Characterization of specific protein-RNA complexes associated with the coupling of polyadenylation and last-intron removal

Polyadenylation and splicing are highly coordinated on substrate RNAs capable of coupled polyadenylation and splicing. Individual elements of both splicing and polyadenylation signals are required for the in vitro coupling of the processing reactions. In order to understand more about the coupling mechanism, we examined specific protein-RNA complexes formed on RNA substrates, which undergo coupled splicing and polyadenylation. We hypothesized that formation of a coupling complex would be adversely affected by mutations of either splicing or polyadenylation elements known to be required for coupling. We defined three specific complexes (A(C)', A(C), and B(C)) that form rapidly on a coupled splicing and polyadenylation substrate, well before the appearance of spliced and/or polyadenylated products. The A(C)' complex is formed by 30 s after mixing, the A(C) complex is formed between 1 and 2 min after mixing, and the B(C) complex is formed by 2 to 3 min after mixing. A(C)' is a precursor of A(C), and the A(C)' and/or A(C) complex is a precursor of B(C). Of the three complexes, B(C) appears to be a true coupling complex in that its formation was consistently diminished by mutations or experimental conditions known to disrupt coupling. The characteristics of the A(C)' complex suggest that it is analogous to the spliceosomal A complex, which forms on splicing-only substrates. Formation of the A(C)' complex is dependent on the polypyrimidine tract. The transition from A(C)' to A(C) appears to require an intact 3'-splice site. Formation of the B(C) complex requires both splicing elements and the polyadenylation signal. A unique polyadenylation-specific complex formed rapidly on substrates containing only the polyadenylation signal. This complex, like the A(C)' complex, formed very transiently on the coupled splicing and polyadenylation substrate; we suggest that these two complexes coordinate, resulting in the B(C) complex. We also suggest a model in which the coupling mechanism may act as a dominant checkpoint in which aberrant definition of one exon overrides the normal processing at surrounding wild-type sites.     

The spliceosome assembly pathway in mammalian extracts.

A mammalian splicing commitment complex was functionally defined by using a template commitment assay. This complex was partially purified and shown to be a required intermediate for complex A formation. The productive formation of this commitment complex required both splice sites and the polypyrimidine tract. U1 small nuclear ribonucleoprotein (snRNP) was the only spliceosomal U snRNP required for this formation. A protein factor, very likely U2AF, is probably involved in the formation of the splicing commitment complex. From the kinetics of appearance of complex A and complex B, it was previously postulated that complex A represents a functional intermediate in spliceosome assembly. Complex A was partially purified and shown to be a required intermediate for complex B (spliceosome) formation. Thus, a spliceosome pathway is for the first time supported by direct biochemical evidence: RNA+U1 snRNP+?U2 auxiliary factor+?Y----CC+U2 snRNP+Z----A+U4/6,5 snRNPs+ beta----B.     

Probing the role of IFT particle complex A and B in flagellar entry and exit of IFT-dynein in Chlamydomonas

Mediating the transport of flagellar precursors and removal of turnover products, intraflagellar transport (IFT) is required for flagella assembly and maintenance. The IFT apparatus is composed of the anterograde IFT motor kinesin II, the retrograde IFT motor IFT-dynein, and IFT particles containing two complexes, A and B. In order to have a balanced two-way transportation, IFT-dynein has to be carried into flagella and transported to the flagellar tip by kinesin II, where it is activated to drive the retrograde IFT back to the flagellar base. In this study, we investigated the role of complex A and complex B in the flagellar entry and exit of IFT-dynein. We showed that regardless of the amount of complex A, IFT-dynein accumulated proportionally to the amount of complex B in the flagella of fla15/ift144 and fla17-1/ift139, two complex A temperature-sensitive mutants. Complex A was depleted from both cellular and flagellar compartments in fla15/ift144 mutant. However, in fla17-1/ift139 mutant, the flagellar level of complex A was at the wild-type level, which was in radical contrast to the significantly reduced cellular amount of complex A. These results support that complex A is not required for the flagellar entry of IFT-dynein, but might be essential for the lagellar exit of IFT-dynein. Additionally, we confirmed the essential role of IFT172, a complex B subunit, in the flagellar entry of IFT-dynein. These results indicate that complexes A and B play complementary but distinct roles for IFT-dynein, with complex B carrying IFT-dynein into the flagella while complex A mediates the flagellar exit of IFT-dynein.     

Association of U2 snRNP with the spliceosomal complex E.

In metazoans, the E complex is operationally defined as an ATP-independent spliceosomal complex that elutes as a single peak on a gel filtration column and can be chased into spliced products in the presence of an excess of competitor pre-mRNA. The A complex is the first ATP-dependent functional spliceosomal complex. U1 snRNP first binds tightly to the 5'splice site in the E complex and U2 snRNP first binds tightly to the branch site in the A complex. In this study, we have generated and characterized a monoclonal antibody (mAb 4G8) directed against SAP 62, a component of U2 snRNP and a subunit of the essential mammalian splicing factor SF3a. We show that this antibody is highly specific for SAP 62, detecting only SAP 62 on Western blots and immunoprecipitating only SAP 62 from nuclear extracts. The anti-SAP 62 antibody also immunoprecipitates U2 snRNP and the A complex. Significantly, however, we find that the E complex is also efficiently immunoprecipitated by the anti-SAP 62 antibody. This antibody does not cross-react with any E complex-specific components, indicating that SAP 62 itself is associated with the E complex. To determine whether other U2 snRNP components are associated with the E complex, we used antibodies to the U2 snRNP proteins B"and SAP 155. These antibodies also specifically immunoprecipitate the E complex. These observations indicate that U2 snRNP is associated with the E complex. However, we find that U2 snRNP is not as tightly bound in the E complex as it is in the A complex. The possible significance of the weak association of U2 snRNP with the E complex is discussed.     

Separation of protein synthesis initiation factor eIF4A from a p220-associated cap binding complex activity

A cap binding complex activity was purified from HeLa cells by a procedure which does not depend on the use of cap-affinity chromatography. The activity co-purified with a Mr 220000 polypeptide (p220), but not with elF4A. The active complex therefore differs from eIF4F, the complex purified by cap analog-affinity chromatography, in that it lacks the Mr 50000 subunit which is antigenically identical to elF4A. The activities of elF4F, CBP I and the eIF4A free complex purified here were compared in a fractionated system translating capped globin mRNA. Results indicate that the two complexes have similar activities and that they perform a function which cannot be provided by CBP I alone. Cap binding complex activity can be partly separated from eIF4A activity on sucrose gradients, thus eIF4A provides a function that is distinct from cap binding complex activity. The results indicate that eIF4A can be physically separated from the cap binding complex without affecting the ability of the remaining structure to function in an in vitro translation system. They suggest that the eIF4A-free complex may provide a function that is not a property of either CBP I or of eIF4A, but may be a property of p220.     

Photoaffinity labeling of the cap-binding protein complex with ATP/dATP. Differential labeling of free eukaryotic initiation factor 4A and the eukaryotic initiation factor 4A component of the cap-binding protein complex with [alpha-32P]ATP/dATP

It has been suggested that the cap-binding protein complex is involved in ATP-mediated melting of 5'-mRNA secondary structure to facilitate ribosome binding during initiation of translation in eukaryotic cells (Edery, I., Lee, K. A. W., and Sonenberg, N. (1984) Biochemistry 23, 2456-2462). Consequently, we have studied the interaction of dATP/ATP with the eukaryotic cap-binding protein complex by UV photoaffinity labeling. UV irradiation of the cap-binding protein complex in the presence of [alpha-32P]dATP/ATP resulted in the cross-linking of this compound to the 50-kDa polypeptide of the complex. This polypeptide is almost identical to the previously characterized eukaryotic initiation factor (eIF) 4A. We examined the ability of dATP/ATP to cross-link to eIF-4A and found that it cross-links less efficiently (approximately 60-fold on a molar basis) compared to the cross-linking obtained for the eIF-4A component of the cap-binding protein complex. Irradiation of purified eIF-4A together with the cap-binding protein complex in the presence of [alpha-32P]dATP resulted in greater than additive labeling of the eIF-4A component of the cap-binding protein complex and purified eIF-4A, suggesting a synergistic interaction between purified eIF-4A, the cap-binding protein complex, and dATP/ATP. We also report that photoaffinity labeling of eIF-4A and the eIF-4A component in the cap-binding protein complex is stimulated by eIF-4B, but not by other initiation factors or mRNA.     

Reconstitution in vitro of V1 complex of Thermus thermophilus V-ATPase revealed that ATP binding to the A subunit is crucial for V1 formation

Vacuolar-type H(+)-ATPase (V-ATPase or V-type ATPase) is a multisubunit complex comprised of a water-soluble V(1) complex, responsible for ATP hydrolysis, and a membrane-embedded V(o) complex, responsible for proton translocation. The V(1) complex of Thermus thermophilus V-ATPase has the subunit composition of A(3)B(3)DF, in which the A and B subunits form a hexameric ring structure. A central stalk composed of the D and F subunits penetrates the ring. In this study, we investigated the pathway for assembly of the V(1) complex by reconstituting the V(1) complex from the monomeric A and B subunits and DF subcomplex in vitro. Assembly of these components into the V(1) complex required binding of ATP to the A subunit, although hydrolysis of ATP is not necessary. In the absence of the DF subcomplex, the A and B monomers assembled into A(1)B(1) and A(3)B(3) subcomplexes in an ATP binding-dependent manner, suggesting that ATP binding-dependent interaction between the A and B subunits is a crucial step of assembly into V(1) complex. Kinetic analysis of assembly of the A and B monomers into the A(1)B(1) heterodimer using fluorescence resonance energy transfer indicated that the A subunit binds ATP prior to binding the B subunit. Kinetics of binding of a fluorescent ADP analog, N-methylanthraniloyl ADP (mant-ADP), to the monomeric A subunit also supported the rapid nucleotide binding to the A subunit.     

Inter-species variation in the oligomeric states of the higher plant Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase

In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein-protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the 'non-regulatory' A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed.     

Components required for in vitro cleavage and polyadenylation of eukaryotic mRNA.

We have studied in vitro cleavage/polyadenylation of precursor RNA containing herpes simplex virus type 2 poly A site sequences and have analyzed four RNA/protein complexes which form during in vitro reactions. Two complexes, A and B, form extremely rapidly and are then progressively replaced by a third complex, C which is produced following cleavage and polyadenylation of precursor RNA. Substitution of ATP with cordycepin triphosphate prevents polyadenylation and the formation of complex C however a fourth complex, D results which contains cleaved RNA. A precursor RNA lacking GU-rich downstream sequences required for efficient cleavage/polyadenylation fails to form complex B and produces a markedly reduced amount of complex A. As these GU-rich sequences are required for efficient cleavage, this establishes a relationship between complex B formation and cleavage/polyadenylation of precursor RNA in vitro. The components required for in vitro RNA processing have been separated by fractionation of the nuclear extract on Q-Sepharose and Biorex 70 columns. A Q-Sepharose fraction forms complex B but does not process RNA. Addition of a Biorex 70 fraction restores cleavage activity at the poly A site but this fraction does not appear to contribute to complex formation. Moreover, in the absence of polyethylene glycol, precursor RNA is not cleaved and polyadenylated, however, complexes A and B readily form. Thus, while complex B is necessary for in vitro cleavage and polyadenylation, it may not contain all the components required for this processing.     

Iron-sulfur cluster N7 of the NADH:ubiquinone oxidoreductase (complex I) is essential for stability but not involved in electron transfer

The NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli is composed of 13 subunits called NuoA through NuoN. It catalyzes the electron transfer from NADH to ubiquinone by a chain of redox groups consisting of one FMN and seven iron-sulfur clusters. The function of the additional, nonconserved cluster N7 located on NuoG is not known. It has been speculated that it is not involved in electron transfer, due to its distance of more than 20 A from the electron transfer chain. Dithionite-reduced minus NADH-reduced EPR difference spectra of complex I and of a soluble fragment containing NuoG revealed for the first time the EPR spectrum of N7 in the complex. Individual mutation of the cysteines ligating this cluster to alanine led to a decreased amount of complex I in the membrane without affecting the electron transfer activity. Sucrose gradient centrifugation revealed that the complex from the C230A and C233A mutants decayed in detergent solution while the C237A and C265A mutant complex was stable. Cluster N7 was detectable in the latter mutants but with shifted g-values, indicating a different ligation of N7. Thus, N7 is essential for the stability of the complex but is not involved in electron transfer.     

Characterization of an RNA-a protein complex isolated from the RNA bacteriophage M12.

The properties of an RNA-A protein complex isolated from the RNA bacteriophage M12 are described. The molar ratio of RNA to A protein in the complex is estimated to be 1:1. In sucrose gradients, the complex sediments like free RNA molecules. In contrast to RNA alone, which can only infect spheroplasts, the RNA-A protein complex infects intact Escherichia coli cells and produces infectious progeny particles like the original phage. Evidence is presented that the infection of the host cells by the complex takes place via F pili. All of the infectivity disappears if the ionic bonds of RNA to A protein in the complex are dissociated in 0.5 M sodium chloride buffer at 37 degrees C. Furthermore, the kinetics of complex dissociation and loss of infectivity are the same, implying that the binding of A protein to the RNA is a prerequisite for infectivity on intact host cells.     

Electrophoretic separation of complexes involved in the splicing of precursors to mRNAs

Splicing complexes were analyzed by electrophoresis on a native low-percentage polyacrylamide gel. Two distinct heparin-resistant complexes, A and B, are assembled specifically on an RNA precursor containing authentic 5' and 3' splice sites. This assembly is ATP-dependent. Kinetic experiments suggest that complex A is converted with time to a larger, slower migrating complex B. Complexes A and B detected by gel electrophoresis correspond to material sedimenting at 25S and 35S, respectively. Substrate RNA containing only the 3' splice site is capable of forming the smaller complex A but not complex B. Complex A protects sequences upstream of the 3' splice site, encompassing the branch site and polypyrimidine tract from digestion by RNAase T1. U2 snRNA, but not U1 snRNA was detected in both complexes A and B by Northern hybridization analysis. Interestingly, an endogenous large complex containing U2 snRNP could be detected in nuclear extracts.     

Binding mode of nucleosome-assembly protein (AP-I) and histones

Studies were made on the binding mode of the nucleosome-assembly protein AP-I with histones H2A + H2B and/or H3 + H4. Histones H2A + H2B bound with AP-I to form a 7-S complex which contained twice as much AP-I (by weight) as histones. Histone H3 + H4 formed an 8-S complex with AP-I. The 7-S and 8-S complexes did not form a new complex when mixed, but significant amounts of two histone pairs were assembled into a 12-S complex on mixing the (H2A + H2B)--AP-I complex (7-S) with free H3 + H4. In contrast, when the (H3 + H4)--AP-I complex (8-S) was incubated with free H2A + H2B, almost no assembly occurred, but the 7-S complex of H2A + H2B was newly formed. Binding studies by enzyme-linked immunosorbent assay showed that AP-I bound with H2A + H2B faster than with H3 + H4. From these results, it is suggested that AP-I has a higher binding affinity for histone H2A + H2B than for H3 + H4, and that the 7-S complex is an intermediate in formation of the 12-S octamer complex (H2A + H2B + H3 + H4)2.     

ADP-binding to origin recognition complex of Saccharomyces cerevisiae

The origin recognition complex (ORC), a possible initiator of chromosomal DNA replication in eukaryotes, binds to ATP through its subunits Orc1p and Orc5p. Orc1p possesses ATPase activity. As for DnaA, the Escherichia coli initiator, the ATP-DnaA complex is active but the ADP-DnaA complex is inactive for DNA replication and, therefore, the ATPase activity of DnaA inactivates the ATP-DnaA complex to suppress the re-initiation of chromosomal DNA replication. We investigated ADP-binding to ORC by a filter-binding assay. The K(d) values for ADP-binding to wild-type ORC and to ORC-1A (ORC containing Orc1p with a defective Walker A motif) were less than 10nM, showing that Orc5p can bind to ADP with a high affinity, similar to ATP. ORC-5A (ORC containing Orc5p with a defective Walker A motif) did not bind to ADP, suggesting that the ADP-Orc1p complex is too unstable to be detected by the filter-binding assay. ADP dissociated more rapidly than ATP from wild-type ORC and ORC-1A. Origin DNA fragments did not stimulate ADP-binding to any type of ORC. In the presence of ADP, ORC could not bind to origin DNA in a sequence-specific manner. Thus, in eukaryotes, the ADP-ORC complex may be unable to initiate chromosomal DNA replication, and in this it resembles the ADP-DnaA complex in prokaryotes. However, overall control may be different. In eukaryotes, the ADP-ORC complex is unstable, suggesting that the ADP-ORC complex might rapidly become an ATP-ORC complex; whereas in prokaryotes, ADP remains bound to DnaA, keeping DnaA inactive, and preventing re-initiation for some periods.     

Multiple host shifts between distantly related plants, Juglandaceae and Ericaceae, in the leaf-mining moth Acrocercops leucophaea complex (Lepidoptera: Gracillariidae)

Insect herbivores such as gall formers and leaf miners are often highly specialized and adapted to their respective natal host plants. Due to the specialization and adaptation, it is presumed that host shifts readily occur among closely related plant species. Leaf-mining moths, the Acrocercops leucophaea complex, consist of three species, A. leucophaea, A. defigurata, and A. transecta. Larvae of all the species of the complex feed on Juglandaceae plants, but A. leucophaea and A. transecta are also associated with an Ericaceae plant, which is quite distantly related to Juglandaceae. Such a host utilization as in this species complex is very rare among phytophagous insects. In the present study, we estimate the history of host shifts by reconstructing the phylogeny of the A. leucophaea complex using molecular data (partial sequence of mitochondrial COI, 12S rDNA, and ND5). Parsimony and maximum likelihood analyses indicated that the common ancestor of the A. leucophaea complex used Juglandaceae only, and that the association with Ericaceae has evolved in A. leucophaea and A. transecta independently. Parametric bootstrap analysis also supported multiple origins of the association with Ericaceae in this complex. These results imply that there are ecological and biochemical factors that promote host shifting between Juglandaceae and Ericaceae despite the two families being not closely related.     

Purification of aspartate transcarbamylase from Drosophila melanogaster.

A purification procedure is described by which aspartate transcarbamylase was obtained from cultured cells of Drosophila melanogaster as part of a high-molecular-weight enzyme complex. The complex is shown to contain several polypeptides. An antiserum directed against the complex enzyme inhibited in vitro the activity of aspartate transcarbamylase, carbamylphosphate synthetase and dihydro-orotase which were shown to copurify on a sucrose gradient and by gel electrophoresis. A fast preparation procedure using this antiserum yielded a 220 000-molecular-weight protein in addition to the polypeptides present in the complex. A purification procedure is also described to obtain aspartate transcarbamylase from second instar larvae of Drosophila. At this stage, the enzyme is not complexed with carbamylphosphate synthetase and dihydro-orotase but exhibits the same molecular weight as the aspartate transcarbamylase moiety found in the high-molecular-weight complex of cultured cells.