Biomedical subjects

William F Marzluff

Publications and source records attributed to William F Marzluff.

At least 19 recordsLinked to original sources

PubMed2006

Genetic and biochemical characterization of Drosophila Snipper: A promiscuous member of the metazoan 3'hExo/ERI-1 family of 3' to 5' exonucleases.

The DnaQ-H family exonuclease Snipper (Snp) is a 33-kDa Drosophila melanogaster homolog of 3'hExo and ERI-1, exoribonucleases implicated in the degradation of histone mRNA in mammals and in the negative regulation of RNA interference (RNAi) in Caenorhabditis elegans, respectively. In metazoans, Snp, Exod1, 3'hExo, ERI-1, and the prpip nucleases define a new subclass of structure-specific 3'-5' exonucleases that bind and degrade double-stranded RNA and/or DNA substrates with 3' overhangs of 2-5 nucleotides (nt) in the presence of Mg2+ with no apparent sequence specificity. These nucleases are also capable of degrading linear substrates. Snp efficiently degrades structured RNA and DNA substrates as long as there exists a minimum 3' overhang of 2 nt to initiate degradation. We identified a Snp mutant and used it to test whether Snp plays a role in regulating histone mRNA degradation or RNAi in vivo. Snp mutant flies are viable, and display no obvious developmental abnormalities. The expression pattern and level of histone H3 mRNA in Snp mutant embryos and third instar imaginal eye discs was indistinguishable from wild type, suggesting that Snp does not play a significant role in the turnover of histone mRNA at the end of the S phase. The loss of Snp was also unable to enhance the silencing capability of two different RNAi transgenes targeting the white and yellow genes, suggesting that Snp does not negatively modulate RNAi. Therefore, Snp is a nonessential exonuclease that is not a functional ortholog of either 3'hExo or ERI-1.

Amino Acid Sequence↗

PubMed2006

The genomic repertoire for cell cycle control and DNA metabolism in S. purpuratus.

A search of the Strongylocentrotus purpuratus genome for genes associated with cell cycle control and DNA metabolism shows that the known repertoire of these genes is conserved in the sea urchin, although with fewer family members represented than in vertebrates, and with some cases of echinoderm-specific gene diversifications. For example, while homologues of the known cyclins are mostly encoded by single genes in S. purpuratus (unlike vertebrates, which have multiple isoforms), there are additional genes encoding novel cyclins of the B and K/L types. Almost all known cyclin-dependent kinases (CDKs) or CDK-like proteins have an orthologue in S. purpuratus; CDK3 is one exception, whereas CDK4 and 6 are represented by a single homologue, referred to as CDK4. While the complexity of the two families of mitotic kinases, Polo and Aurora, is close to that found in the nematode, the diversity of the NIMA-related kinases (NEK proteins) approaches that of vertebrates. Among the nine NEK proteins found in S. purpuratus, eight could be assigned orthologues in vertebrates, whereas the ninth is unique to sea urchins. Most known DNA replication, DNA repair and mitotic checkpoint genes are also present, as are homologues of the pRB (two) and p53 (one) tumor suppressors. Interestingly, the p21/p27 family of CDK inhibitors is represented by one homologue, whereas the INK4 and ARF families of tumor suppressors appear to be absent, suggesting that these evolved only in vertebrates. Our results suggest that, while the cell cycle control mechanisms known from other animals are generally conserved in sea urchin, parts of the machinery have diversified within the echinoderm lineage. The set of genes uncovered in this analysis of the S. purpuratus genome should enhance future research on cell cycle control and developmental regulation in this model.

Amino Acid Sequence↗

PubMed2006

The sea urchin histone gene complement.

The only eukaryotic mRNAs that are not polyadenylated are the replication-dependent histone mRNAs in metazoans. The sea urchin genome contains two sets of histone genes that encode non-polyadenylated mRNAs. One of these sets is a tandemly repeated gene cluster with a 5.6-kb repeat unit containing one copy of each of the five alpha-histone genes and is present as a single large cluster which spans over 1 Mb. There is a second set of genes, consisting of 39 genes, containing two histone H1 genes, 34 genes encoding core histone proteins (H2a, H2b, H3 and H4) and three genes expressed only in the testis. Unlike vertebrates where these genes are clustered, the sea urchin late histone genes, expressed in embryos, larvae and adults, are dispersed throughout the genome. There are also genes encoding polyadenylated histone mRNAs, which encode histone variants, including all variants found in other metazoans, as well as a unique set of five cleavage stage histone proteins expressed in oocytes. The cleavage stage histone H1 is the orthologue of an oocyte-specific histone H1 protein found in vertebrates.

Animals↗

PubMed2006

Genome-wide analysis of mRNAs bound to the histone stem-loop binding protein.

The replication-dependent histone mRNAs are cell-cycle-regulated and expressed only during S phase. In contrast to all other eukaryotic mRNAs, the histone mRNAs end in a highly conserved 16-nucleotide stem-loop rather than a poly(A) tail. The stem-loop is necessary and sufficient for the post-transcriptional regulation of histone mRNA during the cell cycle. The histone mRNA 3' stem-loop is bound by the stem-loop binding protein (SLBP) that is involved in pre-mRNA processing, translation, and stability of histone mRNA. Immunoprecipitation (IP) of RNA-binding proteins (RBPs) followed by microarray analysis has been used to identify the targets of RNA-binding proteins. This method is sometimes referred to as RIP-Chip (RNA IP followed by microarray analysis). Here we introduce a variation on the RIP-Chip method that uses a recombinant RBP to identify mRNA targets in a pool of total RNA; we call this method recombinant, or rRIP-Chip. Using this method, we show that recombinant SLBP binds exclusively to all five classes of histone mRNA. We also analyze the messages bound to the endogenous SLBP on polyribosomes by immunoprecipitation. We use two different microarray platforms to identify enriched mRNAs. Both platforms demonstrate remarkable specificity and consistency of results. Our data suggest that the replication-dependent histone mRNAs are likely to be the sole target of SLBP.

Base Sequence↗

PubMed2006

Characterization of 3'hExo, a 3' exonuclease specifically interacting with the 3' end of histone mRNA.

The 3' end of mammalian histone mRNAs consisting of a conserved stem-loop and a terminal ACCCA interacts with a recently identified human 3' exonuclease designated 3'hExo. The sequence-specific interaction suggests that 3'hExo may participate in the degradation of histone mRNAs. ERI-1, a Caenorhabditis elegans homologue of 3'hExo, has been implicated in degradation of small interfering RNAs. We introduced a number of mutations to 3'hExo to identify residues required for RNA binding and catalysis. To assure that the introduced mutations specifically target one of these two activities of 3'hExo rather than cause global structural defects, the mutant proteins were tested in parallel for the ability both to bind the stem-loop RNA and to degrade RNA substrates. Our analysis confirms that 3'hExo is a member of the DEDDh family of 3' exonucleases. Specific binding to the RNA requires the SAP domain and two lysines located immediately to its C terminus. 3'hExo binds with the highest affinity to the wild-type 3' end of histone mRNA, and any changes to this sequence reduce efficiency of binding. 3'hExo has only residual, if any, 3' exonuclease activity on DNA substrates and localizes mostly to the cytoplasm, suggesting that in vivo it performs exclusively RNA-specific functions. Efficient degradation of RNA substrates by 3'hExo requires 2' and 3' hydroxyl groups at the last nucleotide. 3'hExo removes 3' overhangs of small interfering RNAs, whereas the double-stranded region is resistant to the enzymatic activity.

Amino Acid Sequence↗

PubMed2006

A Golgi-localized hexose transporter is involved in heterotrimeric G protein-mediated early development in Arabidopsis.

Signal transduction involving heterotrimeric G proteins is universal among fungi, animals, and plants. In plants and fungi, the best understood function for the G protein complex is its modulation of cell proliferation and one of several important signals that are known to modulate the rate at which these cells proliferate is D-glucose. Arabidopsis thaliana seedlings lacking the beta subunit (AGB1) of the G protein complex have altered cell division in the hypocotyl and are D-glucose hypersensitive. With the aim to discover new elements in G protein signaling, we screened for gain-of-function suppressors of altered cell proliferation during early development in the agb1-2 mutant background. One agb1-2-dependent suppressor, designated sgb1-1(D) for suppressor of G protein beta1 (agb1-2), restored to wild type the altered cell division in the hypocotyl and sugar hypersensitivity of the agb1-2 mutant. Consistent with AGB1 localization, SGB1 is found at the highest steady-state level in tissues with active cell division, and this level increases in hypocotyls when grown on D-glucose and sucrose. SGB1 is shown here to be a Golgi-localized hexose transporter and acts genetically with AGB1 in early seedling development.

Alleles↗

PubMed2006

Nuclear autoantigenic sperm protein (NASP), a linker histone chaperone that is required for cell proliferation.

A multichaperone nucleosome-remodeling complex that contains the H1 linker histone chaperone nuclear autoantigenic sperm protein (NASP) has recently been described. Linker histones (H1) are required for the proper completion of normal development, and NASP transports H1 histones into nuclei and exchanges H1 histones with DNA. Consequently, we investigated whether NASP is required for normal cell cycle progression and development. We now report that without sufficient NASP, HeLa cells and U2OS cells are unable to replicate their DNA and progress through the cell cycle and that the NASP(-/-) null mutation causes embryonic lethality. Although the null mutation NASP(-/-) caused embryonic lethality, null embryos survive until the blastocyst stage, which may be explained by the presence of stored NASP protein in the cytoplasm of oocytes. We conclude from this study that NASP and therefore the linker histones are key players in the assembly of chromatin after DNA replication.

Animals↗

PubMed2006

Conserved zinc fingers mediate multiple functions of ZFP100, a U7snRNP associated protein.

Formation of the 3' end of replication-dependent histone mRNAs is most robust during S phase and is mediated by both the stem-loop binding protein (SLBP) and the U7 snRNP. We previously identified a 100-kDa zinc finger protein (ZFP100) as a component of U7 snRNP that interacts with the SLBP/pre-mRNA complex. Here, we show that myc- or GFP-tagged ZFP100 overexpressed after transfection is concentrated in Cajal bodies (CBs), and unlike components of the spliceosomal snRNPs, photobleaching experiments demonstrate that ZFP100 is stably associated with CBs. Of the 18 zinc fingers contained within ZFP100, the region encompassing fingers 2-6 is sufficient to maintain CB localization. Zn fingers 5-10 are required for maximal binding of ZFP100 to a 20-amino-acid region of Lsm11, a U7 snRNP core protein. Expression of ZFP100 stimulates histone mRNA processing in vivo, assayed by activation of a reporter gene that encodes a GFP mRNA ending in a histone 3' end. Importantly, the domain that is required for CB localization and Lsm11 binding is also sufficient to stimulate histone pre-mRNA processing in vivo. Comparisons with other mammalian ZFP100 orthologs show that the central Zn fingers sufficient for in vivo activity are most highly conserved, whereas the number and sequence of the Zn fingers in the N- and C-terminal domains vary.

Amino Acid Sequence↗

PubMed2006

Combined top-down and bottom-up proteomics identifies a phosphorylation site in stem-loop-binding proteins that contributes to high-affinity RNA binding.

The stem-loop-binding protein (SLBP) is involved in multiple aspects of histone mRNA metabolism. To characterize the modification status and sites of SLBP, we combined mass spectrometric bottom-up (analysis of peptides) and top-down (analysis of intact proteins) proteomic approaches. Drosophilia SLBP is heavily phosphorylated, containing up to seven phosphoryl groups. Accurate M(r) determination by Fourier transform ion cyclotron resonance (FTICR)-MS and FTICR-MS top-down experiments using a variety of dissociation techniques show there is removal of the initiator methionine and acetylation of the N terminus in the baculovirus-expressed protein, and that T230 is stoichiometrically phosphorylated. T230 is highly conserved; we have determined that this site is also completely phosphorylated in baculovirus-expressed mammalian SLBP and extensively phosphorylated in both Drosophila and mammalian cultured cells. Removal of the phosphoryl group from T230 by either dephosphorylation or mutation results in a 7-fold reduction in the affinity of SLBP for the stem-loop RNA.

Amino Acid Motifs↗

PubMed2006

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.

Animals↗

PubMed2006

U7 snRNA mutations in Drosophila block histone pre-mRNA processing and disrupt oogenesis.

Metazoan replication-dependent histone mRNAs are not polyadenylated, and instead terminate in a conserved stem-loop structure generated by an endonucleolytic cleavage involving the U7 snRNP, which interacts with histone pre-mRNAs through base-pairing between U7 snRNA and a purine-rich sequence in the pre-mRNA located downstream of the cleavage site. Here we generate null mutations of the single Drosophila U7 gene and demonstrate that U7 snRNA is required in vivo for processing all replication-associated histone pre-mRNAs. Mutation of U7 results in the production of poly A+ histone mRNA in both proliferating and endocycling cells because of read-through to cryptic polyadenylation sites found downstream of each Drosophila histone gene. A similar molecular phenotype also results from mutation of Slbp, which encodes the protein that binds the histone mRNA 3' stem-loop. U7 null mutants develop into sterile males and females, and these females display defects during oogenesis similar to germ line clones of Slbp null cells. In contrast to U7 mutants, Slbp null mutations cause lethality. This may reflect a later onset of the histone pre-mRNA processing defect in U7 mutants compared to Slbp mutants, due to maternal stores of U7 snRNA. A double mutant combination of a viable, hypomorphic Slbp allele and a viable U7 null allele is lethal, and these double mutants express polyadenylated histone mRNAs earlier in development than either single mutant. These data suggest that SLBP and U7 snRNP cooperate in the production of histone mRNA in vivo, and that disruption of histone pre-mRNA processing is detrimental to development.

Animals↗

PubMed2005

Differences and similarities between Drosophila and mammalian 3' end processing of histone pre-mRNAs.

We used nuclear extracts from Drosophila Kc cells to characterize 3' end processing of Drosophila histone pre-mRNAs. Drosophila SLBP plays a critical role in recruiting the U 7 snRNP to the pre-mRNA and is essential for processing all five Drosophila histone pre-mRNAs. The Drosophila processing machinery strongly prefers cleavage after a fourth nucleotide following the stem-loop and favors an adenosine over pyrimidines in this position. Increasing the distance between the stem-loop and the HDE does not result in a corresponding shift of the cleavage site, suggesting that in Drosophila processing the U 7 snRNP does not function as a molecular ruler. Instead, SLBP directs the cleavage site close to the stem-loop. The upstream cleavage product generated in Drosophila nuclear extracts contains a 3' OH, and the downstream cleavage product is degraded by a nuclease dependent on the U 7 snRNP, suggesting that the cleavage factor has been conserved between Drosophila and mammalian processing. A 2'O-methyl oligonucleotide complementary to the first 17 nt of the Drosophila U 7 snRNA was not able to deplete the U 7 snRNP from Drosophila nuclear extracts, suggesting that the 5' end of the Drosophila U 7 snRNA is inaccessible. This oligonucleotide selectively inhibited processing of only two Drosophila pre-mRNAs and had no effect on processing of the other three pre-mRNAs. Together, these studies demonstrate that although Drosophila and mammalian histone pre-mRNA processing share common features, there are also significant differences, likely reflecting divergence in the mechanism of 3' end processing between vertebrates and invertebrates.

Animals↗

PubMed2005

The polyadenylation factor CPSF-73 is involved in histone-pre-mRNA processing.

During 3' end processing, histone pre-mRNAs are cleaved 5 nucleotides after a conserved stem loop by an endonuclease dependent on the U7 small nuclear ribonucleoprotein (snRNP). The upstream cleavage product corresponds to the mature histone mRNA, while the downstream product is degraded by a 5'-3' exonuclease, also dependent on the U7 snRNP. To identify the two nuclease activities, we carried out UV-crosslinking studies using both the complete RNA substrate and the downstream cleavage product, each containing a single radioactive phosphate and a phosphorothioate modification at the cleavage site. We detected a protein migrating at 85 kDa that crosslinked to each substrate in a U7-dependent manner. Immunoprecipitation experiments identified this protein as CPSF-73, a known component of the cleavage/polyadenylation machinery. These studies suggest that CPSF-73 is both the endonuclease and 5'-3' exonuclease in histone-pre-mRNA processing and reveal an evolutionary link between 3' end formation of histone mRNAs and polyadenylated mRNAs.

Animals↗

PubMed2005

The stem-loop binding protein regulates translation of histone mRNA during mammalian oogenesis.

Although messenger RNAs encoding the histone proteins are among the most abundant in mammalian oocytes, the mechanism regulating their translation has not been identified. The stem-loop binding protein (SLBP) binds to a highly conserved sequence in the 3'-untranslated region (utr) of the non-polyadenylated histone mRNAs in somatic cells and mediates their stabilization and translation. We previously showed that SLBP, which is expressed only during S-phase of proliferating cells, is expressed in growing oocytes at G2 of the cell cycle and accumulates substantially during meiotic maturation. We report here that elevating the amount of SLBP in immature (G2) oocytes is sufficient to increase translation of a reporter mRNA bearing the histone 3'-utr and endogenous histone synthesis and that this effect is not mediated through increased stability of the encoding mRNAs. We further report that translation of the reporter mRNA increases dramatically during meiotic maturation coincident with the accumulation of SLBP. Conversely, when SLBP accumulation during maturation is prevented using RNA interference, both translation of the reporter mRNA and synthesis of endogenous histones are significantly reduced. This effect is not mediated by a loss of the encoding mRNAs. Moreover, following fertilization, SLBP-depleted oocytes also show a significant decrease in pronuclear size and in the amount of acetylated histone detectable on the chromatin. These results demonstrate that histone synthesis in immature and maturing oocytes is governed by a translational control mechanism that is directly regulated by changes in the amount of SLBP.

3' Untranslated Regions↗

PubMed2005

Regulated degradation of replication-dependent histone mRNAs requires both ATR and Upf1.

Eukaryotic cells coordinately regulate histone and DNA synthesis. In mammalian cells, most of the regulation of histone synthesis occurs post-transcriptionally by regulating the concentrations of histone mRNA. As cells enter S phase, histone mRNA levels increase, and at the end of S phase they are rapidly degraded. Moreover, inhibition of DNA synthesis causes rapid degradation 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 the only cis-acting element required for coupling regulation of histone mRNA half-life with DNA synthesis. Here we show that regulated degradation of histone mRNAs requires Upf1, a key regulator of the nonsense-mediated decay pathway, and ATR, a key regulator of the DNA damage checkpoint pathway activated during replication stress.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2005

Nuclear import of the stem-loop binding protein and localization during the cell cycle.

A key factor involved in the processing of histone pre-mRNAs in the nucleus and translation of mature histone mRNAs in the cytoplasm is the stem-loop binding protein (SLBP). In this work, we have investigated SLBP nuclear transport and subcellular localization during the cell cycle. SLBP is predominantly nuclear under steady-state conditions and localizes to the cytoplasm during S phase when histone mRNAs accumulate. Consistently, SLBP mutants that are defective in histone mRNA binding remain nuclear. As assayed in heterokaryons, export of SLBP from the nucleus is dependent on histone mRNA binding, demonstrating that SLBP on its own does not possess any nuclear export signals. We find that SLBP interacts with the import receptors Impalpha/Impbeta and Transportin-SR2. Moreover, complexes formed between SLBP and the two import receptors are disrupted by RanGTP. We have further shown that SLBP is imported by both receptors in vitro. Three sequences in SLBP required for Impalpha/Impbeta binding were identified. Simultaneous mutation of all three sequences was necessary to abolish SLBP nuclear localization in vivo. In contrast, we were unable to identify an in vivo role for Transportin-SR2 in SLBP nuclear localization. Thus, only the Impalpha/Impbeta pathway contributes to SLBP nuclear import in HeLa cells.

Alanine↗

PubMed2005

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.

Active Transport, Cell Nucleus↗

PubMed2005

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.

3' Untranslated Regions↗