Association of nuclear matrix antigens with exon-containing splicing complexes.

mAbs raised against the human nuclear matrix (anti-NM)1 mAbs have been used to investigate the role of nuclear matrix antigens in pre-mRNA processing. The three anti-NM mAbs used in this study recognize antigens that are highly localized to nuclear matrix speckles. Surprisingly, all three of these mAbs preferentially immunoprecipitate splicing complexes containing exon sequences. The anti-NM mAbs efficiently immunoprecipitate the exon product complex but not complexes containing the lariat product after the second step of splicing. Two of the anti-NM mAbs completely inhibit pre-mRNA splicing in vitro. However, none of the anti-NM mAbs appear to recognize factors stably associated with splicing snRNPs. The three anti-NM mAbs predominantly react with distinct high molecular weight antigens, which belong to a class of nuclear proteins that selectively precipitate with Ser-Arg protein-splicing factors in the presence of high Mg2+ concentrations. Immunological, biochemical, and cell biological data indicate that two of the NM antigens are related to the defined set of Ser-Arg proteins. The results suggest the existence of an extended Ser-Arg family as a component of the nuclear matrix.

Splicing of messenger RNA precursors.

A general mechanism for the splicing of nuclear messenger RNA precursors in eukaryotic cells has been widely accepted. This mechanism, which generates lariat RNAs possessing a branch site, seems related to the RNA-catalyzed reactions of self-splicing introns. The splicing of nuclear messenger RNA precursors involves the formation of a multicomponent complex, the spliceosome. This splicing body contains at least three different small nuclear ribonucleoprotein particles (snRNPs), U2, U5, and U4 + U6. A complex containing precursor RNA and the U2 snRNP particle is a likely intermediate in the formation of the spliceosome.

Tat-SF1: cofactor for stimulation of transcriptional elongation by HIV-1 Tat.

Tat may stimulate transcriptional elongation by recruitment of a complex containing Tat-SF1 and a kinase to the human immunodeficiency virus-type 1 (HIV-1) promoter through a Tat-TAR interaction. A complementary DNA for the cellular activity, Tat-SF1, has been isolated. This factor is required for Tat trans-activation and is a substrate of an associated cellular kinase. Cotransfection with the complementary DNA for Tat-SF1 specifically modulates Tat activation. Tat-SF1 contains two RNA recognition motifs and a highly acidic carboxyl-terminal half. It is distantly related to EWS and FUS/TLS, members of a family of putative transcription factors with RNA recognition motifs that are associated with sarcomas.

Affinity chromatography of splicing complexes: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles in the spliceosome.

The splicing process, which removes intervening sequences from messenger RNA (mRNA) precursors is essential to gene expression in eukaryotic cells. This site-specific process requires precise sequence recognition at the boundaries of an intervening sequence, but the mechanism of this recognition is not understood. The splicing of mRNA precursors occurs in a multicomponent complex termed the spliceosome. Such an assembly of components is likely to play a key role in specifying those sequences to be spliced. In order to analyze spliceosome structure, a stringent approach was developed to obtain splicing complexes free of cellular contaminants. This approach is a form of affinity chromatography based on the high specificity of the biotin-streptavidin interaction. A minimum of three subunits: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles were identified in the 35S spliceosome structure, which also contains the bipartite RNA intermediate of splicing. A 25S presplicing complex contained only the U2 particle. The multiple subunit structure of the spliceosome has implications for the regulation of a splicing event and for its possible catalysis by ribozyme or ribozymes.

Site-specific modification of pre-mRNA: the 2'-hydroxyl groups at the splice sites.

A simple and efficient method for synthesizing long, site-specifically modified RNA molecules was developed whereby segments of RNA were joined with the use of bacteriophage T4 DNA ligase. A single hydrogen or O-methyl group was substituted for the 2'-hydroxyl group at either splice site of a nuclear pre-messenger RNA substrate. Splicing of the modified pre-messenger RNA's in vitro revealed that, although a 2'-hydroxyl is not absolutely required at either splice site, the 2'-hydroxyl at the 3' splice site is important for the second step of splicing. These results are compared to previous studies of analogous 2'-hydroxyl groups in the self-splicing Tetrahymena group I intron.

Structure-based design of transcription factors.

Computer modeling suggested that transcription factors with novel sequence specificities could be designed by combining known DNA binding domains. This structure-based strategy was tested by construction of a fusion protein, ZFHD1, that contained zinc fingers 1 and 2 from Zif268, a short polypeptide linker, and the homeodomain from Oct-1. The fusion protein bound optimally to a sequence containing adjacent homeodomain (TAATTA) and zinc finger (NGGGNG) subsites. When fused to an activation domain, ZFHD1 regulated promoter activity in vivo in a sequence-specific manner. Analysis of known protein-DNA complexes suggests that many other DNA binding proteins could be designed in a similar fashion.

Complementation by SR proteins of pre-mRNA splicing reactions depleted of U1 snRNP.

Individual small nuclear ribonucleoproteins (snRNPs) U1, U2, and U4/U6 were removed from nuclear extracts of HeLa cells by antisense affinity depletion. Addition of a highly purified preparation of SR proteins fully restored splicing activity in reactions depleted of U1 snRNP but did not reconstitute splicing in reactions depleted of the other snRNPs. Affinity selection experiments revealed that spliceosomes lacking U1 snRNA formed in the U1 snRNP-depleted reactions reconstituted with SR proteins. Thus, high concentrations of SR proteins facilitate the assembly of precursor messenger RNA (pre-mRNA) into a spliceosome in the absence of interactions with U1 snRNP.

Heterogeneous nuclear ribonucleoproteins: role in RNA splicing.

Splicing in vitro of a messenger RNA (mRNA) precursor (pre-mRNA) is inhibited by a monoclonal antibody to the C proteins (anti-C) of the heterogeneous nuclear RNA (hnRNA)-ribonucleoprotein (hnRNP) particles. This antibody, 4F4, inhibits an early step of the reaction: cleavage at the 3' end of the upstream exon and the formation of the intron lariat. In contrast, boiled 4F4, or a different monoclonal antibody (designated 2B12) to the C proteins, or antibodies to other hnRNP proteins (120 and 68 kilodaltons) and nonimmune mouse antibodies have no inhibitory effect. The 4F4 antibody does not prevent the adenosine triphosphate-dependent formation of a 60S splicing complex (spliceosome). Furthermore, the 60S splicing complex contains C proteins, and it can be immunoprecipitated with 4F4. Depletion of C proteins from the splicing extract by immunoadsorption with either of the two monoclonal antibodies to the C proteins (4F4 or 2B12) results in the loss of splicing activity, whereas mock-depletion with nonimmune mouse antibodies bodies has no effect. A 60S splicing complex does not form in a C protein-depleted nuclear extract. These results indicate an essential role for proteins of the hnRNP complex in the splicing of mRNA precursors.

Lariat RNA's as intermediates and products in the splicing of messenger RNA precursors.

The splicing of messenger RNA precursors in vitro proceeds through an intermediate that has the 5' end of the intervening sequence joined to a site near the 3' splice site. This lariat structure, which has been characterized for an adenovirus 2 major late transcript, has a branch point, with 2'-5' and 3'-5' phosphodiester bonds emanating from a single adenosine residue. The excised intervening sequence retains the branch site and terminates in a guanosine residue with a 3' hydroxyl group. The phosphate group at the splice junction between the two exons originates from the 3' splice site at the precursor.

The adenovirus major late transcription factor activates the rat gamma-fibrinogen promoter.

The major late transcription factor (MLTF) is a 46-kilodalton polypeptide that specifically binds to and activates transcription from the major late promoter of adenovirus. The presence of this promoter-specific transcription factor in uninfected HeLa cell extracts suggests that MLTF is also involved in the transcription of cellular genes. This report demonstrates that MLTF specifically stimulates transcription of the rat gamma-fibrinogen gene through a high-affinity binding site. Stimulation of transcription by MLTF was not dependent on the exact position of the MLTF binding site with respect either to the transcription initiation site or to adjacent promoter elements. These results suggest that one of the cellular functions of MLTF is to control gamma-fibrinogen gene expression.

Cloning of a lymphoid-specific cDNA encoding a protein binding the regulatory octamer DNA motif.

An octamer DNA sequence plays a critical role in directing transcription of immunoglobulin genes in B lymphocytes. A new technique of direct binding of radioactive DNA was used to screen a complementary DNA expression library from the BJAB cell line in lambda gt11 phage to derive molecular cDNA clones representing a putative B lymphocyte-specific octamer binding protein. The plaques were screened with DNA containing four copies of the octamer sequence and positive phage recombinants were identified. The fusion protein produced on inducing a lysogen of one phage bound to a monomeric octamer probe. The cDNA insert from this phage hybridized to messenger RNA found in B lymphocytes, but not in most other cells. Thus, this cDNA derives from a gene (oct-2) that specifies an octamer binding protein expressed preferentially in B lymphocytes, proving that, for at least one gene, a cell-specific transcription factor exists and its amount is controlled through messenger RNA availability.

Measurement of suppressor transfer RNA activity.

Transfer RNA (tRNA) suppression of nonsense mutations in prokaryotic systems has been widely used to study the structure and function of different prokaryotic genes. Through genetic engineering techniques, it is now possible to introduce suppressor (Su+) tRNA molecules into mammalian cells. A quantitative assay of the suppressor tRNA activity in these mammalian cells is described; it is based on the amount of tRNA-mediated readthrough of a terminating codon in the influenza virus NS1 gene after the cells are infected with virus. Suppressor activity in L cells continuously expressing Su+ (tRNAtyr) was 3.5 percent and that in CV-1 cells infected with an SV40- Su+ (tRNAtyr) recombinant was 22.5 percent.

Improving wheat as a source of iron and zinc for global nutrition.

Wheat is the staple food crop in temperate countries and increasingly consumed in developing countries, displacing traditional foods. However, wheat products are typically low in bioavailable iron and zinc, contributing to deficiencies in these micronutrients in countries where wheat is consumed as a staple food. Two factors contribute to the low contents of bioavailable iron and zinc in wheat: the low concentrations of these minerals in white flour, which is most widely consumed, and the presence of phytates in mineral-rich bran fractions. Although high zinc types of wheat have been developed by conventional plant breeding (biofortification), this approach has failed for iron. However, studies in wheat and other cereals have shown that transgenic (also known as genetically modified; GM) strategies can be used to increase the contents of iron and zinc in white flour, by converting the starchy endosperm tissue into a 'sink' for minerals. Although such strategies currently have low acceptability, greater understanding of the mechanisms which control the transport and deposition of iron and zinc in the developing grain should allow similar effects to be achieved by exploiting naturally induced genetic variation. When combined with conventional biofortification and innovative processing, this approach should provide increased mineral bioavailability in a range of wheat products, from white flour to wholemeal.

DNA-PK phosphorylation sites on Oct-1 promote cell survival following DNA damage.

Octamer transcription factor-1 (Oct-1) has recently been shown to function as a stress sensor that promotes cell survival subsequent to DNA damage. Here, we show that the survival signal imparted by Oct-1 following exposure to ionizing radiation (IR) is dependent upon DNA-dependent protein kinase (DNA-PK)-dependent phosphorylation of a cluster of 13 specific ser/thr residues within the N-terminal transcriptional regulatory domain of Oct-1. Although IR treatment did not affect the recruitment of Oct-1 to the histone H2B promoter, the recruitment of RNA polymerase II, TATA-binding protein and histone H4 acetylation were strongly reduced, consistent with a decrease in Oct-1 transcriptional regulatory potential following IR exposure. Ser/Thr-Ala substitution of 13 sites present in Oct-1 transcriptional regulatory domain eliminated Oct-1 phosphorylation subsequent to IR exposure. Further, these substitutions prevented Oct-1 from rescuing the survival of IR-treated Oct-1-/- murine embryonic fibroblasts, providing a direct link between DNA-PK-dependent phosphorylation and the contribution of Oct-1 to cell survival. These results implicate Oct-1 as a primary effector in a DNA-PK-dependent cell survival pathway that is activated by double-stranded DNA breaks.

Enhancing mineral bioavailability from cereals: Current strategies and future perspectives.

Inadequate intake of essential minerals such as iron and zinc is a public health concern in the UK, particularly for girls and young women. Approximately 30% and 50% of the zinc and iron, respectively, in the UK diet is provided by cereals. In wheat, most of the iron and zinc is contained within the aleurone cell layer; however, aleurone is removed during processing of wheat into white flour. While elemental iron powder is added back into white flour at the milling stage, there is no restoration of zinc. Elemental iron powder has very low bioavailability, and therefore, in our current Biotechnology and Biological Sciences Research Council Diet and Health Research Industry Club-funded project, we are investigating the potential use of aleurone as a bioavailable source of minerals that could be added to wheat-based foods. This work has relevance for the food industry and may establish the use of aleurone as a functional food ingredient for fortification of a range of cereal-based food products.

A helix-loop-helix protein related to the immunoglobulin E box-binding proteins.

A human cDNA encoding a novel protein in the helix-loop-helix family has been isolated by screening a bacteriophage expression library with a probe containing the binding site for major late transcription factor. The protein encoded by this cDNA, TFEB, probably recognizes E-box sequences in the heavy-chain immunoglobulin enhancer.

Requirement for distal upstream sequences for maximal transcription in vitro of early region IV of adenovirus.

A series of deletion mutants spanning the adenovirus early region IV (EIV) promoter were tested for transcription activity in vitro. At least three elements were found to be important for maximal transcription in HeLa whole-cell extracts. Deletion of the TATA box drastically reduced the transcription activity from the EIV promoter. Sequences between nucleotides -58 and -44 are also important for efficient transcription since deletion of this region reduced activity by 50%. More importantly, sequences residing upstream from -140 critically influence the level of EIV transcription. Deletion of sequences between nucleotides -325 (the right terminus of adenovirus genome) and -140 reduced the level of transcription more than 10-fold. It is possible that a specific cellular factor stimulates EIV transcription by recognition of these upstream sequences. The dependence of transcription from the EIV promoter on a distal upstream element may explain some aspects of the regulation of this promoter.

Transcription initiation complexes and upstream activation with RNA polymerase II lacking the C-terminal domain of the largest subunit.

RNA polymerase II assembles with other factors on the adenovirus type 2 major late promoter to generate pairs of transcription initiation complexes resolvable by nondenaturing gel electrophoresis. The pairing of the complexes is caused by the presence or absence of the C-terminal domain of the largest subunit. This domain is not required for transcription stimulation by the major late transcription factor in vitro.

Cell lines established by a temperature-sensitive simian virus 40 large-T-antigen gene are growth restricted at the nonpermissive temperature.

The thermolabile large T antigen, encoded by the simian virus 40 early-region mutant tsA58, was used to establish clonal cell lines derived from rat embryo fibroblasts. These cell lines grew continuously at the permissive temperature but upon shift-up to the nonpermissive temperature showed rapidly arrested growth. The growth arrest occurred in either the G1 or G2 phase of the cell cycle. After growth arrest, the cells remained metabolically active as assayed by general protein synthesis and the ability to exclude trypan blue. The inability of these cell lines to divide at the nonpermissive temperature was not readily complemented by the exogenous introduction of other nuclear oncogenes. This finding suggests that either these genes establish cells via different pathways or that immortalization by one oncogene results in a finely balanced cellular state which cannot be adequately complemented by another establishment gene.

Alternative splicing of the fibronectin EIIIB exon depends on specific TGCATG repeats.

The fibronectin EIIIB exon is alternatively spliced in a cell-type-specific manner, and TGCATG repeats in the intron downstream of EIIIB have been implicated in this regulation. Analysis of the intron sequence from several vertebrates shows that the pattern of repeats in the 3' half of the intron is evolutionarily conserved. Point mutations in certain highly conserved repeats greatly reduce EIIIB inclusion, suggesting that a multicomponent complex may recognize the repeats. Expression of the SR protein SRp40, SRp20, or ASF/SF2 stimulates EIIIB inclusion. Studies of the interplay between mutations in the repeats and SRp40-stimulated inclusion suggest that the repeats are recognized in many, if not all, cell types, and that EIIIB inclusion may be regulated by quantitative changes in multiple factors.