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P D Zamore

Publications and source records attributed to P D Zamore.

15 recordsLinked to original sources
PubMed1999

The PUMILIO-RNA interaction: a single RNA-binding domain monomer recognizes a bipartite target sequence.

Translational repression of hunchback (hb) mRNA in the posterior of the Drosophila embryo requires two copies of a bipartite sequence, the Nanos Response Element (NRE), located in the 3' untranslated region of the mRNA. The PUMILIO (PUM) protein is thought to bind the NREs and thereby repress hb translation. The RNA-binding domain of PUM defines an evolutionarily conserved family of RNA-binding proteins, the PUM-Homology Domain (PUM-HD) proteins, which have been identified in yeast, plants, and animals. The PUM RNA-binding domain, the Drosophila PUM-HD (DmPUM-HD), has been shown previously to recognize nucleotides in both the 5' and 3' halves of the NRE, suggesting that a dimer of PUM might recognize one NRE. Here, we analyze the RNA-binding affinity and stoichiometry of the DmPUM-HD and find that one DmPUM-HD monomer binds independently and with equal affinity to each NRE (KD approximately 0.5 nM). We detect no cooperative interactions between DmPUM-HD monomers bound at adjacent sites. Our results imply that a single DmPUM-HD protein recognizes nucleotides in both the 5' and 3' NRE half-sites. Based on our estimate of the intraembryonic concentration of PUM (>40 nM), we propose that in vivo nearly all NREs are occupied by a PUM monomer.

Animals↗
PubMed1997

A CCHC metal-binding domain in Nanos is essential for translational regulation.

The Drosophila Nanos protein is a localized repressor of hunchback mRNA translation in the early embryo, and is required for the establishment of the anterior-posterior body axis. Analysis of nanos mutants reveals that a small, evolutionarily conserved, C-terminal region is essential for Nanos function in vivo, while no other single portion of the Nanos protein is absolutely required. Within the C-terminal region are two unusual Cys-Cys-His-Cys (CCHC) motifs that are potential zinc-binding sites. Using absorption spectroscopy and NMR we demonstrate that the CCHC motifs each bind one equivalent of zinc with high affinity. nanos mutations disrupting metal binding at either of these two sites in vitro abolish Nanos translational repression activity in vivo. We show that full-length and C-terminal Nanos proteins bind to RNA in vitro with high affinity, but with little sequence specificity. Mutations affecting the hunchback mRNA target sites for Nanos-dependent translational repression were found to disrupt translational repression in vivo, but had little effect on Nanos RNA binding in vitro. Thus, the Nanos zinc domain does not specifically recognize target hunchback RNA sequences, but might interact with RNA in the context of a larger ribonucleoprotein complex.

Amino Acid Sequence↗
PubMed1997

The Pumilio protein binds RNA through a conserved domain that defines a new class of RNA-binding proteins.

Translation of hunchback(mat) (hb[mat]) mRNA must be repressed in the posterior of the pre-blastoderm Drosophila embryo to permit formation of abdominal segments. This translational repression requires two copies of the Nanos Response Element (NRE), a 16-nt sequence in the hb[mat] 3' untranslated region. Translational repression also requires the action of two proteins: Pumilio (PUM), a sequence-specific RNA-binding protein; and Nanos, a protein that determines the location of repression. Binding of PUM to the NRE is thought to target hb(mat) mRNA for repression. Here, we show the RNA-binding domain of PUM to be an evolutionarily conserved, 334-amino acid region at the carboxy-terminus of the approximately 158-kDa PUM protein. This contiguous region of PUM retains the RNA-binding specificity of full-length PUM protein. Proteins with sequences homologous to the PUM RNA-binding domain are found in animals, plants, and fungi. The high degree of sequence conservation of the PUM RNA-binding domain in other far-flung species suggests that the domain is an ancient protein motif, and we show that conservation of sequence reflects conservation of function: that is, the homologous region from a human protein binds RNA with sequence specificity related to but distinct from Drosophila PUM.

Amino Acid Sequence↗
PubMed1993

RNA annealing activity is intrinsically associated with U2AF.

U2AF is a protein that is essential for the formation of the prespliceosome complex during pre-mRNA splicing. It contains two subunits, 65 and 35 kDa, although only the 65-kDa subunit has been shown to be essential for its splicing activity. Here, we show that the 65-kDa subunit mediates the annealing of complementary single-stranded RNAs or single-stranded DNAs. This activity was shown to reverse the action of RNA helicase A, an enzyme that catalyzes the displacement of duplex RNAs. The NH2-terminal region of the 65-kDa subunit of U2AF, containing arginine-serine (RS) dipeptides and basic amino acid sequences, was shown to be essential for the annealing of complementary sequences, RNA binding, and the inhibition of RNA helicase A activity. Thus, through the combined action of U2AF and RNA helicases, duplex RNA regions can be reversibly formed and displaced. Such reactions appear to be critical for pre-mRNA splicing, translation, and transcription.

Base Sequence↗
PubMed1993

The protein Sex-lethal antagonizes the splicing factor U2AF to regulate alternative splicing of transformer pre-mRNA.

Somatic sexual differentiation in Drosophila melanogaster involves a cascade of regulated splicing events and provides an attractive model system for the analysis of alternative splicing mechanisms. The protein Sex-lethal (Sxl) activates a female-specific 3' splice site in the first intron of transformer (tra) pre-mRNA while repressing an alternative non-sex-specific site. We have developed an in vitro system that recapitulates this regulation in a manner consistent with genetic, transfection and fly transformation studies. Using this system, we have determined the molecular basis of the splice site switch. Here we show that Sxl inhibits splicing to the non-sex-specific (default) site by specifically binding to its polypyrimidine tract, blocking the binding of the essential splicing factor U2AF. This enables U2AF to activate the lower-affinity female-specific site. A splicing 'effector' domain present in U2AF but absent from Sxl accounts for the different activities of these two polypyrimidine-tract-binding proteins: addition of the U2AF effector domain to Sxl converts it from a splicing repressor to an activator and renders it unable to mediate splice-site switching.

Alternative Splicing↗
PubMed1992

Cloning and intracellular localization of the U2 small nuclear ribonucleoprotein auxiliary factor small subunit.

U2 small nuclear ribonucleoprotein auxiliary factor (U2AF), an essential mammalian splicing factor, is composed of two subunits: a 65-kDa protein (U2AF65), which binds the pre-mRNA polypyrimidine tract and is required for in vitro splicing, and an associated 35-kDa protein (U2AF35). Here we report the isolation of a cDNA encoding U2AF35. U2AF35 contains sequence motifs found in several mammalian pre-mRNA splicing factors. We show directly that U2AF65 and U2AF35 interact with each other and delineate the regions of both proteins that mediate this interaction. Using anti-peptide antibodies against U2AF35, we show that the protein has the intracellular distribution characteristic of U2AF65. Both U2AF65 and U2AF35 are concentrated in a small number of nuclear foci corresponding to coiled bodies, subnuclear organelles first identified by light microscopy in 1903.

Amino Acid Sequence↗
PubMed1992

Cloning and domain structure of the mammalian splicing factor U2AF.

A complementary DNA clone encoding the large subunit of the essential mammalian pre-messenger RNA splicing component U2 snRNP auxiliary factor (U2AF65) has been isolated and expressed in vitro. It contains two functional domains: a sequence-specific RNA-binding region composed of three ribonucleoprotein-consensus sequence domains, and an arginine/serine-rich motif necessary for splicing but not for binding to pre-mRNA.

Amino Acid Sequence↗
PubMed1991

Mammalian nuclei contain foci which are highly enriched in components of the pre-mRNA splicing machinery.

The organization of the major snRNP particles in mammalian cell nuclei has been analysed by in situ labelling using snRNA-specific antisense probes made of 2'-OMe RNA. U3 snRNA is exclusively detected in the nucleolus while all the spliceosomal snRNAs are found in the nucleoplasm outside of nucleoli. Surprisingly, U2, U4, U5 and U6 snRNAs are predominantly observed in discrete nucleoplasmic foci. U1 snRNA is also present in foci but in addition is detected widely distributed throughout the nucleoplasm. An anti-peptide antibody specific for the non-snRNP splicing factor U2AF reveals it to have a similar distribution to U1 snRNA. Co-localization studies using confocal fluorescence microscopy prove that U2AF is present in the snRNA-containing foci. Antibody staining also shows the foci to contain snRNP-specific proteins and m3G-cap structures. The presence of major components of the nuclear splicing apparatus in foci suggests that these structures may play a role in pre-mRNA processing.

Antisense Elements (Genetics)↗
PubMed1991

Biochemical characterization of U2 snRNP auxiliary factor: an essential pre-mRNA splicing factor with a novel intranuclear distribution.

U2 auxiliary factor (U2AF) is a non-snRNP protein required for the binding of U2 snRNP to the pre-mRNA branch site. Purified U2AF comprises two polypeptides of 65 and 35 kd. We have performed biochemical complementation and immunological assays to characterize U2AF in greater detail. First, we use an extract lacking only U2AF activity to show that U2AF is an essential splicing factor. Second, we show that all U2AF activity in vitro resides in the 65 kd U2AF polypeptide. Third, based upon both immunological and functional criteria, we show that U2AF is evolutionarily conserved. Most significantly, a Drosophila melanogaster nuclear extract contains proteins that are antigenically related to both human U2AF polypeptides and can substitute for human U2AF in vitro. Finally, we show that U2AF has an unexpected intranuclear distribution. Although diffusely present throughout the nucleoplasm, U2AF is also concentrated in a small number (between one and five) of nuclear 'centers.' This localization differs strikingly from that reported for snRNP antigens and splicing factors. Our data, in conjunction with those in the accompanying paper [Carmo-Fonseca et al. (1991) EMBO J., 10, 195-206.], suggest that these centers represent novel aspects of nuclear organization.

Amino Acid Sequence↗
PubMed1989

Identification, purification, and biochemical characterization of U2 small nuclear ribonucleoprotein auxiliary factor.

Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA branch site is an early step in spliceosome assembly and appears to commit a pre-mRNA to the splicing pathway. We have shown previously that this ATP-dependent binding requires a non-rnRNP factor, U2 snRNP auxiliary factor (U2AF), in addition to U2 snRNP. In this report we have identified U2AF, purified it to homogeneity, and characterized its biochemical properties. Purified U2AF comprises roughly equimolar quantities of two polypeptides, approximately 65 kDa and approximately 35 kDa, which appear to be associated. Measured by ultraviolet crosslinking, the 65-kDa polypeptide binds specifically to the polypyrimidine tract/3' splice site region. U2AF binds rapidly at 4 degrees C in the absence of ATP and remains associated with the pre-mRNA following U2 snRNP binding. Thus, the simple binding of U2AF initiates mammalian spliceosome assembly by facilitating the ATP-dependent binding of U2 snRNP.

Adenosine Triphosphate↗
PubMed1988

Triosephosphate isomerase catalysis is diffusion controlled. Appendix: Analysis of triose phosphate equilibria in aqueous solution by 31P NMR.

The rates of the forward and reverse reactions of triosephosphate isomerase catalyzed by the wild-type and by a sluggish mutant enzyme have been studied in the absence and the presence of several viscosogenic agents. For the mutant enzyme, the kcat for which is some 10(3) times less than that for the wild-type enzyme, the value of kcat/Km with glyceraldehyde phosphate as substrate is almost unaffected by the presence of sucrose or glycerol, even though the concentration of the aldehyde form of the substrate is smaller because of hemiacetal formation. [The nature and relative amounts of the various forms of triose phosphate present in solution (free carbonyl forms, hydrates, dimers, hemiacetal adducts) have been evaluated by 31P NMR and are presented in the Appendix.] The viscosogenic agents cause the substrate to bind more tightly to the enzyme, roughly compensating for the lower substrate concentration. With dihydroxyacetone phosphate as substrate, the values of kcat/Km for the mutant enzyme increase with the addition of viscosogenic agent, consistent with the tighter binding of substrate without (in this case) any concomitant loss due to hemiketal formation. These results for the mutant enzyme (known to be limited in rate by an enolization step in the catalytic mechanism) can be used to interpret the behavior of the wild-type enzyme. Plots of the relative values of kcat/Km for catalysis by the wild-type enzyme (normalized with the corresponding data for the mutant enzyme) against the relative viscosity have slopes close to unity, as predicted by the Stokes-Einstein equation for a cleanly diffusive process. In the presence of polymeric viscosogenic additives such as poly(ethylene glycol), polyacrylamide, or ficoll, no effect on kcat/Km is seen for the wild-type enzyme, consistent with the expectation that molecular diffusion rates are unaffected by the macroviscosity and are only slowed by the presence of smaller agents that raise the microviscosity. These results show that the reaction catalyzed by the wild-type triosephosphate isomerase is limited by the rate at which glyceraldehyde phosphate encounters, or departs from, the active site.

Animals↗
PubMed1988

A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly.

Pre-mRNA splicing complex assembly is mediated by two specific pre-mRNA-snRNP interactions: U1 snRNP binds to the 5' splice site and U2 snRNP binds to the branch point. Here we show that unlike a purified U1 snRNP, which can bind to a 5' splice site, a partially purified U2 snRNP cannot interact with its target pre-mRNA sequence. We identify a previously uncharacterized activity, U2AF, that is required for the U2 snRNP-branch point interaction and splicing complex formation. Using RNA substrate exclusion and competition assays, we demonstrate that U2AF binds to the 3' splice site region prior to the U2 snRNP-branch point interaction. This provides an explanation for the necessity of the 3' splice site region in U2 snRNP binding and, hence, the first step of splicing.

Binding Sites↗