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PubMed1997

The alternative splicing factor PSI regulates P-element third intron splicing in vivo.

Splicing of the Drosophila P-element third intron (IVS3) is inhibited in somatic cells, restricting transposase expression to the germ line. Somatic inhibition of IVS3 splicing involves the assembly of a multiprotein complex on a regulatory sequence in the IVS3 5' exon. The P-element somatic inhibitor protein (PSI) is a component of this ribonucleoprotein complex and is required for inhibition of IVS3 splicing in vitro. The soma-specific expression pattern of PSI suggests that its low abundance in the germ line allows IVS3 splicing. We demonstrate that ectopic expression of PSI in the female germ line is sufficient to repress splicing of an IVS3 reporter transgene. We also show that IVS3 splicing is activated in somatic embryonic cells in the presence of an antisense PSI ribozyme. These results support the model that PSI is a tissue-specific regulator of IVS3 splicing in vivo.

Alternative Splicing↗
PubMed1997

SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast.

Yeast core telomeric heterochromatin can silence adjacent genes and requires RAP1, SIR2, SIR3, and SIR4 and histones H3 and H4 for this telomere position effect. SIR3 overproduction can extend the silenced domain. We examine here the nature of these multiprotein complexes. SIR2 and SIR4 were immunoprecipitated from whole-cell extracts. In addition, using formaldehyde cross-linking we have mapped SIR2, SIR4, and RAP1 along telomeric chromatin before and after SIR3 overexpression. Our data demonstrate that SIR2 and SIR4 interact in a protein complex and that SIR2, SIR3, SIR4, and RAP1 map to the same sites along telomeric heterochromatin in wild-type cells. However, when overexpressed, SIR3 spreads along the chromosome and its interactions are dominant to those of SIR4 and especially SIR2, whose detection is decreased in extended heterochromatin. RAP1 binding at the core region is unaffected by SIR3 overproduction and RAP1 shows no evidence of spreading. Thus, we propose that the structure of core telomeric heterochromatin differs from that extended by SIR3.

Blotting, Western↗
PubMed1997

A new regulatory protein, KSRP, mediates exon inclusion through an intronic splicing enhancer.

We have purified and cloned a new splicing factor, KSRP. KSRP is a component of a multiprotein complex that binds specifically to an intronic splicing enhancer element downstream of the neuron-specific c-src N1 exon. This 75-kD protein induces the assembly of five other proteins, including the heterogeneous nuclear ribonucleoprotein F, onto the splicing enhancer. The sequence of the KSRP cDNA indicates that the protein contains four K homology RNA-binding domains and an unusual carboxy-terminal domain. KSRP is similar to two proteins, FUSE-binding protein and P-element somatic inhibitor. KSRP is expressed in both neural and non-neural cell lines, although it is present at higher levels in neural cells. Antibodies specific for KSRP inhibit the splicing of the N1 exon in vitro. Moreover, this inhibition of N1 splicing can be rescued by the addition of purified KSRP. KSRP is likely to regulate splicing from a number of intronic splicing enhancer sequences.

Amino Acid Sequence↗
PubMed2003

Hierarchical assembly of the budding yeast kinetochore from multiple subcomplexes.

Kinetochores are multiprotein complexes that assemble on centromeric DNA and attach chromosomes to spindle microtubules. Over the past six years, the number of proteins known to localize to the Saccharomyces cerevisiae kinetochore has increased from around 10 to over 60. However, relatively little is known about the protein-protein interactions that mediate kinetochore assembly or about the overall structure of microtubule-attachment sites. Here we used biophysical techniques, affinity purification, mass spectrometry, and in vivo assays to examine the state of association of 31 centromere-binding proteins, including six proteins newly identified as kinetochore subunits. We found that yeast kinetochores resemble transcriptional enhancers in being composed of at least 17 discrete subcomplexes that assemble on DNA to form a very large structure with a mass in excess of 5 MD. Critical to kinetochore assembly are proteins that bridge subunits in direct contact with DNA and subunits bound to microtubules. We show that two newly identified kinetochore complexes, COMA (Ctf19p-Okp1p-Mcm21p-Ame1p) and MIND (Mtw1p including Nnf1p-Nsl1p-Dsn1p) function as bridges. COMA, MIND, and the previously described Ndc80 complex constitute three independent and essential platforms onto which outer kinetochore proteins assemble. In addition, we propose that the three complexes have different functions with respect to force generation and MT attachment.

Electrophoresis, Polyacrylamide Gel↗
PubMed1998

Histone deacetylase activity of Rpd3 is important for transcriptional repression in vivo.

Eukaryotic organisms from yeast to human contain a multiprotein complex that includes Rpd3 histone deacetylase and Sin3 corepressor. The Sin3-Rpd3 complex, when recruited to promoters by specific DNA-binding proteins, can direct transcriptional repression of specific classes of target genes. It has been proposed that the histone deacetylase activity of Rpd3 is important for repression, but direct evidence is lacking. Here, we describe four Rpd3 derivatives with mutations in evolutionarily invariant histidine residues in a putative deacetylation motif. These Rpd3 mutants lack detectable histone deacetylase activity in vitro, but interact normally with Sin3 in vivo. In yeast cells, these catalytically inactive mutants are defective for transcriptional repression. They retain some residual Rpd3 function in vivo, however, suggesting that repression by the Sin3-Rpd3 complex may not be attributable exclusively to its intrinsic histone deacetylase activity. Finally, we show that a human Rpd3 homolog can interact with yeast Sin3 and repress transcription when artificially recruited to a promoter. These results suggest that the histone deacetylase activity of Rpd3 is important, but perhaps not absolutely required, for transcriptional repression in vivo.

Acetylation↗
PubMed1999

Reconstitution of a minimal RNA degradosome demonstrates functional coordination between a 3' exonuclease and a DEAD-box RNA helicase.

The RNA degradosome is a multiprotein complex required for the degradation of highly structured RNAs. We have developed a method for reconstituting a minimal degradosome from purified proteins. Our results demonstrate that a degradosome-like complex containing RNase E, PNPase, and RhlB can form spontaneously in vitro in the absence of all other cellular components. Moreover, ATP-dependent degradation of the malEF REP RNA by the reconstituted, minimal degradosome is indistinguishable from that of degradosomes isolated from whole cells. The Rne protein serves as an essential scaffold in the reconstitution process; however, RNase E activity is not required. Rather, Rne coordinates the activation of RhlB dependent on a 3' single-stranded extension on RNA substrates. A model for degradosome-mediated degradation of structured RNA is presented with its implications for mRNA decay in Escherichia coli.

ATP-Binding Cassette Transporters↗
PubMed1990

Interactions of the Oct-1 POU subdomains with specific DNA sequences and with the HSV alpha-trans-activator protein.

Trans-activation by the herpes simplex virus (HSV) protein, alpha TIF (VP16), is dependent on an inducible enhancer sequence that contains a homolog of the octamer element. An ordered series of multiprotein complexes can be assembled on this enhancer, requiring the interactions of Oct-1, alpha TIF, and two additional cellular factors (C1 and C2). Oct-1 binds to the octamer homolog, whereas alpha TIF, also a sequence-specific DNA-binding protein, recognizes sequences within the HSV enhancer core. The partially purified C1 factor interacts directly with alpha TIF in the absence of DNA and is required to form a stabile Oct-1/alpha TIF/C1 factor complex. The POU domain of Oct-1 is a bipartite sequence recognition structure, as both the POU-specific box and the POU homeo box contribute directly to the recognition of the octamer element. Surprisingly, the POU homeo box alone is sufficient to direct the cooperative binding of alpha TIF and to assemble the Oct-1/alpha TIF/C1 factor complex.

Animals↗
PubMed1992

Recognition of the surface of a homeo domain protein.

Homeo domain proteins exhibit distinct biological functions with specificities that cannot be predicted by their sequence specificities for binding DNA. Recognition of the surface of the Oct-1 POU homeo domain provides a general model for the contribution of selective protein-protein interactions to the functional specificity of the homeo domain family of factors. The assembly of Oct-1 into a multiprotein complex on the herpes simplex virus alpha/IE enhancer is specified by the interactions of its homeo domain with ancillary factors. This complex (C1 complex) is composed of the viral alpha TIF protein (VP16), Oct-1, and one additional cellular component, the C1 factor. Variants of the Oct-1 POU homeo domain were generated by site-directed mutagenesis, which altered the residues predicted to form the exposed surface of the domain-DNA complex. Proteins with single amino acid substitutions on the surface of either helix 1 or 2 of the Oct-1 POU homeo domain had decreased abilities to form the C1 complex. The behavior of these mutants in a cooperative DNA-binding assay with alpha TIF suggested that the Oct-1 POU homeo domain is principally recognized by alpha TIF in the C1 complex. The preferential recognition of Oct-1 over the closely related Oct-2 protein is critically influenced by a single residue on the surface of helix 1 because the introduction of this residue into the Oct-2 POU homeo domain significantly enhanced its ability to form a C1 complex.

Amino Acid Sequence↗
PubMed1994

Regulation of tissue-specific P-element pre-mRNA splicing requires the RNA-binding protein PSI.

Binding of a multiprotein complex to a 5' exon inhibitory element appears to repress splicing of the Drosophila P-element third intron (IVS3) in the soma. We have purified 97- and 50-kD proteins that interact specifically with the inhibitory element using RNA affinity chromatography. Antibodies specific for the 97-kD protein relieve inhibition of IVS3 splicing in somatic extracts, providing direct evidence that inhibition requires this protein, P-element somatic inhibitor (PSI). We identify the 50-kD protein as hrp48, a protein similar to the mammalian splicing factor hnRNP A1, and show that hrp48 recognizes specific nucleotides in a pseudo-5' splice site within the inhibitory element. The results indicate that PSI is an alternative splicing factor that regulates tissue-specific splicing, probably through interactions with generally expressed factors such as hrp48.

Alternative Splicing↗
PubMed1995

Synergistic interactions between two distinct elements of a regulated splicing enhancer.

Regulated alternative splicing of doublesex (dsx) pre-mRNA requires a splicing enhancer designated the dsx repeat element (dsxRE) that contains six copies of a 13-nucleotide repeat sequence. Previous studies have shown that the activity of the dsxRE requires the splicing regulators Transformer (Tra) and Transformer 2 (Tra2), and one or more members of the SR family of general splicing factors. In this paper we identify a purine-rich enhancer (PRE) sequence within the dsxRE, and show that this element functionally synergizes with the repeat sequences. In vitro binding studies show that the PRE is required for specific binding of Tra2 to the dsxRE, and that Tra and SR proteins bind cooperatively to the dsxRE in the presence or absence of the PRE. Thus positive control of dsx pre-mRNA splicing requires the Tra- and Tra2-dependent assembly of a multiprotein complex on at least two distinct enhancer elements.

Alternative Splicing↗
PubMed1998

Transcription regulation by repressosome and by RNA polymerase contact.

The original model of repression of transcription initiation is steric interference of RNA polymerase binding to a promoter by its repressor protein bound to a DNA site that overlaps the promoter. From the results described here, we propose two other mechanisms of repressor action, both of which involve formation of higher-order DNA-multiprotein complexes. These models also explain the problem of RNA polymerase gaining access to a promoter in the condensed nucleoid in response to an inducing signal to initiate transcription.

Amino Acid Sequence↗
PubMed2006

The fertilization-induced DNA replication factor MCM6 of maize shuttles between cytoplasm and nucleus, and is essential for plant growth and development.

The eukaryotic genome is duplicated exactly once per cell division cycle. A strategy that limits every replication origin to a single initiation event is tightly regulated by a multiprotein complex, which involves at least 20 protein factors. A key player in this regulation is the evolutionary conserved hexameric MCM2-7 complex. From maize (Zea mays) zygotes, we have cloned MCM6 and characterized this essential gene in more detail. Shortly after fertilization, expression of ZmMCM6 is strongly induced. During progression of zygote and proembryo development, ZmMCM6 transcript amounts decrease and are low in vegetative tissues, where expression is restricted to tissues containing proliferating cells. The highest protein amounts are detectable about 6 to 20 d after fertilization in developing kernels. Subcellular localization studies revealed that MCM6 protein shuttles between cytoplasm and nucleoplasm in a cell cycle-dependent manner. ZmMCM6 is taken up by the nucleus during G1 phase and the highest protein levels were observed during late G1/S phase. ZmMCM6 is excluded from the nucleus during late S, G2, and mitosis. Transgenic maize was generated to overexpress and down-regulate ZmMCM6. Plants displaying minor antisense transcript amounts were reduced in size and did not develop cobs to maturity. Down-regulation of ZmMCM6 gene activity seems also to affect pollen development because antisense transgenes could not be propagated via pollen to wild-type plants. In summary, the transgenic data indicate that MCM6 is essential for both vegetative as well as reproductive growth and development in plants.

Active Transport, Cell Nucleus↗
PubMed2006

The Arabidopsis tetratricopeptide repeat-containing protein TTL1 is required for osmotic stress responses and abscisic acid sensitivity.

Mutations in the Arabidopsis (Arabidopsis thaliana) TETRATRICOPEPTIDE-REPEAT THIOREDOXIN-LIKE 1 (TTL1) cause reduced tolerance to NaCl and osmotic stress that is characterized by reduced root elongation, disorganization of the root meristem, and impaired osmotic responses during germination and seedling development. Expression analyses of genes involved in abscisic acid (ABA) biosynthesis and catabolism suggest that TTL1 is not involved in the regulation of ABA levels but is required for ABA-regulated responses. TTL1 regulates the transcript levels of several dehydration-responsive genes, such as the transcription factor DREB2A, and genes encoding dehydration response proteins, such as ERD1 (early response to dehydration 1), ERD3, and COR15a. The TTL1 gene encodes a novel plant protein with tetratricopeptide repeats and a region with homology to thioredoxin proteins. Based on homology searches, there are four TTL members in the Arabidopsis genome with similar intron-exon structure and conserved amino acid domains. Proteins containing tetratricopeptide repeat motifs act as scaffold-forming multiprotein complexes and are emerging as essential elements for plant hormonal responses (such as gibberellin responses and ethylene biosynthesis). In this report, we identify TTL1 as a positive regulator of ABA signaling during germination and seedling development under stress.

Abscisic Acid↗
PubMed2003

The COP9 signalosome interacts physically with SCF COI1 and modulates jasmonate responses.

The COP9 signalosome (CSN) is an evolutionarily conserved, nucleus-enriched multiprotein complex. CSN plays roles in photomorphogenesis, auxin response, and floral organ formation, possibly via the regulation of ubiquitin-proteasome-mediated protein degradation. COI1 encodes an F-box protein, which is a subunit of SCF(COI1) E3 ubiquitin ligase, and is required for jasmonate (JA) responses. Here, we demonstrate using coimmunoprecipitation and gel-filtration analyses that endogenous as well as epitope-tagged COI1 forms SCF(COI1) and associates directly with CSN in vivo. Like the coi1-1 mutant, CSN reduction-of-function plants exhibited a JA-insensitive root elongation phenotype and an absence of JA-induced-specific gene expression. Genome expression profile analyses indicated that JA-triggered genome expression is critically dependent on COI1 dosage. More importantly, most of the COI1-dependent JA-responsive genes also required CSN function, and CSN abundance was shown to be important for JA responses. Furthermore, we showed that both COI1 and CSN are essential for modulating the expression of genes in most cellular pathways responsive to JA. Thus, CSN and SCF(COI1) work together to control genome expression and promote JA responses.

Arabidopsis↗
PubMed2001

Crystallization and preliminary crystallographic study of the peptidoglycan-associated lipoprotein from Escherichia coli.

The peptidoglycan-associated lipoprotein (Pal) from Escherichia coli is part of the Tol--Pal multiprotein complex used by group A colicins to penetrate and kill cells. Pal homologues are found in many Gram-negative bacteria and the Tol--Pal system is thought to play a role in bacterial envelope integrity. The Pal protein comprises 152 amino acids. Crystals of the C-terminal 109-amino-acid fragment of the Pal protein have been produced. The crystals belong to the tetragonal space group I4(1), with unit-cell parameters a = b = 89.3, c = 67.2 A. There are two molecules in the asymmetric unit. Frozen crystals diffract to at least 2.8 A resolution using synchrotron radiation. Selenomethionine-substituted truncated Pal protein is currently being produced in order to use multiwavelength anomalous dispersion (MAD) for phasing.

Bacterial Outer Membrane Proteins↗
PubMed2001

Crystallization and preliminary crystallographic study of the periplasmic domain of the Escherichia coli TolR protein.

The TolR protein from Escherichia coli is part of the Tol-Pal multiprotein complex used by group A colicins to penetrate and kill cells. All genes of the Tol-Pal system are conserved in Gram-negative bacteria and this system is thought to play a role in the maintenance of the bacterial envelope integrity, although its exact function is not known. The TolR protein comprises 142 amino acids. The periplasmic domain of the TolR protein has been expressed, purified and crystallized. The crystals belong to the tetragonal space group P4(1)22, with unit-cell parameters a = 46.3, c = 178.0 A. There are one or two molecules in the asymmetric unit. Frozen crystals diffract to at least 3.2 A resolution using synchrotron radiation. Selenomethionine-substituted periplasmic TolR protein is currently being produced in order to use multiwavelength anomalous dispersion (MAD) for phasing.

Bacterial Proteins↗
PubMed2002

A CH domain-containing N terminus in NuMA?

Nuclear mitotic apparatus protein (NuMA) is an essential vertebrate component in organizing microtubule ends at spindle poles. The NuMA-dynactin/dynein motor multiprotein complex not only explains the transport of NuMA along spindle fibers but also is linked to the process of microtubule focusing. The interaction sites of NuMA to dynein/dynactin have not been mapped. In the yet functionally uncharacterized N terminus of NuMA, we predict a calponin-homology (CH) domain, a motif with binding activity for actin-like molecules. We substantiate the primary sequence analysis-based prediction with secondary structure and fold recognition analysis, and we propose the N-terminal CH domain of NuMA as a likely interaction site for actin-related protein 1 (Arp1) protein of the dynactin/dynein complex.

Amino Acid Sequence↗