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F M Rottman

Publications and source records attributed to F M Rottman.

At least 19 recordsLinked to original sources
PubMed2000

Mapping the SF2/ASF binding sites in the bovine growth hormone exonic splicing enhancer.

Splicing of the last intron (intron D) of the bovine growth hormone pre-mRNA requires the presence of a downstream exonic splicing enhancer (ESE). This enhancer is contained within a 115-nucleotide FspI-PvuII (FP) fragment located in the middle of the last exon (exon 5). Previous work showed that the splicing factor SF2/ASF binds to this FP region and stimulates splicing of intron D in vitro. However, the precise sequences recognized by SF2/ASF within the FP region had not been determined. Here we used multiple strategies to map the SF2/ASF binding sites and determine their importance for ESE function. Taking advantage of the fact that SF2/ASF ultraviolet (UV) cross-links specifically to RNA containing the FP sequence, we first mapped a major SF2/ASF binding site by UV cross-linking and reverse transcription. This strategy identified a 29-nucleotide SF2/ASF binding region in the middle of the FP sequence containing the 7-nucleotide purine-rich motif described previously. Interestingly, this binding region is neither sufficient, nor absolutely required for SF2/ASF-mediated splicing, suggesting that additional SF2/ASF binding sites are present. The location of these additional sites was determined by electrophoretic mobility shift analysis of various subfragments of the FP sequence. Antisense 2'-O-methyl oligoribonucleotides complementary to selected SF2/ASF binding sites block bovine growth hormone intron D splicing. Thus, multiple SF2/ASF binding sites within the exonic splicing enhancer contribute to maximal enhancer activity.

Animals↗
PubMed2000

SR proteins Asf/SF2 and 9G8 interact to activate enhancer-dependent intron D splicing of bovine growth hormone pre-mRNA in vitro.

The alternative splicing of the last intron (intron D) of bovine growth hormone (bGH) pre-mRNA requires a down-stream exonic splicing enhancer (FP/ESE). The presence of at least one SR protein has been shown to be essential for FP/ESE function and splicing of intron D in in vitro splicing assays. However, in vitro reconstitution of splicing using individual purified SR proteins may not accurately reflect the true complexity of alternative splicing in an intact nucleus, where multiple SR proteins in varying amounts are likely to be available simultaneously. Here, a panel of recombinant baculovirus-expressed SR proteins was produced and tested for the ability to activate FP/ESE-dependent splicing. Individual recombinant SR proteins differed significantly in their activity in promoting intron D splicing. Among the recombinant SR proteins tested, SRp55 was the most active, SC35 showed very little activity, and ASF/SF2 and 9G8 individually had intermediate activity. At least one SR protein (ASF/SF2) bound to the FP/ESE with characteristics of a cooperative interaction. Most interestingly, low concentrations of ASF/SF2 and 9G8 acted synergistically to activate intron D splicing. This was due in part to synergistic binding to the FP/ESE. Splicing of bGH intron D is inherently complex, and is likely controlled by an interaction of the FP/ESE with several trans-acting protein factors acting both independently and cooperatively. This level of complexity may be required for precise control of alternative splicing by an exon sequence, which simultaneously is constrained to maintain translational integrity of the mature mRNA.

Alternative Splicing↗
PubMed1997

Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase.

The methylation of internal adenosine residues in eukaryotic mRNA, forming N6-methyladenosine (m6A), is catalyzed by a complex multicomponent enzyme. Previous studies suggested that m6A affects the efficiency of mRNA processing or transport, although the mechanism by which this occurs is not known. As a step toward better understanding the mechanism and function of this ubiquitous posttranscriptional modification, we have shown that HeLa mRNA (N6-adenosine)-methyltransferase requires at least two separate protein factors, MT-A and MT-B, and MT-A contains the AdoMet binding site on a 70-kDa subunit (MT-A70). MT-A70 was purified by conventional chromatography and electrophoresis, and was microsequenced. The peptide sequence was used to design a degenerate oligodeoxynucleotide that in turn was used to isolate the cDNA clone coding for MT-A70 from a HeLa cDNA library. Recombinant MT-A70 was expressed as a fusion protein in bacteria and was used to generate anti-MT-A70 antisera in rabbits. These antisera recognize MT-A70 in HeLa nuclear extracts by western blot and are capable of depleting (N6-adenosine)-methyltransferase activity from HeLa nuclear extract, confirming that MT-A70 is a critical subunit of (N6-adenosine)-methyltransferase. Northern blot analysis reveals that MT-A70 mRNA is present in a wide variety of human tissues and may undergo alternative splicing. MT-A70 cDNA probe hybridizes to a 2.0-kilobase (kb) polyadenylated RNA isolated from HeLa cells, whereas it hybridizes to two predominant RNA species (approximately 2.0 kb and 3.0 kb) using mRNA isolated from six different human tissues. Analysis of the cDNA sequence indicates that it codes for a 580-amino acid protein with a predicted MW = 65 kDa. The predicted protein contains sequences similar to consensus methylation motifs I and II identified in prokaryotic DNA (N6-adenosine)-methyltransferases, suggesting the functional conservation of peptide motifs. MT-A70 also contains a long region of homology to the yeast protein SPO8, which is involved in induction of sporulation by an unknown mechanism.

Amino Acid Sequence↗
PubMed1995

Multiple splicing signals control alternative intron retention of bovine growth hormone pre-mRNA.

A fraction of bovine growth hormone (bGH) pre-mRNA undergoes alternative splicing in which the last intron is retained and transported to the cytoplasm. Our goal was to characterize the cis-acting signals in bGH pre-mRNA that collectively determine the distribution between intron splicing and intron retention. We now demonstrate that the balance between splicing and intron retention in cytoplasmic mRNA is primarily determined by the interaction of three splicing signals and the degree to which these signals deviate from consensus splicing signals. Intron retention requires the presence of both suboptimal 5'- and 3'-splice sites. Mutation of either splice site toward consensus leads to complete splicing of the intron. In the presence of both wild-type, suboptimal splice sites, efficient splicing of this intron is ensured by the presence of a third splicing element, a purine-rich exonic splicing enhancer (ESE). Although strong ESEs can be contained within very small sequences, the bGH ESE activity appears to be composed of multiple sequences spread throughout a 115-nucleotide region of exon 5. Consequently, the final ratio of splicing to intron retention depends on the balance between the relative strengths of each of these three splicing signals, which still allow intron-containing coding sequences to be transported to the cytoplasm.

Alternative Splicing↗
PubMed1994

A purine-rich exon sequence enhances alternative splicing of bovine growth hormone pre-mRNA.

A previous study has demonstrated that deletion of a region within the last exon of bovine growth hormone (bGH) pre-mRNA results in almost complete retention of the upstream intron (Hampson, R. K., LaFollette, L., and Rottman, F. M. (1989) Mol. Cell. Biol. 9, 1604-1610). We now demonstrate that insertion of a simple purine-rich element (GGAAG), which is present within the deleted region, activates intron splicing upon expression in transfected cells. Moreover, several repeats of the GGAA(G) sequence restore splicing to near wild-type levels and direct the binding of a factor present in HeLa cell nuclear extracts. Mutation of the 5'-splice site toward U1 small nuclear RNA complementarity eliminates dependence on the downstream exon sequence for splicing. These results support a model for alternative intron retention in which purine-rich sequences function as part of an "exonic splicing enhancer" to complement a weak 5'-splice site and thereby facilitate intron removal. As a result, the majority of bGH mRNA is processed to remove intron D while still allowing a fraction of bGH mRNA containing the intact intron to reach the cytoplasm.

Alternative Splicing↗
PubMed1994

Context effects on N6-adenosine methylation sites in prolactin mRNA.

The methylation of internal adenosine residues in mRNA only occurs within GAC or AAC sequences. Although both of these sequence motifs are utilized, a general preference has been noted for the extended sequence RGACU. Not all RGACU sequences in an mRNA are methylated and the mechanisms that govern the selection of methylation sites in mRNA remain unclear. To address this problem we have examined the methylation of transcripts containing sequences of a natural mRNA, namely, bovine prolactin mRNA. In this mRNA, a specific AGACU sequence in the 3' untranslated region is the predominant site of methylation both in vivo and in vitro. The degree to which N6-adenosine methyltransferase recognizes the sequence context of the consensus methylation site was explored by mutational analysis of the nucleotides adjacent to the core sequence as well as the extended regions in which the core element was found. Our results indicate that efficient methylation depends on the extended five nucleotide consensus sequence but is strongly influenced by the context in which the consensus sequence occurs within the overall mRNA molecule. Furthermore, consensus methylation sites present in an RNA duplex are not recognized by the methyltransferase.

Adenine↗
PubMed1994

N6-adenosine methylation in mRNA: substrate specificity and enzyme complexity.

The N6-methylation of internal adenosine residues is a common post-transcriptional modification of eukaryotic pre-mRNA sequences. An in vitro methylation system which retains the precise selectivity of in vivo methylation sites has been used to further define the nature of RNA site recognition. In addition to short consensus sequences, other structural features or context effects contribute to the selection of methylation sites in pre-mRNAs. Partial purification of the mRNA N6-adenosine methyltransferase revealed unexpected levels of complexity. The methyltransferase is composed of three separate components with molecular masses of 30, 200 and 875 kDa, respectively. These components are readily separated under non-denaturing conditions and each is required for mRNA methylation activity.

Adenosine↗
PubMed1993

In vitro analysis of bovine growth hormone pre-mRNA alternative splicing. Involvement of exon sequences and trans-acting factor(s).

Bovine growth hormone (bGH) pre-mRNA is alternatively spliced, resulting in retention of the last intron (intron D) in a fraction of the cytosolic bGH mRNA. To study the mechanism of this alternative splicing event, we examined the splicing of bGH pre-mRNA in vitro. The splicing of bGH intron D in vitro required a 115-base pair segment of exon 5, reflecting the positive influence of exon sequences observed in transfected cells. No detectable spliceosome complex formation was observed using bGH pre-mRNA containing the 115-base pair deletion in exon 5. The in vitro splicing of the wild type bGH pre-mRNA was inhibited by the addition of RNA containing the 115-nucleotide exon sequence, but not by nonspecific RNAs. UV irradiation of the in vitro splicing reaction resulted in specific cross-linking of a 35-kDa protein(s) to the 115-nucleotide bGH exon sequence. These results suggest that terminal exon sequences are required at an early step of spliceosome complex formation and are consistent with a mechanism in which saturable, trans-acting factor(s) bind to these exon sequences to activate spliceosome complex formation and splicing of bGH intron D.

Alternative Splicing↗
PubMed1993

General splicing factor SF2/ASF promotes alternative splicing by binding to an exonic splicing enhancer.

The general splicing factor SF2/ASF binds in a sequence-specific manner to a purine-rich exonic splicing enhancer (ESE) in the last exon of bovine growth hormone (bGH) pre-mRNA. More importantly, SF2/ASF stimulates in vitro splicing of bGH intron D through specific interaction with the ESE sequences. However, another general splicing factor, SC35, does not bind the ESE sequences and has no effect on bGH intron D splicing. Thus, one possible function of SF2/ASF in alternative and, perhaps, constitutive pre-mRNA splicing is to recognize ESE sequences. The stimulation of bGH intron D splicing by SF2/ASF is counteracted by the addition of hnRNP A1. The relative levels of SF2/ASF and hnRNP A1 influence the efficiency of bGH intron D splicing in vitro and may be the underlying mechanism of this alternative pre-mRNA processing event in vivo.

Alternative Splicing↗
PubMed1992

Application of an immune-tolerizing procedure to generate monoclonal antibodies specific to an alternate protein isoform of bovine growth hormone.

An immune-tolerizing protocol was employed to generate monoclonal antibodies to a variant protein isoform of bovine growth hormone arising from alternative pre-mRNA processing. Variant bovine growth hormone used for immunization was obtained by expression in bacteria and electroelution of the protein from preparative sodium dodecyl sulfate-polyacrylamide gels. Balb/c mice were first immunized with wild-type bovine growth hormone in the presence of the cytotoxic drug cyclophosphamide, thereby tolerizing the mouse to common epitopes shared among the two proteins. Subsequently, the mice were immunized with variant bovine growth hormone to produce antibodies specific to variant epitopes. Comparisons of fusions resulting from standard and tolerizing immunization protocols resulted in a significantly enhanced production of variant bovine growth hormone-specific antibodies as a result of the immunotolerizing protocol. The specificity of the antibodies to the variant growth hormone was substantiated by differential enzyme-linked immunosorbent assay and Western blot. Nearly all hybridomas positive for variant growth hormone were negative for wild-type growth hormone. Finally, the antibodies were used to demonstrate intracytoplasmic staining of COS I cells transiently transfected with a variant growth hormone-producing plasmid. Given the power of the polymerase chain reaction to conveniently clone alternatively processed mRNA species, followed by expression in bacteria to provide antigen, the immunotolerizing protocol provides a convenient general method for producing antibodies specific to desired protein isoforms.

Amino Acid Sequence↗
PubMed1992

The 3'-flanking sequence of the bovine growth hormone gene contains novel elements required for efficient and accurate polyadenylation.

In addition to the conserved AAUAAA hexanucleotide, GU- and U-rich sequences in the 3'-flanking region are thought to be critical for efficient polyadenylation. The 3'-flanking sequence requirements for efficient and accurate polyadenylation of the bovine growth hormone (bGH) gene were determined by quantitative S1 nuclease analysis of transcripts derived from various bGH 3' deletions and block mutations transiently transfected into COS-1 cells. Though the bGH 3'-flanking sequence contains a portion of the putative GU efficiency element, we find that mutation of this element leads to a marginal decrease in efficiency similar to that from mutation of other sequences that do not contain recognizable GU- or U-rich motifs. The data are consistent with a diffuse efficiency element in the bGH polyadenylation signal rather than a discrete element as is thought to exist in other mammalian signals. We have also determined that a region from 18 to 27 nucleotides downstream of the cleavage site contains sequences required for correctly positioning the cleavage site.

Animals↗
PubMed1990

Characterization of an inducible promoter system to investigate decay of stable mRNA molecules.

We have developed a system in which the decay of stable mRNAs can be studied without the use of inhibitors of transcription. The Drosophila hsp70 heat shock promoter linked to the bovine growth hormone (BGH) gene was used to establish stable cell lines in which the BGH gene is transcribed in a conditional manner. The BGH mRNA is synthesized only after induction at 43 degrees C. Following a brief period of re-equilibration at 37 degrees C during which transcription of the heat shock-driven gene ceases, the stable BGH mRNA decays with typical first-order kinetics. Hence, the decay of the mRNA can be studied without assumptions regarding radioactive labeling of precursor pools or transcriptional inhibitors. The system is applicable to any stable mRNA.

Animals↗
PubMed1990

A spliced leader is present on a subset of mRNAs from the human parasite Schistosoma mansoni.

We present evidence that a subset of mRNAs in the human parasitic trematode Schistosoma mansoni contain an identical 36-nucleotide spliced leader (SL) sequence at their 5' termini. The SL is derived from a 90-nucleotide nonpolyadenylylated RNA (SL RNA), presumably by trans-splicing. Neither the SL nor the SL RNA share significant sequence identity with previously described trans-spliced leaders and SL RNAs in trypanosomatid protozoans or nematodes. However, several features, such as predicted secondary structure, trimethylguanosine cap, and potential Sm binding site, suggest similarities among SL RNAs in widely divergent organisms. Our evidence also indicates that the exon 3 acceptor site of the 3-hydroxy-3-methylglutaryl-CoA reductase gene can be spliced either to the SL by trans-splicing or to an upstream exon, 2, by cis-splicing. The presence of a SL sequence in S. mansoni, a member of the phylum Platyhelminthes, suggests that transplicing may be a common feature of other lower invertebrates.

Animals↗
PubMed1990

N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA.

N6-methyladenosine (m6A) residues occur at internal positions in most cellular and viral RNAs; both heterogeneous nuclear RNA and mRNA are involved. This modification arises by enzymatic transfer of a methyl group from S-adenosylmethionine to the central adenosine residue in the canonical sequence G/AAC. Thus far, m6A has been mapped to specific locations in eucaryotic mRNA and viral genomic RNA. We have now examined an intron-specific sequence of a modified bovine prolactin precursor RNA for the presence of this methylated nucleotide by using both transfected-cell systems and a cell-free system capable of methylating mRNA transcripts in vitro. The results indicate the final intron-specific sequence (intron D) of a prolactin RNA molecule does indeed possess m6A residues. When mapped to specific T1 oligonucleotides, the predominant site of methylation was found to be within the consensus sequence AGm6ACU. The level of m6A at this site is nonstoichiometric; approximately 24% of the molecules are modified in vivo. Methylation was detected at markedly reduced levels at other consensus sites within the intron but not in T1 oligonucleotides which do not contain either AAC or GAC consensus sequences. In an attempt to correlate mRNA methylation with processing, stably transfected CHO cells expressing augmented levels of bovine prolactin were treated with neplanocin A, an inhibitor of methylation. Under these conditions, the relative steady-state levels of the intron-containing nuclear precursor increased four to six times that found in control cells.

Adenosine↗
PubMed1990

Production of transgenic pigs harbouring a rat phosphoenolpyruvate carboxykinase-bovine growth hormone fusion gene.

Over the past 5 years, reports detailing the production of transgenic pigs have focussed on enhanced growth performance. Phenotypic side-effects observed in pigs harbouring chimaeric constructs containing metallothionein or Moloney murine leukaemia virus transcriptional activators fused to growth hormone (GH) structural genes have been attributed to chronic overexpression of GH. In an effort to regulate a transgene product more effectively, a liver specific 460 bp 5' flanking sequence of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene was ligated to a BamHI site of the first exon of the genomic bovine GH (bGH) structural gene. Following micro-injection of the PEPCK/bGH construct into 1- and 2-cell pig zygotes. 124 offspring were produced of which 7 pigs were determined to be transgenic by dot-blot and Southern analysis. The PEPCK gene expression, in terms of tissue and developmental specificity, appears similar to that observed in PEPCK/bGH transgenic mice. Germ-line transmission was identified in 1 of 3 mated founders. Dramatic influences on backfat thickness were observed including a 41% reduction in backfat depth when compared to non-transgenic sex-matched littermate control pigs. Both the regulation and characterization of gene expression in PEPCK/bGH transgenic pigs are under investigation.

Animals↗
PubMed1989

Molecular cloning and sequence analysis of 3-hydroxy-3-methylglutaryl-coenzyme A reductase from the human parasite Schistosoma mansoni.

cDNA clones encoding the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase [(S)-mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34] from the human parasite Schistosoma mansoni have been isolated and characterized. The composite 3459 base pairs of cDNA sequence contains a 2844-base-pair open reading frame corresponding to a protein of 948 amino acids. The predicted S. mansoni HMG-CoA reductase protein contains a hydrophobic amino terminus consisting of seven potential transmembrane domains that are structurally conservative but are not identical in amino acid sequence with HMG-CoA reductases from other species. The hydrophilic carboxyl terminus of the S. mansoni HMG-CoA reductase protein, however, shares 48-52% sequence identity with the carboxyl termini of other HMG-CoA reductases in a region that contains the catalytic domain. When expressed as a fusion protein in Escherichia coli, the carboxyl-terminal domain of the schistosome protein exhibits HMG-CoA reductase enzyme activity.

Amino Acid Sequence↗