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Biomedical subjects

M Wickens

Publications and source records attributed to M Wickens.

49 records · Page 3Linked to original sources
PubMed1990

Poly(A) removal during oocyte maturation: a default reaction selectively prevented by specific sequences in the 3' UTR of certain maternal mRNAs.

Certain maternal mRNAs lose their poly(A) tails during early development and concomitantly become translationally inactive. In this report we analyze the mechanism of poly(A) removal during frog oocyte maturation by injecting short synthetic RNAs. We demonstrate that removal of poly(A) during oocyte maturation is a default reaction: In the absence of any specific sequence information, poly(A) is removed. However, poly(A) removal can be prevented by specific sequences in the 3'-untranslated regions of certain maternal mRNAs. These sequences are also required for poly(A) addition during oocyte maturation and include AAUAAA and a nearby U-rich element. Mutations in either AAUAAA or the U-rich element cause loss of poly(A) and not merely a failure to extend the poly(A) tail. We infer that poly(A) addition is required to escape poly(A) loss. The enzyme that removes the poly(A) during oocyte maturation appears to be a 3'----5' nuclease that prefers a 3'-terminal poly(A) segment. We discuss possible mechanisms by which poly(A) addition might circumvent default poly(A) removal and consider whether poly(A) removal is also a default reaction in somatic cells. Finally, we consider the possible implications of our results for the selectivity of poly(A) addition and removal, and for translational regulation during early development.

Animals↗
PubMed1990

Polyadenylation-specific complexes undergo a transition early in the polymerization of a poly(A) tail.

We have analyzed several properties of the complex that forms between RNAs that end at the poly(A) site of simian virus 40 late mRNA and factors present in a HeLa cell nuclear extract. Formation of this polyadenylation-specific complex requires the sequence AAUAAA and a proximal 3' end. We have observed three changes in the polyadenylation complex early in the addition of the poly(A) tail. First, the complex becomes heparin sensitive after the addition of approximately 10 adenosines. Second, a 68-kilodalton protein present in the complex, which can be cross-linked by UV light to the RNA before polyadenylation has begun, no longer can be cross-linked after approximately 10 adenosines have been added. Third, after 30 adenosines have been added, the AAUAAA sequence becomes accessible to a complementary oligonucleotide and RNase H. This accessibility gradually increases with longer poly(A) tail lengths until, with the addition of 60 A's, all substrates are accessible at AAUAAA. Sheets and Wickens (Genes Dev. 3:1401-1412, 1989) have recently demonstrated two phases in the addition of a poly(A) tail: the first requires AAUAAA, whereas the second is independent of AAUAAA but requires a short oligo(A) primer. The data reported here further support a biphasic model for poly(A) addition and may indicate disengagement of specific factors from AAUAAA after the initiation phase.

Base Sequence↗
PubMed1990

The enzyme that adds poly(A) to mRNAs is a classical poly(A) polymerase.

Virtually all mRNAs in eucaryotes end in a poly(A) tail. This tail is added posttranscriptionally. In this report, we demonstrate that the enzyme that catalyzes this modification is identical with an activity first identified 30 years ago, the function of which was previously unknown. This enzyme, poly(A) polymerase, lacks any intrinsic specificity for its mRNA substrate but gains specificity by interacting with distinct molecules: a poly(A) polymerase from calf thymus, when combined with specificity factor(s) from cultured human cells, specifically and efficiently polyadenylates only appropriate mRNA substrates. Our results thus demonstrate that this polymerase is responsible for the addition of poly(A) to mRNAs and that its interaction with specificity factors is conserved.

Animals↗
PubMed1990

Polyadenylation of mRNA: minimal substrates and a requirement for the 2' hydroxyl of the U in AAUAAA.

mRNA-specific polyadenylation can be assayed in vitro by using synthetic RNAs that end at or near the natural cleavage site. This reaction requires the highly conserved sequence AAUAAA. At least two distinct nuclear components, an AAUAAA specificity factor and poly(A) polymerase, are required to catalyze the reaction. In this study, we identified structural features of the RNA substrate that are critical for mRNA-specific polyadenylation. We found that a substrate that contained only 11 nucleotides, of which the first six were AAUAAA, underwent AAUAAA-specific polyadenylation. This is the shortest substrate we have used that supports polyadenylation: removal of a single nucleotide from either end of this RNA abolished the reaction. Although AAUAAA appeared to be the only strict sequence requirement for polyadenylation, the number of nucleotides between AAUAAA and the 3' end was critical. Substrates with seven or fewer nucleotides beyond AAUAAA received poly(A) with decreased efficiency yet still bound efficiently to specificity factor. We infer that on these shortened substrates, poly(A) polymerase cannot simultaneously contact the specificity factor bound to AAUAAA and the 3' end of the RNA. By incorporating 2'-deoxyuridine into the U of AAUAAA, we demonstrated that the 2' hydroxyl of the U in AAUAAA was required for the binding of specificity factor to the substrate and hence for poly(A) addition. This finding may indicate that at least one of the factors involved in the interaction with AAUAAA is a protein.

Base Sequence↗
PubMed1989

Two phases in the addition of a poly(A) tail.

The addition of a poly(A) tail has been examined in a HeLa cell nuclear extract using SV40 late RNAs that end at or near the natural poly(A) site. We find that the addition of a full-length, 200-nucleotide poly(A) tail occurs in two discrete phases. In the first phase, the addition of each adenosine is dependent on the highly conserved sequence AAUAAA. Mutations in that sequence result in an accumulation of products that contain 9 or fewer adenosine residues. In the second phase, poly(A) addition no longer requires AAUAAA but, instead, requires the oligo(A) primer synthesized during the first phase. Thus, RNAs carrying an AAUAAA mutation and a 3'-terminal oligo(A) segment are extended efficiently to full-length poly(A). The transition between phases occurs with the addition of the tenth adenosine residue. An activity exists that limits the length of poly(A) added in the extract to approximately 200 nucleotides. The two phases share at least one component and are likely to involve the same poly(A) polymerase.

HeLa Cells↗
PubMed1988

A functionally redundant downstream sequence in SV40 late pre-mRNA is required for mRNA 3'-end formation and for assembly of a precleavage complex in vitro.

In eukaryotes, mRNA 3' termini are formed by endonucleolytic cleavage of a long primary transcript and polyadenylation of the new end. Here we show that sequences downstream of the poly(A) site are required for cleavage of simian virus 40 (SV40) late pre-mRNAs in vitro in a crude nuclear extract from HeLa cells. The critical sequences are functionally redundant: extensive deletions or substitutions of downstream sequences prevent cleavage, but small substitutions do not. This functional redundancy is not due to a repetition of the same sequence. Either two or more different sequences can promote cleavage, or a single element exists which is long and diffuse. Although pre-mRNAs transcribed from certain genes require a U- or UG-rich sequence downstream of the poly(A) site for efficient cleavage, SV40 does not. Removal of these sequences from SV40 late pre-mRNAs does not significantly reduce cleavage efficiency. Downstream sequences also are required for formation of a specific precleavage complex between SV40 pre-mRNA and components present in the extract. Mutant RNAs that are cleaved efficiently form such complexes, while those that are cleaved inefficiently do not. Based on these and previous results (Zarkower, D., and Wickens, M. (1987b) EMBO J. 6, 4185-4192), we propose that a critical role of the region downstream of the poly(A) site is to facilitate formation of a specific precleavage complex in which cleavage subsequently occurs.

Animals↗
PubMed1987

Analysis of mRNA 3' end formation by modification interference: the only modifications which prevent processing lie in AAUAAA and the poly(A) site.

A modification interference method is described in which chemically modified transcripts are used to identify bases required for any reaction for which synthetic RNA is a substrate. This technique provides information analogous to that obtained from the analysis of a complete set of point mutants. Using SV40 late pre-mRNAs, we determine that modification of any base in the AAUAAA sequence prevents cleavage, polyadenylation and formation of pre-cleavage complexes in vitro. Modification of the A to which poly(A) is added prevents polyadenylation, but does not interfere with formation of the pre-cleavage complex. No single modification downstream of the poly(A) site significantly affects cleavage efficiency. Since the region downstream of the poly(A) site is required for cleavage and complex formation (Conway and Wickens, 1985; Zarkower and Wickens, 1987b), we infer that the critical features of this downstream region are either diffuse or redundant.

Base Sequence↗
PubMed1987

Specific pre-cleavage and post-cleavage complexes involved in the formation of SV40 late mRNA 3' termini in vitro.

Complexes form between processing factors present in a crude nuclear extract from HeLa cells and a simian virus 40 (SV40) late pre-mRNA which spans the polyadenylation [poly(A)] site. A specific 'pre-cleavage complex' forms on the pre-mRNA before cleavage. Formation of this complex requires the highly conserved sequence AAUAAA: it is prevented by mutations in AAUAAA, and by annealing DNA oligonucleotides to that sequence. After cleavage, the 5' half-molecule is found in a distinct 'post-cleavage complex'. In contrast, the 3' half-molecule is released. After cleavage and polyadenylation, polyadenylated RNA also is released. De novo formation of the post-cleavage complex requires AAUAAA and a nearby 3' terminus. Competition experiments suggest that a component which recognizes AAUAAA is required for formation of both pre- and post-cleavage complexes.

Base Sequence↗
PubMed1987

Formation of mRNA 3' termini: stability and dissociation of a complex involving the AAUAAA sequence.

Formation of the 3' termini of mRNAs in animal cells involves endonucleolytic cleavage of a pre-mRNA, followed by polyadenylation of the newly formed end. Here we demonstrate that, during cleavage in vitro, the highly conserved AAUAAA sequence of the pre-mRNA forms a complex with a factor present in a crude nuclear extract. This complex is required for cleavage and polyadenylation. It normally is transient, but is very stable on cleaved RNA to which a single terminal cordycepin residue has been added. The complex can form either during the cleavage reaction, or on a synthetic RNA that ends at the polyadenylation site. Mutations which prevent cleavage also prevent complex formation. The complex dissociates during or after polyadenylation, enabling the released activities to catalyze a second round of cleavage.

Base Sequence↗
PubMed1986

The AAUAAA sequence is required both for cleavage and for polyadenylation of simian virus 40 pre-mRNA in vitro.

The sequence AAUAAA is found near the polyadenylation site of eucaryotic mRNAs. This sequence is required for accurate and efficient cleavage and polyadenylation of pre-mRNAs in vivo. In this study we show that synthetic simian virus 40 late pre-mRNAs are cleaved and polyadenylated in vitro in a HeLa cell nuclear extract, and that cleavage in vitro is abolished by each of four different single-base changes in AAUAAA. In this same extract, precleaved RNAs (RNAs with 3' termini at the polyadenylation site) are efficiently polyadenylated. This in vitro polyadenylation reaction also requires the AAUAAA sequence.

DNA Restriction Enzymes↗
PubMed1985

A sequence downstream of A-A-U-A-A-A is required for formation of simian virus 40 late mRNA 3' termini in frog oocytes.

The 3' terminus of simian virus 40 late mRNA is formed by nucleolytic cleavage of an mRNA precursor. In this report we show that efficient cleavage requires a sequence in the 3' flanking region, "downstream" of the highly conserved A-A-U-A-A-A sequence. This sequence is not required for polyadenylylation itself or for accurately positioning the 3' terminus that is formed.

Animals↗