The activity of a single-stranded promoter of plasmid ColIb-P9 depends on its secondary structure.

The leading region of the conjugal bacterial plasmid ColIb-P9 contains three dispersed repeats of a 328 bp sequence homologous to Frpo, a sequence from plasmid F that acts as a promoter in single-stranded DNA. One of these sequences, ssi3, inactive in the double-stranded form, promoted in vitro transcription exclusively from the single strand that is transferred during conjugation. Promoter activity was dependent on the presence of RNA polymerase holoenzyme containing sigma 70. Transcription initiated from the position predicted from folding the single-stranded DNA to form a pseudo double-stranded hairpin structure containing recognizable -35 and -10 promoter elements. Footprinting of RNA polymerase holoenzyme on single-stranded ssi3 DNA was consistent with this suggestion. Mutagenesis of the putative -35 region inactivated the promoter, but random mutations in the -10 region had little effect. The putative -10 region is a poor match to the consensus sequence and contains mismatched bases. Elimination of these mismatches invariably destroyed single-strand promoter activity. These observations reveal the crucial contribution of the unpaired bases in the -10 region in potentiating the formation of the productive open complex with RNA polymerase.

A double reporter assay for detecting changes in the ratio of spliced and unspliced mRNA in mammalian cells.

Current methods for measuring the efficiency of splicing in mammalian cells rely on either direct analysis of the RNA, which does not lend itself to rapid assays, or on single reporter functions that are subject to numerous intrinsic variables. If two protein activities are encoded within a single reading frame but on separate exons, with an intervening sequence containing termination codons, then the expression of the second activity is dependent on removal of the intervening sequence by pre-mRNA splicing. Thus, the ratio of the activities encoded by exon 2 to exon 1 reflects the ratio of expression from spliced mRNA to the total expression of spliced and unspliced RNA. This provides a rapid and convenient assay for the effects on splicing efficiency of trans-acting factors or of alterations in the sequences of the intron and surrounding exon sequences.

Selection of alternative 5' splice sites: role of U1 snRNP and models for the antagonistic effects of SF2/ASF and hnRNP A1.

The first component known to recognize and discriminate among potential 5' splice sites (5'SSs) in pre-mRNA is the U1 snRNP. However, the relative levels of U1 snRNP binding to alternative 5'SSs do not necessarily determine the splicing outcome. Strikingly, SF2/ASF, one of the essential SR protein-splicing factors, causes a dose-dependent shift in splicing to a downstream (intron-proximal) site, and yet it increases U1 snRNP binding at upstream and downstream sites simultaneously. We show here that hnRNP A1, which shifts splicing towards an upstream 5'SS, causes reduced U1 snRNP binding at both sites. Nonetheless, the importance of U1 snRNP binding is shown by proportionality between the level of U1 snRNP binding to the downstream site and its use in splicing. With purified components, hnRNP A1 reduces U1 snRNP binding to 5'SSs by binding cooperatively and indiscriminately to the pre-mRNA. Mutations in hnRNP A1 and SF2/ASF show that the opposite effects of the proteins on 5'SS choice are correlated with their effects on U1 snRNP binding. Cross-linking experiments show that SF2/ASF and hnRNP A1 compete to bind pre-mRNA, and we conclude that this competition is the basis of their functional antagonism; SF2/ASF enhances U1 snRNP binding at all 5'SSs, the rise in simultaneous occupancy causing a shift in splicing towards the downstream site, whereas hnRNP A1 interferes with U1 snRNP binding such that 5'SS occupancy is lower and the affinities of U1 snRNP for the individual sites determine the site of splicing.

An evolutionarily conserved germ cell-specific hnRNP is encoded by a retrotransposed gene.

The gene encoding heterogeneous ribonucleoprotein (hnRNP) G recently has been mapped to the X chromosome. All mammals have a Y chromosome-encoded homologue of HNRNP G called RBMY, which is implicated with a role in male fertility and is a candidate for the azoospermia factor gene. We have identified a new member of this gene family, HNRNP G-T, and have mapped it as a single-copy gene on chromosome 11. This gene contains an uninterrupted open reading frame and no introns, consistent with derivation from a retroposon. However, unlike many retroposon-derived genes, HNRNP G-T is not a pseudogene. An antiserum raised to the conceptual reading frame of HNRNP G-T showed that it encodes a protein that is highly expressed in germ cells and in particular in the nuclei of meiotic spermatocytes. Surprisingly, although this antiserum was raised against human hnRNP G-T protein, it can also detect a similar protein in the testis of several mammals. This suggests that the protein is highly conserved and that the retrotransposition event generating the HNRNP G-T gene pre-dated at least the common ancestor of mouse and man. The existence of an additional testis-specific hnRNP G family member provides evidence for the importance of these proteins in normal germ cell development.

RBMY, a probable human spermatogenesis factor, and other hnRNP G proteins interact with Tra2beta and affect splicing.

The RBMY gene family is found on the Y chromosome of all mammals, and microdeletions are strongly associated with infertility in men. RBMY expresses RBM only in the nuclei of germ cells, whereas its X chromosome homologue, RBMX, expresses hnRNP G ubiquitously. We show here that RBM, hnRNP G and a novel testis-specific relative, termed hnRNP G-T, interact with Tra2beta, an activator of pre-mRNA splicing that is ubiquitous but highly expressed in testis. Endogenous hnRNP G and Tra2beta proteins are associated in HeLa nuclear extracts. RBM and Tra2beta co-localize in two major domains in human spermatocyte nuclei. Phosphorylation enhanced the interaction and reduced competing RNA binding to the interaction domains. Incubation with the protein interaction domain of RBM inhibited splicing in vitro of a specific pre-mRNA substrate containing an essential enhancer bound by Tra2beta. The RNA-binding domain of RBM affected 5' splice site selection. We conclude that the hnRNP G family of proteins is involved in pre-mRNA splicing and infer that RBM may be involved in Tra2beta-dependent splicing in spermatocytes.

An exonic splicing silencer in the testes-specific DNA ligase III beta exon.

Alternative pre-mRNA splicing of two terminal exons (alpha and beta) regulates the expression of the human DNA ligase III gene. In most tissues, the alpha exon is expressed. In testes and during spermatogenesis, the beta exon is used instead. The alpha exon encodes the interaction domain with a scaffold DNA repair protein, XRCC1, while the beta exon-encoded C-terminal does not. Sequence elements regulating the alternative splicing pattern were mapped by in vitro splicing assays in HeLa nuclear extracts. Deletion of a region beginning in the beta exon and extending into the downstream intron derepressed splicing to the beta exon. Two silencing elements were found within this 101 nt region: a 16 nt exonic splicing silencer immediately upstream of the beta exon polyadenylation signal and a 45 nt intronic splicing silencer. The exonic splicing silencer inhibited splicing, even when the poly-adenylation signal was deleted or replaced by a 5' splice site. This element also enhanced polyadenylation under conditions unfavourable to splicing. The splicing silencer partially inhibited assembly of spliceo-somal complexes and functioned in an adenoviral pre-mRNA context. Silencing of splicing by the element was associated with cross-linking of a 37 kDa protein to the RNA substrate. The element exerts opposite functions in splicing and polyadenylation.

Exon repetition in mRNA.

The production of different transcripts (transcript heterogeneity) is a feature of many genes that may result in phenotypic variation. Several mechanisms, that occur at both the DNA and RNA level have been shown to contribute to this transcript heterogeneity in mammals, all of which involve either the rearrangement of sequences within a genome or the use of alternative signals in linear, contiguous DNA or RNA. Here we describe tissue-specific repetition of selective exons in transcripts of a rat gene (SA) with a normal exon-intron organization. We conclude that nonlinear mRNA processing can generate tissue-specific transcripts.

T-STAR/ETOILE: a novel relative of SAM68 that interacts with an RNA-binding protein implicated in spermatogenesis.

RBM is an RNA-binding protein encoded on the Y chromosome in mammals and is expressed only in the nuclei of male germ cells. Genetic evidence from infertile men implicates it in spermatogenesis, but its function is unknown. Of a number of potential partners for RBM identified by a yeast two-hybrid screen with testis cDNA, the most frequent isolates encoded a novel RNA-binding protein, termed T-STAR, that is closely related to SAM68, an Src-associated protein of unknown function. The mouse homologue was also cloned and designated étoile. It mapped to chromosome 15, while T-STAR mapped to the syntenic region on human chromosome 8. T-STAR/étoile is expressed primarily in the testis; in rat germ cells, the expression of both T-STAR/étoile and SAM68 is regulated during meiosis. Transfection of T-STAR/étoile fused with green fluorescent protein into HeLa cells caused an accumulation of protein in a novel compartment of the nucleus, adjacent to the nucleolus but distinct from the peri-nucleolar compartment. RBM and other hnRNP G family members are candidate downstream targets for regulation by T-STAR/ETOILE and SAM68.

The pre-mRNA 5' cap determines whether U6 small nuclear RNA succeeds U1 small nuclear ribonucleoprotein particle at 5' splice sites.

Efficient splicing of the 5'-most intron of pre-mRNA requires a 5' m7G(5')ppp(5')N cap, which has been implicated in U1 snRNP binding to 5' splice sites. We demonstrate that the cap alters the kinetic profile of U1 snRNP binding, but its major effect is on U6 snRNA binding. With two alternative wild-type splice sites in an adenovirus pre-mRNA, the cap selectively alters U1 snRNA binding at the site to which cap-independent U1 snRNP binding is stronger and that is used predominantly in splicing; with two consensus sites, the cap acts on both, even though one is substantially preferred for splicing. However, the most striking quantitative effect of the 5' cap is neither on U1 snRNP binding nor on the assembly of large complexes but on the replacement of U1 snRNP by U6 snRNA at the 5' splice site. Inhibition of splicing by a cap analogue is correlated with the loss of U6 interactions at the 5' splice site and not with any loss of U1 snRNP binding.

Dynamic changes in the subnuclear organisation of pre-mRNA splicing proteins and RBM during human germ cell development.

RBM is a germ-cell-specific RNA-binding protein encoded by the Y chromosome in all mammals, implying an important and evolutionarily conserved (but as yet unidentified) function during male germ cell development. In order to address this function, we have developed new antibody reagents to immunolocalise RBM in the different cell types in the human testis. We find that RBM has a different expression profile from its closest homologue hnRNPG. Despite its ubiquitous expression in all transcriptionally active germ cell types, RBM has a complex and dynamic cell biology in human germ cells. The ratio of RBM distributed between punctate nuclear structures and the remainder of the nucleoplasm is dynamically modulated over the course of germ cell development. Moreover, pre-mRNA splicing components are targeted to the same punctate nuclear regions as RBM during the early stages of germ cell development but late in meiosis this spatial association breaks down. After meiosis, pre-mRNA splicing components are differentially targeted to a specific region of the nucleus. While pre-mRNA splicing components undergo profound spatial reorganisations during spermatogenesis, neither heterogeneous ribonucleoproteins nor the transcription factor Sp1 show either developmental spatial reorganisations or any specific co-localisation with RBM. These results suggest dynamic and possibly multiple functions for RBM in germ cell development.

Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides.

Deletions and point mutations in the gene encoding the cytoskeletal protein dystrophin and its isoforms cause either the severe progressive myopathy Duchenne muscular dystrophy (DMD) or the milder Becker muscular dystrophy (BMD), largely depending on whether the reading frame is lost or maintained respectively. Frameshift mutations tend to result in a lack of dystrophin at the sarcolemma, destabilization of the membrane and degeneration of skeletal muscle. The mdx mouse is a valuable animal model of DMD as it bears a nonsense point mutation in exon 23 of the murine DMD gene leading to an absence of dystrophin expression in the muscle sarcolemma and muscular dystrophy. This report represents a novel approach to correct dystrophin deficiency at the post-transcriptional level by transfection of muscle cells with antisense RNA. Essentially, 2'- O -methyl oligoribonucleotides (2'OMeRNA) were delivered to the nuclei of primary mdx myoblasts in culture. Dystrophin expression was observed in the sarcolemma of transfected mdx myotubes after transfection by an oligonucleotide complementary to the 3' splice site of murine dystrophin intron 22. Direct sequencing of RT-PCR products from these cells revealed precise splicing of exon 22 to exon 30, skipping the mutant exon and creating a novel in-frame dystrophin transcript. As patients with comparable in-frame internal deletions show relatively mild myopathic symptoms, this may in the future offer a therapeutic approach for DMD, as well as for other inherited disorders.

Selective amplification of T-cell receptor variable region species is demonstrable but not essential in early lesions of psoriasis vulgaris: analysis by anchored polymerase chain reaction and hypervariable region size spectratyping.

Several groups have investigated the role of T cells in the pathogenesis of psoriasis by determination of T-cell receptor (TCR) B-chain variable (V) region usage, both in chronic plaque (psoriasis vulgaris) and guttate forms, with various results. Because there are no data on TCR expression in early psoriasis vulgaris, when specific cellular immune events may be expected to be most pronounced, we have analyzed early lesions (less than 3 wk old) of ten patients, with highly reproducible results. We have developed a highly controlled anchored polymerase chain reaction (PCR) method in which TCR beta chain species are all amplified with the same primer pair and products are quantified by dot blot hybridization with BV family-specific oligonucleotide probes. Overexpression of certain TCR BV genes was observed in the majority of lesional biopsies, but in samples in which the expanded BV family formed more than 10% of total lesional BV (half of the samples analyzed), BV2 and BV6 predominated. The consistency of overexpression of these BV species between patients was much less than in previous studies of TCRBV usage in established chronic plaque psoriasis lesions. Complementarity-determining region 3 (CDR3) size spectratyping demonstrated evidence for selective clonal T cell accumulation in less than half of the lesional samples showing BV expansion. These results indicate that selective amplification of TCRBV species occurs in early psoriasis vulgaris but is not essential to the pathogenic process and may be more important in the maintenance or expansion of chronic lesions.

The complex of DNA gyrase and quinolone drugs with DNA forms a barrier to transcription by RNA polymerase.

The effects of DNA gyrase and quinolone drugs on in vitro transcription of a template containing a preferred gyrase cleavage site have been investigated. We have found that gyrase-quinolone complexes with DNA lead to blocking of transcription by Escherichia coli and bacteriophage T7 RNA polymerases. Either gyrase or quinolone alone has no effect on transcription. With DNA gyrase containing a point mutation in the gyrase A protein, known to confer quinolone resistance, blocking was found to occur only at much higher concentrations of the drug. Other agents that inhibit gyrase-catalysed supercoiling (novobiocin and 5'-adenylyl-beta,gamma-imidodiphosphate) do not arrest transcription in the presence of gyrase. Mapping of the transcription termination sites in the presence of gyrase and quinolones shows that blocking occurs about 10 to 20 base-pairs upstream of the gyrase cleavage site. Analysis of transcription in the absence of drug suggests that RNA polymerase does not displace gyrase from the template. These results are discussed in the light of models for the bactericidal effects of quinolone drugs.

Pathways for selection of 5' splice sites by U1 snRNPs and SF2/ASF.

We have used protection against ribonuclease H to investigate the mechanisms by which U1 small nuclear ribonucleoprotein particles (snRNPs) determine the use of two alternative 5' splice sites. The initial binding of U1 snRNPs to alternative consensus splice sites was indiscriminate, and on a high proportion of pre-mRNA molecules both sites were occupied simultaneously. When the sites were close, this inhibited splicing. We propose that double occupancy leads to the use of the downstream site for splicing and that this is the cause of the proximity effect seen with strong alternative splice sites. This model predicts that splicing to an upstream site of any strength requires a low affinity of U1 snRNPs for the downstream site. This prediction was tested both by cleaving the 5' end of U1 snRNA and by altering the sequence of the downstream site of an adenovirus E1A gene. The enhancement of downstream 5' splice site use by splicing factor SF2/ASF appears to be mediated by an increase in the strength of U1 snRNP binding to all sites indiscriminately.

Alternative splicing of a human alpha-tropomyosin muscle-specific exon: identification of determining sequences.

The human alpha-tropomyosin gene hTMnm has two mutually exclusive versions of exon 5 (NM and SK), one of which is expressed specifically in skeletal muscle (exon SK). A minigene construct expresses only the nonmuscle (NM) isoform when transfected into COS-1 cells and both forms when transfected into myoblasts. Twenty-four mutants were produced to determine why the SK exon is not expressed in COS cells. The results showed that exons NM and SK are not in competition for splicing to the flanking exons and that there is no intrinsic barrier to splicing between the exons. Instead, exon SK is skipped whenever there are flanking introns. Splicing of exon SK was induced when the branch site sequence 70 nucleotides upstream of the exon was mutated to resemble the consensus and when the extremities of the exon itself were changed to the corresponding NM sequence. Precise swaps of the NM and SK exon sequences showed that the exon sequence effect was dominant to that of intron sequences. The mechanism of regulation appears to be unlike that of other tropomyosin genes. We propose that exclusion of exon SK arises because its 3' splicing signals are weak and are prevented by an exon-specific repressor from competing for splice site recognition.

Mutually exclusive splicing of calcium-binding domain exons in chick alpha-actinin.

We have determined the complete sequence of chick brain alpha-actinin (892 amino acids; 107,644 Da). The sequence differs from that of smooth muscle alpha-actinin only in the region of the first EF-hand calcium-binding motif, where 27 residues in brain alpha-actinin are replaced by just 22 residues in the smooth muscle isoform. This probably accounts for the different calcium sensitivities of the two isoforms with respect to actin binding. Analysis of the gene structure showed that this region of sequence divergence is encoded by two separate exons whose incorporation is mutually exclusive. We have determined the proportion of the two transcripts in various tissues and cell lines using poly(A)+ RNA and a quantitative assay based on the polymerase chain reaction. MRC-5 fibroblasts and HeLa cells express mRNAs encoding both isoforms, whereas Namalwa lymphoblastoid cells, which lack actin stress fibers, express only the non-muscle mRNA. Both isoforms of alpha-actinin became incorporated into stress fibers and cell-matrix junctions when full-length chick alpha-actinin cDNAs were expressed in monkey COS cells. The levels of chick alpha-actinin mRNAs were found to be serum-inducible, suggesting that alpha-actinin may be an early response gene.

Multiple forms of mRNA encoding human pregnancy-associated endometrial alpha 2-globulin, a beta-lactoglobulin homologue.

Human pregnancy-associated endometrial alpha 2-globulin (alpha 2-PEG) is the major secretory protein product of the endometrium during embryo implantation and the first few weeks of pregnancy. It is a homologue of beta-lactoglobulin, a retinol binding protein, but unlike beta-lactoglobulin it is not found in the mammary gland. The cloning and sequencing of 34 alpha 2-PEG clones has revealed several minor variant forms indicative of alternatively spliced alpha 2-PEG pre-mRNA. These minor forms have also been detected amongst uncloned cDNA after PCR amplification. Some of these mRNAs would give rise to forms of alpha 2-PEG protein lacking internal sequences, whereas others affect the mRNA sequences on the 3' boundary of the presumed termination codon. Sequences within the cDNA clones are consistent with the existence of splice sites, and together with similarities found between alpha 2-PEG cDNA and beta-lactoglobulin gene sequences there is good evidence in support of an unusual scheme for the alternative splicing of alpha 2-PEG pre-mRNA involving both alternative 5' splice sites and alternative 3' splice sites. This scheme suggests that the alpha 2-PEG and beta-lactoglobulin genes share a similar structure in at least two regions, and it is likely that beta-lactoglobulin pre-mRNA would show a similar pattern of alternative splicing for one of these regions.

Co-purification of a small RNA species with multicatalytic proteinase (proteasome) from rat liver.

Previous studies have come to different conclusions about the presence of RNA in particles known variously as prosomes, proteasomes or multicatalytic proteinase (MCP). To determine the reason for this, MCP was isolated from rat liver by 4 different purification protocols. One major band of RNA, about 80 nucleotides in length, co-purified in all preparations. The amount of RNA detected was less than one molecule per MCP particle suggesting that there may be more than one population of MCP in rat liver cells.

Influences of separation and adjacent sequences on the use of alternative 5' splice sites.

Single nucleotide changes to the sequence between two alternative 5' splice sites, separated by 25 nucleotides in a beta-globin gene derivative, caused substantial shifts in pre-mRNA splicing preferences, both in vivo and in vitro. An activating sequence for splicing was located. Models for the recognition by U1 small nuclear ribonucleoproteins (snRNPs) of competing 5' splice sites were tested by altering the distance separating the two sites. Use of the upstream splice site declined sharply when it was separated from the downstream (natural) site by distances of 40 nucleotides or more. This effect was reversed in vivo, but not in vitro, by altering the upstream sequence to that of a consensus 5' splice site sequence. Dilution of an extract used for splicing in vitro shifted preferences when the sites were close towards the downstream site. We conclude that the mechanism of selection depends on the distance apart of the potential splice sites and that with close sites steric interference between factors bound to both sites may impede splicing and affect splicing preferences.