication protein A, Rad51, and 53BP1 to damaged regions. SET8 depletion causes DNA damage specifically during replication, which induces a Chk1-mediated S-phase checkpoint. Furthermore, we find that SET8 interacts with proliferating cell nuclear antigen through a conserved motif, and SET8 is required for DNA replication fork progression. Finally, codepletion of Rad51, an important homologous recombination repair protein, abrogates the DNA damage after SET8 depletion. Overall, we show that SET8 is essential for genomic stability in mammalian cells and that decreased expression of SET8 results in DNA damage and Chk1-dependent S-phase arrest.     

Physical evidence for cotranslational regulation of beta-tubulin mRNA degradation.

Tubulin synthesis is controlled by an autoregulatory mechanism through which an increase in the intracellular concentration of tubulin subunits leads to specific degradation of tubulin mRNAs. The sequence necessary and sufficient for the selective degradation of a beta-tubulin mRNA in response to changes in the level of free tubulin subunits resides within the first 13 translated nucleotides that encode the amino-terminal sequence of beta-tubulin, Met-Arg-Glu-Ile (MREI). Previous results have suggested that the sequence responsible for autoregulation resides in the nascent peptide rather than in the mRNA per se, raising the possibility that the regulation of the stability of tubulin mRNA is mediated through binding of tubulin or some other cellular factor to the nascent amino-terminal tubulin peptide. We now show that this putative cotranslational interaction is not mediated by tubulin alone, as no meaningful binding is detectable between tubulin subunits and the amino-terminal beta-tubulin polypeptide. However, microinjection of a monoclonal antibody that binds to the beta-tubulin nascent peptide selectively disrupts the regulation of beta-tubulin, but not alpha-tubulin, synthesis. This finding provides direct evidence for cotranslational degradation of beta-tubulin mRNA mediated through binding of one or more cellular factors to the beta-tubulin nascent peptide.     

FLASH is required for the endonucleolytic cleavage of histone pre-mRNAs but is dispensable for the 5' exonucleolytic degradation of the downstream cleavage product

3'-end cleavage of histone pre-mRNAs is catalyzed by CPSF-73 and requires the interaction of two U7 snRNP-associated proteins, FLASH and Lsm11. Here, by using scanning mutagenesis we identify critical residues in human FLASH and Lsm11 that are involved in the interaction between these two proteins. We also demonstrate that mutations in the region of FLASH located between amino acids 50 and 99 do not affect binding of Lsm11. Interestingly, these mutations convert FLASH into an inhibitory protein that reduces in vitro processing efficiency of highly active nuclear extracts. Our results suggest that this region in FLASH in conjunction with Lsm11 is involved in recruiting a yet-unknown processing factor(s) to histone pre-mRNA. Following endonucleolytic cleavage of histone pre-mRNA, the downstream cleavage product (DCP) is degraded by the 5'-3' exonuclease activity of CPSF-73, which also depends on Lsm11. Strikingly, while cleavage of histone pre-mRNA is stimulated by FLASH and inhibited by both dominant negative mutants of FLASH and anti-FLASH antibodies, the 5'-3' degradation of the DCP is not affected. Thus, the recruitment of FLASH to the processing complex plays a critical role in activating the endonuclease mode of CPSF-73 but is dispensable for its 5'-3' exonuclease activity. These results suggest that CPSF-73, the catalytic component in both reactions, can be recruited to histone pre-mRNA largely in a manner independent of FLASH, possibly by a separate domain in Lsm11.     

hMOF histone acetyltransferase is required for histone H4 lysine 16 acetylation in mammalian cells

Reversible histone acetylation plays an important role in regulation of chromatin structure and function. Here, we report that the human orthologue of Drosophila melanogaster MOF, hMOF, is a histone H4 lysine K16-specific acetyltransferase. hMOF is also required for this modification in mammalian cells. Knockdown of hMOF in HeLa and HepG2 cells causes a dramatic reduction of histone H4K16 acetylation as detected by Western blot analysis and mass spectrometric analysis of endogenous histones. We also provide evidence that, similar to the Drosophila dosage compensation system, hMOF and hMSL3 form a complex in mammalian cells. hMOF and hMSL3 small interfering RNA-treated cells also show dramatic nuclear morphological deformations, depicted by a polylobulated nuclear phenotype. Reduction of hMOF protein levels by RNA interference in HeLa cells also leads to accumulation of cells in the G(2) and M phases of the cell cycle. Treatment with specific inhibitors of the DNA damage response pathway reverts the cell cycle arrest caused by a reduction in hMOF protein levels. Furthermore, hMOF-depleted cells show an increased number of phospho-ATM and gammaH2AX foci and have an impaired repair response to ionizing radiation. Taken together, our data show that hMOF is required for histone H4 lysine 16 acetylation in mammalian cells and suggest that hMOF has a role in DNA damage response during cell cycle progression.