Potential limitations of transcription terminators used as transgene insulators in adenoviral vectors.

The presence of adenoviral cis-elements interfering with the activity of tissue-specific promoters has seriously impaired the use of transcriptional targeting adenoviruses for gene therapy purposes. As an approach to overcome this limitation, transcription terminators were previously employed in cultured cells to insulate a transgene promoter from viral activation. To extend these studies in vivo, we have injected into heart and skeletal muscle, adenoviruses containing the human growth hormone terminator and the cardiac-specific alpha-myosin heavy chain promoter (alphaMyHC) driving the chloramphenicol acetyltransferase (CAT) reporter gene. Promoterless CAT constructs were also tested to study interfering viral transcription and terminator activity. Here we demonstrate that the presence of a terminator can produce undesirable effects on the activity of heterologous promoters. Our analysis shows that in particular conditions, a terminator can reduce the tissue specificity of the transgene promoter. By RNAse protection assay performed on cardiac myocytes, we also show that adenoviral elements can direct high levels of autonomous transcription within the E1A enhancer region. This finding supports the model that passive readthrough of the transgene promoter is responsible for loss of selective expression.

Enhanced detection of tRNA isoacceptors by combinatorial oligonucleotide hybridization.

A method that greatly enhances the detection of tRNA by oligodeoxyribonucleotide probe hybridization has been developed. Because highly structured tRNA regions often preclude heteroduplex formation, we have tested the ability of cold oligodeoxyribonucleotides called unfolders to disrupt the tRNA secondary/tertiary structures and promote hybridization of a second labeled oligonucleotide complementary to the anticodon loop. Here we show that an excess of unfolders in the pre/hybridization reaction can enhance a barely detectable hybridization signal by more than 200-fold without affecting probe specificity. This sensitive assay makes it possible to easily study and monitor changes in tRNA isoacceptor expression.

Suppression of nonsense mutations in cell culture and mice by multimerized suppressor tRNA genes.

We demonstrate here the first experimental suppression of a premature termination codon in vivo by using an ochre suppressor tRNA acting in an intact mouse. Multicopy tRNA expression plasmids were directly injected into skeletal muscle and into the hearts of transgenic mice carrying a reporter gene with an ochre mutation. A strategy for modulation of suppressor efficiency, applicable to diverse systems and based on tandem multimerization of the tRNA gene, is developed. The product of suppression (chloramphenicol acetyltransferase) accumulates linearly with increases in suppressor tRNA concentration to the point where the ochre-suppressing tRNA(Ser) is in four- to fivefold excess over the endogenous tRNA(Ser). The subsequent suppressor activity plateau seems to be attributable to accumulation of unmodified tRNAs. These results define many salient variables for suppression in vivo, for example, for tRNA suppression employed as gene therapy for nonsense defects.

Enhancer elements activate the weak 3' splice site of alpha-tropomyosin exon 2.

We have identified four purine-rich sequences that act as splicing enhancer elements to activate the weak 3' splice site of alpha-tropomyosin exon 2. These elements also activate the splicing of heterologous substrates containing weak 3' splice sites or mutated 5' splice sites. However, they are unique in that they can activate splicing whether they are placed in an upstream or downstream exon, and the two central elements can function regardless of their position relative to one another. The presence of excess RNAs containing these enhancers could effectively inhibit in vitro pre-mRNA splicing reactions in a substrate-dependent manner and, at lower concentrations of competitor RNA, the addition of SR proteins could relieve the inhibition. However, when extracts were depleted by incubation with biotinylated exon 2 RNAs followed by passage over streptavidin agarose, SR proteins were not sufficient to restore splicing. Instead, both SR proteins and fractions containing a 110-kD protein were necessary to rescue splicing. Using gel mobility shift assays, we show that formation of stable enhancer-specific complexes on alpha-tropomyosin exon 2 requires the presence of both SR proteins and the 110-kD protein. By analogy to the doublesex exon enhancer elements in Drosophila, our results suggest that assembly of mammalian exon enhancer complexes requires both SR and non-SR proteins to activate selection of weak splice sites.

Ochre suppressor transfer RNA restored dystrophin expression in mdx mice.

The mdx mouse is an animal model for human Duchenne muscular dystrophy. The lack of dystrophin in mdx mice is caused by an ochre mutation in exon 23 of the dystrophin gene. This study tested the feasibility of inhibiting translational termination as an approach for genetic therapy for diseases caused by nonsense mutations. We evaluated both the in vitro and in vivo efficiencies of readthrough of ochre codons in 2 genes with the tRNA suppressor gene. The first target was a CAT reporter gene bearing an ochre mutation at the 5' end (CATochre). The second target was the dystrophin gene in mdx mice. The readthrough efficiencies were about 20% in COS cells and 5.5% in rat hearts. At four weeks after a direct injection of plasmid DNA encoding the tRNA suppressor into mdx mice, dystrophin positive fibers were detected by sarcolemmal immunostaining. This is the first convincing data that a tRNA suppressor gene might be a useful in vivo treatment for the genetic disorders caused by nonsense mutations.

Functional crosstalk between exon enhancers, polypyrimidine tracts and branchpoint sequences.

We recently identified enhancer elements that activate the weak 3' splice site of alpha-tropomyosin exon 2 as well as a variety of heterologous weak 3' splice sites. To understand their mechanism of action, we devised an iterative selection strategy to identify functional pyrimidine tracts and branchpoint sequences in the presence or absence of enhancer elements. Surprisingly, we found that strong pyrimidine tracts were selected regardless of the presence of enhancer elements. However, the presence of enhancer elements resulted in the selection of multiple, non-consensus branchpoint sequences. Thus, enhancer elements apparently activate weak 3' splice sites primarily by increasing the efficiency of splicing of introns containing branchpoint sequences with less than optimal U2-branchpoint pairing arrangements. Comparison of consensus sequences from both our selection strategy and compilations of published intron sequences suggests that exon enhancer elements could be widespread and play an important role in the selection of 3' splice sites.

Specific protein-protein interactions between the essential mammalian spliceosome-associated proteins SAP 61 and SAP 114.

Spliceosome-associated proteins (SAPs) 61, 62, and 114 can be UV-crosslinked to pre-mRNA in purified spliceosomal complexes and are associated with U2 small nuclear ribonucleoproteins (snRNP). These proteins also compose the essential heterotrimeric splicing factor SF3a, and products of yeast pre-mRNA processing genes PRP9, PRP11, and PRP21 are their likely yeast counterparts. We report the isolation of a cDNA encoding SAP 61 and find that it is 30% identical in amino acid sequence to PRP9. A C-terminal Cys2His2 zinc-finger-like motif, which could be involved in the pre-mRNA binding, is the most highly conserved region of the protein. We also demonstrate specific protein-protein interactions between SAPs 61 and 114 and show that the N terminus of SAP 61 is required for this interaction. Significantly, the corresponding proteins are also known to interact in yeast: PRP9 interacts with PRP21, and the N-terminal portion of PRP9 is required. Previous work showed that direct interactions also occur between SAPs 62 and 114 and between the corresponding PRPs 11 and 21. These observations indicate that the specific protein-protein interactions that occur between the three prespliceosomal factors have been conserved between yeast and mammals.

Human hnRNP protein A1 gene expression. Structural and functional characterization of the promoter.

hnRNP protein A1 (34 kDa, pl 9.5) is a prominent member of the family of proteins (hnRNP proteins) that associate with the nascent transcripts of RNA polymerase II and that accompany the hnRNA through the maturation process and the export to the cytoplasm. New evidence suggests an active and specific role for some of these proteins, including protein A1, in splicing and transport. Contrary to the other hnRNP proteins, the intracellular level of protein A1 was reported to change as a function of proliferation state and cell type. In this work we analyse the A1 gene expression in different cells under different growth and differentiation conditions. Proliferation dependent expression was observed in lymphocytes and fibroblasts while purified neurons express high A1 mRNA levels both in the proliferative (before birth) and in the quiescent (after birth) state. Transformed cell lines exhibit very high (proliferation independent) A1 mRNA levels compared to differentiated tissues. A structural and functional characterization of the A1 gene promoter was carried out by means of DNase I footprinting and CAT assays. The observed promoter features can account for both elevated and regulated mRNA transcription. At least 12 control elements are contained in the 734 nucleotides upstream of the transcription start site. Assays with the deleted and/or mutated promoter indicate a co-operation of multiple transcriptional elements, distributed over the entire promoter, in determining the overall activity and the response to proliferative stimuli (serum).

Phosphorylation of human hnRNP protein A1 abrogates in vitro strand annealing activity.

In HeLa cells metabolically labeled in vivo with [32P] orthophosphate in the presence of okadaic acid the concentration of phosphorylated A1 protein was increased significantly as compared to controls. Purified recombinant hnRNP protein A1 served as an excellent substrate in vitro for the catalytic subunit of cAMP-dependent protein kinase (PKA) and for casein kinase II (CKII). Thin layer electrophoresis of A1 acid hydrolysates showed the protein to be phosphorylated exclusively on serine residue by both kinases. V8 phosphopeptide maps revealed that the target site(s) of in vitro phosphorylation are located in the C-terminal region of A1. Phosphoamino acid sequence analysis and site directed mutagenesis identified Ser 199 as the sole phosphoamino acid in the protein phosphorylated by PKA. Phosphorylation introduced by PKA resulted in the suppression of the ability of protein A1 to promote strand annealing in vitro, without any detectable effect on its nucleic acid binding capacity. This finding indicates that phosphorylation of a single serine residue in the C-terminal domain may significantly alter the properties of protein A1.

Interaction of hnRNP A1 with snRNPs and pre-mRNAs: evidence for a possible role of A1 RNA annealing activity in the first steps of spliceosome assembly.

The in vitro interaction of recombinant hnRNP A1 with purified snRNPs and with pre-mRNAs was investigated. We show that protein A1 can stably bind U2 and U4 snRNP but not U1. Oligo-RNAse H cleavage of U2 nucleotides involved in base pairing with the branch site, totally eliminates the A1-U2 interaction. RNase T1 protection and immunoprecipitation experiments demonstrate that recombinant protein A1 specifically binds the 3'-end regions of both beta-globin and Ad-2 introns. However, while on the beta-globin intron only binding to the polypyrimidine tract was observed, on the Ad-2 intron a 32 nt fragment encompassing the branch point and the AG splice-site dinucleotide was bound and protected. Such protection was drastically reduced in the presence of U2 snRNP. Altogether these results indicate that protein A1 can establish a different pattern of association with different pre-mRNAs and support the hypothesis that this protein could play a role in the annealing of U2 to the branch site and hence in the early events of pre-splicing complex assembly.

Recombinant hnRNP protein A1 and its N-terminal domain show preferential affinity for oligodeoxynucleotides homologous to intron/exon acceptor sites.

The reported binding preference of human hnRNP protein A1 for the 3'-splice site of some introns (Swanson and Dreyfuss (1988) EMBO J. 7, 3519-3529; Mayrand and Pederson (1990) Nucleic Acids Res. 18, 3307-3318) was tested by assaying in vitro the binding of purified recombinant A1 protein (expressed in bacteria) to synthetic oligodeoxynucleotides (21-mers) of suitable sequence. In such a minimal system we find preferential binding of protein A1 to oligodeoxynucleotide sequences corresponding to the 3'-splice site of IVS1 of human beta-globin pre-mRNA and of IVS1 of Adenovirus type 2 major late transcript. Mutation studies demonstrate that the binding specificity is dependent on the known critical domains of this intron region, the AG splice site dinucleotide and polypyrimidine tract, and resides entirely in the short oligonucleotide sequence. Moreover specific binding does not require the presence of other hnRNP proteins or of snRNP particles. Studies with a truncated recombinant protein demonstrated that the minimal protein sequence determinants for A1 recognition of 3'-splice acceptor site reside entirely in the N-terminal 195 aa of the unmodified protein.

Alternative splicing in the human gene for the core protein A1 generates another hnRNP protein.

The human hnRNP core protein A1 (34 kd) is encoded by a 4.6 kb gene split into 10 exons. Here we show that the A1 gene can be differentially spliced by the addition of an extra exon. The new transcript encodes a minor protein of the hnRNP complex, here defined A1B protein, with a calculated mol. wt of 38 kd, that coincides with a protein previously designated as B2 by some authors. In vitro translation of the mRNAs selected by hybridization with A1 cDNA produced two proteins of 34 and 38 kd; Northern blot analysis of poly(A)+ RNA from HeLa cells revealed that the abundance of the A1B mRNA was approximately 5% that of A1. The A1B protein was detected by Western blotting with an anti-A1 monoclonal antibody both in enriched preparations of basic hnRNP proteins and in 40S hnRNP particles. The A1B protein exhibits a significantly higher affinity than A1 for ssDNA. The recombinant A1B protein, expressed in Escherichia coli, shows the same electrophoretic mobility and charge as the cellular one.

Isolation of an active gene encoding human hnRNP protein A1. Evidence for alternative splicing.

Heterogeneous nuclear ribonucleoprotein (hnRNP) core protein A1 is a major component of mammalian hnRNP 40 S particles. We describe the structure of an active A1 gene and report on the partial characterization of the A1 gene family. About 30 A1-specific sequences are present per haploid human genome: 15 such sequences were isolated from a human genomic DNA library. Many corresponded to pseudogenes of the processed type but by applying a selection for actively transcribed regions we isolated an active A1 gene. The gene spans a region of 4.6 x 10(3) base-pairs and it is split into ten exons that encode the 320 amino acid residues of the protein. The amino acid sequence derived from the exon sequences is identical with that deduced from cDNA and reported for the protein. One intron exactly separates the two structural domains that constitute the protein. Each of the two RNA-binding domains in protein A1 is encoded by one exon. Experimental evidence indicates that the A1 gene can encode for more than one protein by alternative splicing. The gene is preceded by a strong promoter that contains at least two CCAAT boxes and two possible Sp1 binding sites, but it lacks a TATA box.

cDNA cloning of human hnRNP protein A1 reveals the existence of multiple mRNA isoforms.

Protein A1 is one of the major component of mammalian ribonucleoprotein particles (hnRNP). Human protein A1 cDNA cloning and sequencing revealed the existence of at least two protein isoforms. Among the cDNAs examined, sequence differences were found both in the structural portion, leading to aminoacid changes (Tyr to Phe or Arg to Lys) and in the non translated 3'-region where two T-stretches of different length were observed. Interestingly one of the aminoacid substitutions falls into a consensus sequence common to many RNA binding proteins. Northern blot analysis of poly A+ RNAs from five human tissues revealed two mRNA forms of 1500 and 1900 n due to alternative polyadenylation. Analysis of genomic DNA showed at least 30 A1-specific sequences, some of which correspond to processed pseudogenes. These results suggest that protein A1 is encoded by a multigene family.

Heterologous expression in Bacillus subtilis. II. In vitro removal of the attenuator sequence of the Escherichia coli his operon allows expression of the cloned hisG gene in B. subtilis.

The promoter-proximal region of the Escherichia coli histidine (his) operon, including the promoter, the attenuator and the hisG gene, as well as the first of the nine structural genes of the his operon, have been cloned in Bacillus subtilis. In this host, the hisG gene could not be expressed because its transcription appeared to be irreversibly terminated at the attenuator (Ferretti et al., 1984). When the attenuator plus various lengths of the two bordering regions were removed, one of the attenuatorless sequences cloned in B. subtilis allowed the progression of transcription and complementation of the corresponding hisA mutation in this Gram-positive host. The deletion removed a 349-bp segment which contained the his attenuator and promoter. In B. subtilis, the productive transcription of the hisG gene started at a site in pAT153 and terminated in pC194. Sequence analysis of the deletion indicates that the E. coli ribosome-binding site of the his operon was used for the translation of the E. coli hisG gene mRNA in B. subtilis cells, which can thus grow in the absence of histidine.