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M P Wickens

Publications and source records attributed to M P Wickens.

18 recordsLinked to original sources
PubMed1999

The cleavage and polyadenylation specificity factor in Xenopus laevis oocytes is a cytoplasmic factor involved in regulated polyadenylation.

During early development, specific mRNAs receive poly(A) in the cytoplasm. This cytoplasmic polyadenylation reaction correlates with, and in some cases causes, translational stimulation. Previously, it was suggested that a factor similar to the multisubunit nuclear cleavage and polyadenylation specificity factor (CPSF) played a role in cytoplasmic polyadenylation. A cDNA encoding a cytoplasmic form of the 100-kDa subunit of Xenopus laevis CPSF has now been isolated. The protein product is 91% identical at the amino acid sequence level to nuclear CPSF isolated from Bos taurus thymus. This report provides three lines of evidence that implicate the X. laevis homologue of the 100-kDa subunit of CPSF in the cytoplasmic polyadenylation reaction. First, the protein is predominantly localized to the cytoplasm of X. laevis oocytes. Second, the 100-kDa subunit of X. laevis CPSF forms a specific complex with RNAs that contain both a cytoplasmic polyadenylation element (CPE) and the polyadenylation element AAUAAA. Third, immunodepletion of the 100-kDa subunit of X. laevis CPSF reduces CPE-specific polyadenylation in vitro. Further support for a cytoplasmic form of CPSF comes from evidence that a putative homologue of the 30-kDa subunit of nuclear CPSF is also localized to the cytoplasm of X. laevis oocytes. Overexpression of influenza virus NS1 protein, which inhibits nuclear polyadenylation through an interaction with the 30-kDa subunit of nuclear CPSF, prevents cytoplasmic polyadenylation, suggesting that the cytoplasmic X. laevis form of the 30-kDa subunit of CPSF is involved in this reaction. Together, these results indicate that a distinct, cytoplasmic form of CPSF is an integral component of the cytoplasmic polyadenylation machinery.

Amino Acid Sequence↗
PubMed1998

mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation.

Translation and mRNA stability are enhanced by the presence of a poly(A) tail. In vivo, the tail interacts with a conserved polypeptide, poly(A) binding protein (Pab1p). To examine Pab1p function in vivo, we have tethered Pab1p to the 3' UTR of reporter mRNAs by fusing it to MS2 coat protein and placing MS2 binding sites in the 3' UTR of the reporter. This strategy allows us to uncouple Pab1p function from its RNA binding activity. We show that mRNAs that lack a poly(A) tail in vivo are stabilized by Pab1p, and that the portions of Pab1p required for stabilization are genetically distinct from those required for poly(A) binding. In addition, stabilization by Pab1p requires ongoing translation of the mRNA. We conclude that the primary, or sole, function of poly(A) with respect to mRNA stability is simply to bring Pab1p to the mRNA, and that mRNA stabilization is an intrinsic property of Pab1p. The approach we describe may be useful in identifying and assaying 3' UTR regulatory proteins, as it uncouples analysis of function from RNA binding.

3' Untranslated Regions↗
PubMed1997

A conserved RNA-binding protein that regulates sexual fates in the C. elegans hermaphrodite germ line.

The nematode Caenorhabditis elegans has two sexes, males and hermaphrodites. Hermaphrodites Initially produce sperm but switch to producing oocytes. This switch appears to be controlled by the 3' untranslated region of fem-3 messenger RNA. We have now identified a binding factor (FBF) which is a cytoplasmic protein that binds specifically to the regulatory region of fem-3 3'UTR and mediates the sperm/oocyte switch. The RNA-binding domain of FBF consists of a stretch of eight tandem repeats and two short flanking regions. This structural element is conserved in several proteins including Drosophila Pumilio, a regulatory protein that controls pattern formation in the fly by binding to a 3'UTR. We propose that FBF and Pumilio are members of a widespread family of sequence-specific RNA-binding proteins.

Amino Acid Sequence↗
PubMed1990

Point mutations in AAUAAA and the poly (A) addition site: effects on the accuracy and efficiency of cleavage and polyadenylation in vitro.

Three sequences in the vicinity of poly (A) addition sites are conserved among vertebrate mRNAs. We analyze the effects of single base changes in each position of AAUAAA and in the nucleotide to which poly (A) is added on 3' end formation in vitro. All 18 possible single base changes of the AAUAAA sequence greatly reduce addition of poly (A) to RNAs that end at the poly (A) addition site, and prevent cleavage of RNAs that extend beyond. The magnitude of reduction varies greatly with the position changed and the base introduced. For any given mutation, cleavage and polyadenylation are reduced to similar extents, strongly suggesting that the same factor interacts with AAUAAA in both reactions. Mutations at and near the conserved adenosine to which poly (A) is added disturb the accuracy, but not the efficiency, of 3' end formation. For example, point mutations at the conserved adenosine shift the 3' end of the most abundant 5' half-molecule downstream by a single nucleotide. The mechanism by which these mutations might exert their effects on the precision of 3' end formation are discussed.

Base Sequence↗
PubMed1990

Splicing of a C. elegans myosin pre-mRNA in a human nuclear extract.

Splicing of mammalian introns requires that the intron possess at least 80 nucleotides. This length requirement presumably reflects the constraints of accommodating multiple snRNPs simultaneously in the same intron. In the free-living nematode, C. elegans, introns typically are 45 to 55 nucleotides in length. In this report, we determine whether C. elegans introns can obviate the mammalian length requirement by virtue of their structure or sequence. We demonstrate that a 53 nucleotide intron from the unc-54 gene of C. elegans does not undergo splicing in a mammalian (HeLa) nuclear extract. However, insertion of 31 nucleotides of foreign, prokaryotic sequence into the same intron results in efficient splicing. The observed splicing proceeds by the same two-step mechanism observed with mammalian introns, and exploits the same 3' and 5' splice sites as are used in C. elegans. The branch point used lies in the inserted sequence. We conclude that C. elegans splicing components are either fewer in number or smaller than their mammalian counterparts.

Animals↗
PubMed1989

Poly(A) addition during maturation of frog oocytes: distinct nuclear and cytoplasmic activities and regulation by the sequence UUUUUAU.

In frog oocytes, certain maternal mRNAs receive poly(A) in the cytoplasm during progesterone-induced maturation. To analyze this reaction and to compare it to poly(A) addition in the nucleus, we injected short, synthetic RNA substrates into Xenopus oocytes. These RNAs contain only portions of the 3'-untranslated regions of appropriate mRNAs and end at the natural poly(A) site. We demonstrate that the nuclear and maturation-specific polyadenylation activities are distinct in substrate specificity and subcellular location. The sequence AAUAAA, contained in virtually all pre-mRNAs, is necessary for both activities. A second sequence element, UUUUUAU, activates poly(A) addition during maturation. UUUUUAU and AAUAAA are both necessary and virtually sufficient for maturation-specific polyadenylation: Poly(A) tails of between 50 and 300 nucleotides are added during maturation to RNAs containing both sequences but not to RNAs that lack either sequence. Before maturation, RNAs that contain AAUAAA are extended by just 10 nucleotides, presumably adenosines. The maturation-specific activity first appears within 1 hr of the time the nucleus breaks down but apparently does not require a nuclear component, as it is unaffected by enucleation. These observations, combined with those of others, lead us to speculate that polyadenylation may be responsible for the translational activation of a family of mRNAs essential for maturation.

Animals↗
PubMed1987

Products of in vitro cleavage and polyadenylation of simian virus 40 late pre-mRNAs.

Formation of mRNA 3' termini involves cleavage of an mRNA precursor and polyadenylation of the newly formed end. Cleavage of simian virus 40 late pre-mRNA in a crude nuclear extract generated two RNAs, 5' and 3' half-molecules. These RNAs were unmodified and linear. The 5' half-molecule contained sequences upstream but not downstream of the poly(A) site and ended in a 3'-terminal hydroxyl. The 3' half-molecules comprised a family of RNAs, each of which contains only sequences downstream of the poly(A) site, and ends in a 5'-terminal phosphate. These RNAs differed only in the locations of their 5' terminus. The 3' terminus of the 5' half-molecule was the adenosine 10 nucleotides downstream of AAUAAA, at the +1 position. The 5' terminus of the longest 3' half-molecule was at +2. Thus, these two RNAs contain every nucleoside and phosphate of the precursor. The existence of these half-molecules demonstrates that endonucleolytic cleavage occurs near the poly(A) site. 5' half-molecules generated in the presence of EDTA (which blocks polyadenylation, but not cleavage) ended at the adenosine at position +1 of the precursor. When incubated in the extract under suitable conditions, they became polyadenylated. 5' half-molecules formed in 3'-dATP-containing reactions contained a single 3'-deoxyadenosine (cordycepin) residue added onto the +1 adenosine and were poor polyadenylation substrates. We infer that the +1 adenosine of the precursor becomes the first A of the poly(A) tract and provides a 3' hydroxyl group to which poly(A) is added posttranscriptionally.

Alkaline Phosphatase↗
PubMed1983

Post-transcriptional processing of simian virus 40 late transcripts in injected frog oocytes.

The capacity of fully grown Xenopus oocytes to process messenger RNA precursors has been assessed using transcripts synthesized from simian virus 40 (SV40) DNA microinjected into the oocyte nucleus. In oocytes, stable transcripts of the SV40 virion protein genes have undergone at least four post-transcriptional maturation steps: cleavage at 3' splice sites, formation of a mature 3' terminus, addition of poly(A), and selective intracellular partitioning, such that only those RNAs with a mature 3' terminus and poly(A) are located in the cytoplasm. Apparently unspliced transcripts with mature 3' termini are transported into the oocyte cytoplasm. A prominent transcript of roughly the full length of SV40 DNA, bearing a 5' terminus in the same region as late mRNA and confined to the nucleus, is found in oocytes injected with SV40 DNA. The possibility that this transcript may serve as a precursor to late mRNA is discussed.

Adenoviridae↗
PubMed1980

Expression of a chicken chromosomal ovalbumin gene injected into frog oocyte nuclei.

Ovalbumin is synthesized by Xenopus oocytes injected with a plasmid containing either the chicken chromosomal ovalbumin gene or a plasmid from which the 5' region of the chromosomal gene has been deleted. However, oocytes injected with a plasmid containing full-length ovalbumin cDNA do not synthesize ovalbumin, despite the fact that at least as much stable, ovalbumin-specific RNA is transcribed from the cDNA as from the chromosomal gene.

Animals↗
PubMed1979

Restriction map of the region surrounding the EcoRI site in the pCR1 plasmid and analysis of an inserted ovalbumin gene.

We have determined a restriction map of a 1650 base pair region surrounding the EcoRI site of the bacterial plasmid, pCR1. We have used pCR1 as a vector in cloning synthetic ovalbumin double-stranded cDNA. Using the pCR1 restriction map, we have characterized the ovalbumin sequences inserted in one recombinant plasmid, pOvE12. POvE12 appears to contain all, or nearly all, of the sequences found in full length, double-stranded cDNA synthesized in vitro.

Base Sequence↗
PubMed1978

Synthesis of full length cDNAs from four partially purified oviduct mRNAs.

Total poly(A)-containing RNA prepared from hen oviduct and centrifuged on an isokinetic sucrose gradient displays four peaks of optical absorbance. These have been identified by translation in vitro as lysozyme, ovomucoid, ovalbumin, and conalbumin mRNAs. Isolation and recentrifugation of the peaks results in partial purification of each mRNA. Molecular weights have been determined for the mRNAs on agarose gels containing 20 mM methylmercury hydroxide. Each mRNA possesses a number of apparently untranslated nucleotides ranging from approximately 900 bases for ovalbumin and conalbumin mRNAs to 200 bases for ovomucoid and lysozyme mRNAs. The mRNAs have been copied with avian myeloblastosis virus reverse transcriptase. Each mRNA with the exception of conalbumin gives rise to a high proportion of full length cDNA. Several parameters previously reported to influence the size distribution of cDNA had no effect on the length of cDNA made from any mRNA fraction. The proportion of full length copy does depend on the reverse transcriptase lot.

Animals↗
PubMed1978

Synthesis of double-stranded DNA complementary to lysozyme, ovomucoid, and ovalbumin mRNAs. Optimization for full length second strand synthesis by Escherichia coli DNA polymerase I.

Sequential reverse transcriptase, DNA polymerase, and S1 nuclease reactions can be employed to synthesize double-stranded DNA representing messenger RNA. Using reverse transcriptase products made from partially purified lysozyme, ovomucoid, and ovalbumin messengers from hen oviduct, we have characterized the Escherichia coli DNA polymerase I reaction. We have optimized for a high yield of full length second strands under conditions which require only a small amount of mRNA. The effects of several parameters (time, enzyme levels, salt concentration, monovalent cation, and temperature) on the length of products synthesized by DNA polymerase I have been investigated. Each has a significant influence on the proportion of products which are full length. Under our conditions the three reactions are efficient in synthesizing full length duplex DNA from partially purified mRNA fractions or from total poly(A)-containing RNA.

Animals↗