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Boston University
2023
University of Wisconsin–Madison
2013-2022
San Francisco General Hospital
1998-2001
University of San Francisco
2001
University of California, Berkeley
1995-1998
University of California, San Francisco
1998
Purdue University West Lafayette
1982-1983
University of California, San Diego
1983
La Roche College
1983
Journal Article Point mutations in AAUAAA and the poly (A) addition site: effects on accuracy efficiency of cleavage polyadenylation vitro Get access Michael D. Sheets, Sheets Search for other works by this author on: Oxford Academic PubMed Google Scholar Stephen C. Ogg, Ogg 1Department Biochemistry, College Agriculture Life Sciences, University WisconsinMadison, WI 53706, USA Marvin P. Wickens * *To whom correspondence should be addressed Nucleic Acids Research, Volume 18, Issue 19, 11...
A large library of phage-displayed human single-chain Fv antibodies (scFv), containing 6.7 × 10 9 members, was generated by improving the steps construction. Fourteen different protein antigens were used to affinity select from this library. panel specific isolated with each antigen, and contained an average 8.7 scFv. Measurements antibody–antigen interactions revealed several affinities below 1 nM, comparable observed during secondary murine immune response. In particular, four scFv...
C A Fox, M D Sheets and P Wickens Department of Biochemistry, College Agriculture Life Sciences, University Wisconsin, Madison 53706.
Early in the development of many animals, before transcription begins, any change pattern protein synthesis is attributable to a translational activity or stability an mRNA egg. As result, control critical for variety developmental decisions, including axis formation Drosophila and sex determination Caenorhabditis elegans. Previous work demonstrated that increases poly(A) length can activate translation, whereas removal prevent it. In this report we focus on c-mos cyclin A1, B1, B2 mRNAs...
We show that microRNA-427 (miR-427) mediates the rapid deadenylation of maternal mRNAs after midblastula transition (MBT) Xenopus laevis embryogenesis. By MBT, stage when embryonic cell cycle is remodeled and zygotic transcription initiated, each embryo has accumulated ∼10 9 molecules miR-427 processed from multimeric pri-miR-427 transcripts synthesized fertilization. demonstrate for cyclins A1 B2 contain a single target sequence, spanning less than 30 nucleotides, both necessary sufficient...
The sequence AAUAAA is found near the polyadenylation site of eucaryotic mRNAs. This required for accurate and efficient cleavage pre-mRNAs in vivo. In this study we show that synthetic simian virus 40 late are cleaved polyadenylated vitro a HeLa cell nuclear extract, abolished by each four different single-base changes AAUAAA. same precleaved RNAs (RNAs with 3' termini at site) efficiently polyadenylated. reaction also requires sequence.
An undecanucleotide extended hybridization probe has been used to screen a rat medullary thyroid carcinoma cDNA library for clones which contain preprosomatostatin sequences.The nucleotide sequence encoding is reported.The of contains 6 7 nucleotides in the 3'noncoding region, 8 4 5"untranslated and 458 bases corresponding coding region.The mRNA codes somatostatin precursor 116 amino acids length (Mr = 12,773).The hydrophobic at NH2 terminus, followed by peptide approximately 78 residues,...
We show that, in Xenopus laevis oocytes and early embryos, double-stranded exogenous siRNAs cannot function as microRNA (miRNA) mimics either deadenylation or guided mRNA cleavage (RNAi). Instead, saturate inactivate maternal Argonaute (Ago) proteins, which are present low amounts but needed for Dicer processing of pre-miRNAs at the midblastula transition (MBT). Consequently, impair accumulation newly made miRNAs, such abundant embryonic pre-miR-427, inhibition dissipates upon synthesis...
The addition of a poly(A) tail has been examined in HeLa cell nuclear extract using SV40 late RNAs that end at or near the natural site. We find full-length, 200-nucleotide occurs two discrete phases. In first phase, each adenosine is dependent on highly conserved sequence AAUAAA. Mutations result an accumulation products contain 9 fewer residues. second no longer requires AAUAAA but, instead, oligo(A) primer synthesized during phase. Thus, carrying mutation and 3'-terminal segment are...
Abstract Rotating cilia at the vertebrate left-right organizer (LRO) generate an asymmetric leftward flow, which is sensed by cells left LRO margin. Ciliary activity of calcium channel Pkd2 crucial for flow sensing. How this signal further processed and relayed to laterality-determining Nodal cascade in lateral plate mesoderm (LPM) largely unknown. We previously showed that down-regulates mRNA expression inhibitor Dand5 sensory cells. De-repression co-expressed Nodal, complexed with TGFß...
The sequence AAUAAA is found near the polyadenylation site of eucaryotic mRNAs. This required for accurate and efficient cleavage pre-mRNAs in vivo. In this study we show that synthetic simian virus 40 late are cleaved polyadenylated vitro a HeLa cell nuclear extract, abolished by each four different single-base changes AAUAAA. same precleaved RNAs (RNAs with 3' termini at site) efficiently polyadenylated. reaction also requires sequence.
Translational control of many mRNAs in developing metazoan embryos is achieved by alterations their poly(A) tail length. A family cytoplasmic poly(A)-binding proteins (PABPs) bind the and can regulate mRNA translation stability. However, despite extensive biochemical characterization one member (PABP1), surprisingly little known about vivo roles or functional relatedness. Because no information available vertebrates, we address biological roles, establishing that each PABPs conserved Xenopus...
Formation of mRNA 3' termini involves cleavage an precursor and polyadenylation the newly formed end. Cleavage simian virus 40 late pre-mRNA in a crude nuclear extract generated two RNAs, 5' half-molecules. These RNAs were unmodified linear. The half-molecule contained sequences upstream but not downstream poly(A) site ended 3'-terminal hydroxyl. half-molecules comprised family each which contains only site, ends 5'-terminal phosphate. differed locations their terminus. terminus was...
Length-dependent axonopathy of the corticospinal tract causes lower limb spasticity and is characteristic several neurological disorders, including hereditary spastic paraplegia (HSP) amyotrophic lateral sclerosis. Mutations in Trk-fused gene (TFG) have been implicated both diseases, but pathomechanisms by which these alterations cause neuropathy remain unclear. Here, we biochemically genetically define impact a mutation within TFG coiled-coil domain, underlies early-onset forms HSP. We find...
Oocyte maturation and early embryonic development require the cytoplasmic polyadenylation concomitant translational activation of stored maternal mRNAs. ePAB [embryonic poly(A)-binding protein, also known as ePABP PABPc1-like] is a multifunctional post-transcriptional regulator that binds to poly(A) tails. In present study we find dynamically modified phosphoprotein in Xenopus laevis oocytes show by mutation phosphorylation at four residue cluster required for oocyte maturation. We further...
The Xenopus Cripto-1 protein is confined to the cells of animal hemisphere during early embryogenesis where it regulates formation anterior structures. accumulates only in because cripto-1 mRNA vegetal translationally repressed. Here, we show that RNA binding protein, Bicaudal-C (Bic-C), functioned directly this cell-specific repression. While Bic-C normally cells, ectopic expression repressed a reporter and associated with endogenous mRNA. Repression by required its N-terminal domain,...
We describe a new RNA binding protein from Xenopus we have named ePABP2 (embryonic poly(A) type II). Based on amino acid similarity, is closely related to the ubiquitously expressed nuclear PABP2 that directs elongation of mRNA tails during pre-mRNA processing. However, in contrast known proteins, cytoplasmic predominantly early stages development and adult ovarian tissue. Biochemical experiments indicate binds with specificity this requires RRM domain. Mouse human proteins were also...
mRNA-specific polyadenylation can be assayed in vitro by using synthetic RNAs that end at or near the natural cleavage site. This reaction requires highly conserved sequence AAUAAA. At least two distinct nuclear components, an AAUAAA specificity factor and poly(A) polymerase, are required to catalyze reaction. In this study, we identified structural features of RNA substrate critical for polyadenylation. We found a contained only 11 nucleotides, which first six were AAUAAA, underwent...