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W F Marzluff

Publications and source records attributed to W F Marzluff.

At least 19 recordsLinked to original sources
PubMed1996

The protein that binds the 3' end of histone mRNA: a novel RNA-binding protein required for histone pre-mRNA processing.

Replication-dependent histone mRNAs are not polyadenylated but end in a conserved 26-nucleotide structure that contains a stem-loop. Much of the cell cycle regulation of histone mRNA is post-transcriptional and is mediated by the 3' end of histone mRNA. The stem-loop binding protein (SLBP) that binds the 3' end of histone mRNA is a candidate for the factor that participates in most, if not all, of the post-transcriptional regulatory events. We have cloned the cDNA for the SLBP from humans, mice, and frogs, using the recently developed yeast three-hybrid system. The human SLBP is a 31-kD protein and contains a novel RNA-binding domain, which has been mapped to a 73-amino-acid region of the protein. The cloned SLBP is the protein bound to the 3' end of histone mRNA as antibodies specific for the SLBP remove all specific binding activity from nuclear and polyribosomal extracts. These depleted extracts do not cleave histone pre-mRNA efficiently, demonstrating that the SLBP is required for efficient histone pre-mRNA processing.

Amino Acid Sequence↗
PubMed1996

Increasing the distance between the snRNA promoter and the 3' box decreases the efficiency of snRNA 3'-end formation.

Chimeric genes which contained the mouse U1b snRNA promoter, portions of the histone H2a or globin coding regions and the U1b 3'-end followed by a histone 3'-end were constructed. The distance between the U1 promoter and the U1 3' box was varied between 146 and 670 nt. The chimeric genes were introduced into CHO cells by stable transfection or into Xenopus oocytes by microinjection. The efficiency of utilization of the U1 3' box, as measured by the relative amounts of transcripts that ended at the U1 3' box and the histone 3'-end, was dependent on the distance between the promoter and 3'-end box. U1 3'-ends were formed with >90% efficiency on transcripts shorter than 200 nt, with 50-70% efficiency on transcripts of 280-400 nt and with only 10-20% efficiency on transcripts >500 nt. Essentially identical results were obtained after stable transfection of CHO cells or after injecting the genes into Xenopus oocytes. The distance between the U1 promoter and the U1 3' box must be <280 nt for efficient transcription termination at the U1 3' box, regardless of the sequence transcribed.

Animals↗
PubMed1996

3' Processing and termination of mouse histone transcripts synthesized in vitro by RNA polymerase II.

The highly expressed mouse histone H2a-614 gene is located 800 nt 5' of the histone H3-614 gene. There is a 140 nt sequence located 500 nt from the end of the H2-614 mRNA which has been defined as a transcription termination site for RNA polymerase II. We established an in vitro transcription system in which both 3' end processing and transcription termination occur. A template containing the adenovirus major late promoter, a portion of the histone H2a-614 coding region, its 3' processing signal, followed by the transcription termination site was transcribed in a nuclear extract prepared from mouse myeloma cells. Some of the transcripts synthesized in the extract were cleaved at the histone processing site in a reaction which was dependent both on the hairpin binding factor and the U7 snRNP. The efficiency of histone 3' end formation was similar both on synthetic transcripts and transcripts synthesized by RNA polymerase II. Defined transcripts, which were not processed and which mapped to the transcription termination site, were released from the template, suggesting that they were formed by transcription termination. Termination in vitro was dependent on a functional histone processing signal.

Animals↗
PubMed1996

Efficient expression of protein coding genes from the murine U1 small nuclear RNA promoters.

Few promoters are active at high levels in all cells. Of these, the majority encode structural RNAs transcribed by RNA polymerases I or III and are not accessible for the expression of proteins. An exception are the small nuclear RNAs (snRNAs) transcribed by RNA polymerase II. Although snRNA biosynthesis is unique and thought not to be compatible with synthesis of functional mRNA, we have tested these promoters for their ability to express functional mRNAs. We have used the murine U1a and U1b snRNA gene promoters to express the Escherichia coli lacZ gene and the human alpha-globin gene from either episomal or integrated templates by transfection, or infection into a variety of mammalian cell types. Equivalent expression of beta-galactosidase was obtained from < 250 nucleotides of 5'-flanking sequence containing the complete promoter of either U1 snRNA gene or from the 750-nt cytomegalovirus promoter and enhancer regions. The mRNA was accurately initiated at the U1 start site, efficiently spliced and polyadenylylated, and localized to polyribosomes. Recombinant adenovirus containing the U1b-lacZ chimeric gene transduced and expressed beta-galactosidase efficiently in human 293 cells and airway epithelial cells in culture. Viral vectors containing U1 snRNA promoters may be an attractive alternative to vectors containing viral promoters for persistent high-level expression of therapeutic genes or proteins.

Animals↗
PubMed1996

The histone 3'-terminal stem-loop is necessary for translation in Chinese hamster ovary cells.

The metazoan cell cycle-regulated histone mRNAs are the only known cellular mRNAs that do not terminate in a poly(A) tall. Instead, mammalian histone mRNAs terminate in a highly conserved stem-loop structure which is required for 3'-end processing and regulates mRNA stability. The poly(A) tail not only regulates translational efficiency and mRNA stability but is required for the function of the cap in translation (m(7)GpppN). We show that the histone terminal stem-loop is functionally similar to a poly(A) tail in that it enhances translational efficiency and is co-dependent on a cap in order to establish an efficient level of translation. The histone stem-loop is sufficient and necessary to increase the translation of reporter mRNA in transfected Chinese hamster ovary cells but must be positioned at the 3'-terminus in order to function optimally. Mutations within the conserved stem or loop regions reduced its ability to facilitate translation. All histone mRNAs in higher plants are polyadenylated. The histone stem-loop did not function to influence translational efficiency or mRNA stability in plant protoplasts. These data demonstrate that the histone stem/loop directs efficient translation and that it is functionally analogous to a poly(A) tail.

Animals↗
PubMed1996

Structure of a cluster of mouse histone genes.

The structure of a 25 kilobase region of mouse DNA containing 6 functional histone genes and an H2a pseudogene has been determined. The sequences and levels of expression of the H3 and H2b gene as well as the sequence of the H2a pseudogene have been determined.

Animals↗
PubMed1996

Efficient extraction and partial purification of the polyribosome-associated stem-loop binding protein bound to the 3' end of histone mRNA.

Replication-dependent histone mRNAs end in a highly conserved stem-loop sequence rather than a polyA sequence. A 45-kDa stem-loop binding protein (SLBP), which specifically binds the stem-loop of histone mRNA, is present in both polyribosomes and nuclei. An identical 45-kDa protein, as determined by partial protease digestion, is cross-linked to a 30 nt RNA containing the 3' stem-loop from both nuclei and polyribosomes. The SLBP can also be detected by a Northwestern blot procedure using the 30 nt RNA as a probe. As judged from the Northwestern assay, more than 90% of the SLBP in the cell is found in the polyribosomes with the remaining SLBP localized to the nucleus. Only 5-10% of the SLBP could be extracted from the polyribosomes with salt. Treatment of the polyribosomes with micrococcal nuclease prior to salt extraction solubilized 5-10 times more SLBP as an RNA-protein complex. The SLBP could be subsequently partially purified from this complex.

Animals↗
PubMed1996

Characterization of the mouse histone gene cluster on chromosome 13: 45 histone genes in three patches spread over 1Mb.

The histone gene cluster on mouse chromosome 13 has been isolated and characterized. Using overlapping YAC clones containing histone genes from chromosome 13, a contig of approximately 2 Mb has been defined. It contains 45 histone genes, organized in three patches containing tightly clustered genes. An 80-kb patch (patch III) containing 12 histone genes is near one end of the contig, and a similar-sized patch (patch I) containing 15 histone genes is near the other end of the contig, located at least 500 kb from the central patch (patch II) of histone genes. The entire cluster contains six histone H1 genes, including the testis-specific histone H1t gene that maps to the middle of the cluster. All nine histone H3 genes in this cluster have been sequenced, and their level of expression determined. Each histone H3 gene is distinct, with five genes encoding the H3.2 protein subtype and four genes encoding the H3.1 protein. They are all expressed, with each histone H3 gene accounting for a small proportion of the total histone H3 mRNA.

Amino Acid Sequence↗
PubMed1996

Characterization of the 55-kb mouse histone gene cluster on chromosome 3.

The histone gene cluster on mouse chromosome 3 has been isolated as a series of overlapping P1 clones, covering 110-120 kb, by probing with the histone H3-614 gene that had been mapped previously to mouse chromosome 3. There are genes for 10 core histone proteins present in a 55-kb cluster within this contig. There are three histone H3 genes, two of which are identical; four histone H2a genes, two of which are identical, one histone H4 gene; and two histone H2b genes. These histone H3 and H2a genes encode approximately 40% of the total H3 and H2a mRNA, whereas the histone H4 and histone H2b genes encode < 10% of the total H4 and H2b mRNA. There are no histone H1 genes present in this cluster. All of the histone H2a genes encode histone H2a.2 proteins (or variants of H2a.2), and account for all the H2a.2 genes in the mouse genome. All three histone H3 genes encode the histone H3.2 protein. A 21-kb region containing the adjacent H3-614 and H2a-614 genes has been duplicated and is present in an inverted repeat separated by 4.5 kb. The other two H2a genes are adjacent, with the 3' ends of their mRNAs separated by only 49 nucleotides in the DNA and the U7 snRNP binding sites separated by only 20 nucleotides. One of the histone H2b genes has lost the stem-loop sequence characteristic of the replication-dependent histone mRNAs and encodes only polyadenylated mRNAs.

Amino Acid Sequence↗
PubMed1996

Common factors direct transcription through the proximal sequence elements (PSEs) of the embryonic sea urchin U1, U2, and U6 genes despite minimal similarity among the PSEs.

The proximal sequence element (PSE) for the sea urchin U6 small nuclear RNA gene has been defined. The most critical nucleotides for expression, located 61 to 64 nucleotides (nt) from the transcription start site, are 4 nt, AACT, at the 5' end of the PSE. Two nucleotide mutations in this region abolish transcription of the sea urchin U6 gene in vitro. The same two nucleotide mutations greatly reduce the binding of specific factors detected by an electrophoretic mobility shift assay. There is also a conserved AC dinucleotide 57 nt from the start site of the sea urchin U1 and U2 PSEs. The sea urchin U1 and U2 PSEs were substituted for the sea urchin U6 PSE, with the conserved AC sequences aligned with those of the U6 PSE. Both of these genes were expressed at levels higher than those observed with the wild-type U6 gene. Similar complexes are formed on the U1 and U2 PSEs, and formation of the complexes is inhibited efficiently by the U6 PSE. In addition, the E-box sequence present upstream of the PSE enhances U6 transcription from both the U1 and U2 PSEs. Finally, depletion of a nuclear extract with a DNA affinity column containing the U6 PSE sequence reduces expression of the U6 genes driven by the U6, U1, or U2 PSE but does not affect expression of the 5S rRNA gene. These data support the possibility that the same factor(s) interacts with the PSE sequences of the U1, U2, and U6 small nuclear RNA genes expressed in early sea urchin embryogenesis.

Animals↗
PubMed1995

The sequence of the stem and flanking sequences at the 3' end of histone mRNA are critical determinants for the binding of the stem-loop binding protein.

Complexes of different electrophoretic mobility containing the stem-loop binding protein, a 45 kDa protein, bound to the stem-loop at the 3' end of histone mRNA, are present in both nuclear and cytoplasmic extracts from mammalian cells. We have determined the effect of changes in the loop, in the stem and in the flanking sequences on the affinity of the SLBP for the 3' end of histone mRNA. The sequence of the stem is particularly critical for SLBP binding. Specific sequences both 5' and 3' of the stem-loop are also required for high-affinity binding. Expanding the four base loop by one or two uridines reduced but did not abolish SLBP binding. RNA footprinting experiments show that the flanking sequences on both sides of the stem-loop are critical for efficient binding, but that cleavages in the loop do not abolish binding. Thus all three regions of the RNA sequence contribute to SLBP binding, suggesting that the 26 nt at the 3' end of histone mRNA forms a defined tertiary structure recognized by the SLBP.

Animals↗
PubMed1995

The polyribosomal protein bound to the 3' end of histone mRNA can function in histone pre-mRNA processing.

Cell cycle-regulated histone mRNAs end in a conserved 26-nt sequence that can form a stem-loop with a six-base stem and a four-base loop. The 3' end of histone mRNA has distinct functions in the nucleus and in the cytoplasm. In the nucleus it functions in pre-mRNA processing and transport, whereas in the cytoplasm it functions in translation and regulation of histone mRNA stability. The stem-loop binding protein (SLBP), present in both nuclei and polyribosomes, is likely the trans-acting factor that binds to the 3' end of mature histone mRNA and mediates its function. A nuclear extract that efficiently processes histone pre-mRNA was prepared from mouse myeloma cells. The factor(s) that bind to the 3' end of histone mRNA can be depleted from this extract using a biotinylated oligonucleotide containing the conserved stem-loop sequence. Using this depleted extract which is deficient in histone pre-mRNA processing, we show that SLBP found in polyribosomes can restore processing, suggesting that SLBP associates with histone pre-mRNA in the nucleus, participates in processing, and then accompanies the mature mRNA to the cytoplasm.

Animals↗
PubMed1995

The polyribosomal protein bound to the 3' end of histone mRNA can function in histone pre-mRNA processing.

Histone mRNAs end in a conserved 26 nt sequence which can form a stem-loop with a six-base stem and a four base loop. The 3' end of histone mRNA functions in the nucleus in pre-mRNA processing and mRNA transport and in the cytoplasm in translation and regulation of histone mRNA stability. The stem-loop binding protein (SLBP), found in both the polyribosomes and the nucleus, binds to the 3' end of histone mRNA. A nuclear extract which efficiently processes histone pre-mRNA has been prepared from mouse myeloma cells. The factors which bind the 3' end of histone mRNA can be depleted from this extract using a biotinylated oligonucleotide. Using the depleted extract, we show that the SLBP found in the polyribosomes can function in histone pre-mRNA processing, suggesting that the SLBP associates with histone pre-mRNA in the nucleus and accompanies the mature mRNA to the cytoplasm.

Animals↗
PubMed1995

Formation and metabolism of histone mRNAs with mutant 3' ends formed by snRNA termination signals.

Histone mRNAs are the only non-polyadenylated mRNAs, ending in a conserved 26 nt sequence which can form a stem-loop. It has not been possible to make histone mRNAs with mutant stem-loops since most mutations in the stem-loop interfere with the 3' processing reaction. The snRNA genes transcribed by RNA polymerase II form their 3' ends by transcription termination directed by a signal which is located entirely 3' of the snRNA coding sequence. Chimeric genes which express RNAs ending in a histone 3' end or in mutant histone 3' ends formed by snRNA termination signals were constructed. The mRNAs from these genes were efficiently transported from the nucleus after injection of the genes into frog oocytes. This was true even for RNAs which end in mutant stem-loops suggesting that the snRNA termination signals promote transport of the transcripts.

Animals↗
PubMed1994

Changes in the stem-loop at the 3' terminus of histone mRNA affects its nucleocytoplasmic transport and cytoplasmic regulation.

The stem-loop structure at the 3' end of replication-dependent histone mRNA is required for efficient pre-mRNA processing, localization of histone mRNA to the polyribosomes, and regulation of histone mRNA degradation. A protein, the stem-loop binding protein (SLBP), binds the 3' end of histone mRNA and is thought to mediate some or all of these processes. A mutant histone mRNA with two nucleotide changes in the loop was constructed and found to be transported inefficiently to the cytoplasm. The mutant histone mRNA, unlike the wild-type histone mRNA, was not rapidly degraded when DNA synthesis is inhibited, and was not stabilized upon inhibition of protein synthesis. The stem-loop binding protein (SLBP) has between a 20-50 fold greater affinity for the wild type histone stem-loop structure than for the mutant stem-loop structure, suggesting that the alteration in the efficiency of transport and the normal degradation pathway in histone mRNA may be due to the reduced affinity of the mutant stem-loop for the SLBP.

Animals↗
PubMed1994

Selection on silent sites in the rodent H3 histone gene family.

Selection promoting differential use of synonymous codons has been shown for several unicellular organisms and for Drosophila, but not for mammals. Selection coefficients operating on synonymous codons are likely to be extremely small, so that a very large effective population size is required for selection to overcome the effects of drift. In mammals, codon-usage bias is believed to be determined exclusively by mutation pressure, with differences between genes due to large-scale variation in base composition around the genome. The replication-dependent histone genes are expressed at extremely high levels during periods of DNA synthesis, and thus are among the most likely mammalian genes to be affected by selection on synonymous codon usage. We suggest that the extremely biased pattern of codon usage in the H3 genes is determined in part by selection. Silent site G + C content is much higher than expected based on flanking sequence G + C content, compared to other rodent genes with similar silent site base composition but lower levels of expression. Dinucleotide-mediated mutation bias does affect codon usage, but the affect is limited to the choice between G and C in some fourfold degenerate codons. Gene conversion between the two clusters of histone genes has not been an important force in the evolution of the H3 genes, but gene conversion appears to have had some effect within the cluster on chromosome 13.

Animals↗