The untranslated leader of nuclear COX4 gene of Saccharomyces cerevisiae contains an intron.

The nuclear gene for subunit IV of cytochrome oxidase (COX4) in Saccharomyces cerevisiae contains a 342 bp intron which is contained entirely within the 5' leader of the message. Splicing of the intron results in removal of several small open reading frames; subsequently, the COX4 AUG becomes the 5' proximal initiation codon. A strain with an rna2- mutation fails to splice mRNA efficiently at restrictive temperature and was used to map the intron splice junctions by RNase protection. Two major mRNA initiation sites were mapped by primer extension of synthetic oligodeoxynucleotides. The splice junctions and internal TACTAAC box conform to consensus sequences previously determined from other yeast introns. One gene for subunit V of cytochrome oxidase (COX5b) has also been shown to contain an intron. The significance of introns in two nuclear genes encoding subunits of cytochrome oxidase is discussed.     

Regulation of splicing: the importance of being translatable

RNA sequences that conform to the consensus sequence of 5' splice sites but are not used for splicing occur frequently in protein coding genes. Mutational analyses have shown that suppression of splicing at such latent sites may be dictated by the necessity to maintain an open reading frame in the mRNA. Here we show that stop codon frequency in introns having latent 5' splice sites is significantly greater than that of introns lacking such sites and significantly greater than the expected occurrence by chance alone. Both observations suggest the occurrence of a general mechanism that recognizes the mRNA reading frame in the context of pre-mRNA.     

Effect of the tripartite leader on synthesis of a non-viral protein in an adenovirus 5 recombinant.

The EIa region of an Adenovirus 5 recombinant has been substituted by a modular gene encoding dihydrofolate reductase (DHFR). In this recombinant, the mouse DHFR cDNA was positioned behind sequences of the major late promoter and the complete tripartite leader. The leader sequences end in the normal 5' splice site (SS) of the third leader, so that RNA splicing joins the tripartite leader to a 3' splice site immediately upstream of the DHFR cDNA. At late stages of infection, high levels of DHFR mRNAs were synthesized. At early times in the late stage, this mRNA was efficiently translated; however, at later times translation of DHFR decreased probably due to poor competition with other late mRNAs. Synthesis of DHFR protein from an analogous Adenovirus 5 recombinant containing only the first late leader was studied in parallel. Equivalent levels of DHFR mRNA were expressed after infection with this recombinant virus; however, the efficiency of DHFR translation was at least 20 fold lower than that of the DHFR mRNA containing the tripartite leader. This suggests that the tripartite leader sequence is important for translation in the late stage of infection. As reported previously, the Ad5 recombinant containing only the first leader vastly overexpresses polypeptide IX from a novel mRNA, formed by the splicing of the first leader in the modular DHFR gene to the 3' splice site in the EIb region. Cells infected with this recombinant synthesize very little normal mRNA from the EIb region. Here, we demonstrated that coinfection of 293 cells with this recombinant and wild type Adenovirus 5 also results in decreased EIb mRNA synthesis. We propose that the overproduction of polypeptide IX suppresses mRNA expression from the EIb and IX promoter sites, probably by an autoregulation loop active during lytic growth.     

Polyoma virus early and late mRNAs in productively infected mouse 3T6 cells.

We mapped polyoma virus-specific mRNAs isolated from productively infected mouse 3T6 cells on the viral genome by analyzing nuclease S1-resistant RNA-DNA hybrids. The polyoma early mRNAs, which code for the three T antigens, have several 5' ends near 73 map units (m.u.). During the late phase of infection an additional 5' end is found near 71 m.u. All of the major early mRNAs have common 3' ends at 26.01 m.u. There is a minor species of early mRNA with a 3' end at 99.05 m.u. There are two proximal and two distal splice junctions in the early region which are used to generate three different spliced early mRNAs. There are three late mRNAs encoding the three virion proteins, VP1, VP2, and VP3. The late mRNAs have common 3' ends at 25.34 m.u. The late mRNAs have heterogeneous 5' leader sequences derived from the region between 65.53 and 68.42 m.u. The leader sequences are joined to the bodies of the messages coding for VP2, VP3, and VP1 at 66.59, 59.62, and 48.57 m.u., respectively. These results confirm and extend previous analyses of the fine structure of polyoma mRNAs.     

The sequence of the 3'' non-coding region of the hexon mRNA discloses a novel adenovirus gene.

We report the sequence of a 1164 nucleotide long DNA segment, located between map positions 59.5 and 62.8 on the adenovirus type 2 genome. The sequence comprises the 701 nucleotides long 3' non-coding region of the hexon mRNA as well as several important processing signals. The sequence revealed unexpectedly that the 3' non-coding region of the hexon mRNA contains a 609 nucleotide long uninterrupted translational reading frame following a potential initiator AUG. A late 14S mRNA, corresponding to the open reading frame, could be identified by S1 nuclease mapping and electronmicroscopy. The mRNA shares a poly(A) addition site with the hexon and pVI mRNAs, and carries a leader sequence which is related, and probably identical, to the tripartite leader, found in late adenovirus mRNAs. The junction between the leader and the body of this novel mRNA is located within the coding part of the hexon gene.     

Nucleotide sequence of the EcoRI D fragment of adenovirus 2 genome

The entire nucleotide sequence of the Ad. 2 EcoRI D fragment has been determined using the Maxam and Gilbert method. This sequence of 2678 bp contains informations relative to late mRNAs ending at position 78 and for which an AATAAA sequence corresponding to their 3' ends is found at residue number 833. Position of the PVIII mRNA is determined thus allowing deduction of the probable amino acid sequence of the PVIII protein. The position and the sequence of the first leader of early 3 mRNAs is determined as well as the sequence and position of the second early leader of region 3 mRNAs, which also correspond to the "y" leader of the fiber mRNA. Following the localization of an open reading frame in which an ATG could initiate protein synthesis it can be predicted that 3a, b, c mRNAs code for the 16K early protein and the probable amino acid sequence of this protein can be deduced. The CAGTTT sequence frequently present at the 5' end of a leader or of a mRNA body as well as the GGTGAG sequence which is found at the 3' end of several leaders were used to postulate the position of various early mRNAs of region 3 and to suggest the existence of an additional splicing event during the processing of mRNAs 3a, b and c. They were also used to predict the position of the additional "x" late leaders. The imbrication of information concerning (i) the family of late mRNAs ending at position 78, (ii) the position of the "x" leader and the "y" leader and (iii) the beginning of early region 3 is also depicted.     

Proto-splice site model of intron origin

It is proposed that nuclear pre-mRNA introns (classical introns) were first generated as by-products during the evolution of alternative splicing. They were formed whenever two splice sites within the coding sequence of ancestral genes were used at a frequency that removed the coding constraint from the intervening sequence. Once introns had evolved, it is suggested that they were spread by the splicing machinery which inserted them into proto or cryptic-splice sites of other genes by reverse splicing, so giving rise to genes that have introns yet are not alternatively spliced. It is argued that 5' and 3' splice sites evolved from common ancestral splice sites, referred to as proto-splice sites, that were bidirectional and had a core consensus sequence of C or A, A, G, R, which remains today as the immediate flanking sequence of most introns. The ancestral splicing machinery, although inefficient, would have been capable of generating vast mRNA diversity by splicing between proto-splice sites. Natural selection would be expected to have preserved mutations that increased the amounts of advantageously spliced mRNA. It is argued that this process drove the evolution of present 5' and 3' splice sites from a subset of proto-splice sites and also drove the evolution of a more efficient splicing machinery. The positions of most introns that evolved directly from the coding sequence would be expected to correlate with protein structure.     

The nucleotide sequence of the genes encoded in early region 2b of human adenovirus type 7

The nucleotide sequence of a cloned DNA segment encoding the early region 2b from the group B human adenovirus Ad7 has been determined. When compared to Ad2, a group C adenovirus, these sequences were found to be approx. 80% homologous within the l-strand gene-coding regions. Most changes are transitions or transversions, although several deletions/insertions also occur within the N-terminal domain of one of the coding regions. The substantial nucleotide homology results in a high degree of amino acid conservation in the predicted polypeptides encoded by the early region 2b genes. Two major open reading frames, corresponding to the Mr 87000 and Mr 140000 polypeptides of Ad2, are found in the l strand of Ad7 between genome coordinates 28.5 to 23.1 and 13.8, respectively. The r strand of the DNA in this region encodes the three leader segments joined to the 5' end of the most late viral mRNAs, and also encodes the i-leader segment found between the second and third leaders on some mRNAs. The positions of the donor and acceptor splice sites of the three leaders are conserved and can be identified by homology to Ad2. Only two of the unidentified open reading frames (URF) in Ad2 (Gingeras et al., J. Biol. Chem., in press) can be found in Ad7. URF1, encoding an Mr 13500 polypeptide at genome coordinate 17, is predominantly conserved in nucleotide and amino acid sequence, but contains one half as many arginine amino acids as does URF1 of Ad2. URF2, encoding an Mr 13600 protein which lies within the i-leader region, is not well conserved in either nucleotide or amino acid sequence.     

Secondary structure analysis of adenovirus tripartite leader

RNA secondary structure analysis was performed to understand the translation function of the adenovirus tripartite leader, a 200-nucleotide 5' noncoding region found on all late viral mRNAs. The tripartite leader facilitates the translation of viral mRNAs at late but not early times after infection and eliminates the normal requirement for the eukaryotic initiation factor 4F or cap binding protein complex. Secondary structures were determined by probing 5' or 3' end-labeled tripartite leader RNAs under nondenaturing conditions with various single strand-specific nucleases, and the information was used to generate a potential model structure. The resulting structure is attractive since it may explain the unusual translation behavior conferred by the tripartite leader. We demonstrate that the first leader segment is predominantly single-stranded, a property consistent with the ability to enhance translation and provide independence from cap binding protein complex. In contrast, the remaining two leader segments form a moderately stable base-paired structure, except for a large hairpin loop. To confirm these findings, the secondary structure of the tripartite leader was also probed when it was attached to a large segment of a messenger RNA and was found to be very similar to that of the individual leader RNA. These findings suggest several possible mechanisms to account for the translation activity of the tripartite leader.     

Characterization of the major mRNAs from adenovirus 2 early region 4 by cDNA cloning and sequencing.

The sequences at the splice junctions of many early region 4 (E4) mRNAs from adenovirus 2 (Ad2) were determined by analysis of cDNA clones. The cDNAs were synthesized from poly(A)+ mRNA isolated from HeLa cells early during Ad2 infection. A standard library was constructed, in pBR322, from double stranded cDNAs initiated by oligo-dT priming. Approximately 1% of total recombinants contained E4 sequences, however among eighty clones analyzed in detail, only four contained the 5' leader sequence. A second library was prepared using a new method that led to a greatly increased representation of desired clones. This method employed oligo-dT to prime the synthesis of the first strand and an oligonucleotide ligated to pBR322, whose sequence was present in the 5' leader, to prime the synthesis of the second strand. With this method the percentage of recombinants containing E4 sequences ranged between 15 and 50% of the total colonies. Virtually all of these E4 cDNA clones contained the 5' leader sequence and several hundred were analyzed by comparing the results from single channel dideoxy sequencing reactions. Nine unique sequence patterns were identified and representative clones were completely sequenced.     

Exon-intron boundary sliding in the generation of two mRNAs coding for porcine urokinase-like plasminogen activator

We have analyzed four full-length cDNA clones to porcine urokinase-like plasminogen activator (uPA) mRNA. DNA sequencing revealed a deletion of 27 nucleotides in one cDNA. The comparison of cDNA and genomic sequences indicated that this length polymorphism was due to an alternative splicing of two potential 5' splice sites to a unique 3' splice site. As the difference was 27 nucleotides (corresponding to 9 amino acids) and there was no termination codon within the same reading frame in this region, the two different mRNAs might be equally biologically active.     

Identification of an alternatively spliced seprase mRNA that encodes a novel intracellular isoform

Seprase is a homodimeric 170-kDa integral membrane gelatinase that is related to the ectoenzyme dipeptidyl peptidase IV. We have identified an alternatively spliced seprase messenger from the human melanoma cell line LOX that encodes a novel truncated isoform, seprase-s. The splice variant mRNA is generated by an out-of-frame deletion of a 1223-base pair exonic region that encodes part of the cytoplasmic tail, transmembrane, and the membrane proximal-central regions of the extracellular domain (Val(5) through Ser(412)) of the seprase 97-kDa subunit (seprase-l). The seprase-s mRNA has an elongated 5' leader (548 nucleotides) that harbors at least two upstream open reading frames that inhibit seprase-s expression from a downstream major open reading frame. Deletion mutagenesis of the wild type splice variant cDNA confirms that initiation of the seprase-s coding sequence begins with an ATG codon that corresponds to Met(522) of seprase-l. The seprase-s open reading frame encodes a 239-amino acid polypeptide with an M(r) approximately 27,000 that precisely overlaps the carboxyl-terminal catalytic region of seprase-l.     

Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus.

Dicistronic mRNA expression vectors efficiently translate a 5' open reading frame (ORF) and contain a selectable marker within the 3' end which is inefficiently translated. In these vectors, the efficiency of translation of the selectable 3' ORF is reduced approximately 100-fold and is highly dependent on the particular sequences inserted into the 5' cloning site. Upon selection for expression of the selection marker gene product, deletions within the 5' ORF occur to yield more efficient translation of the selectable marker. We have generated improved dicistronic mRNA expression vectors by utilization of a putative internal ribosomal entry site isolated from encephalomyocarditis (EMC) virus. Insertion of the EMC virus leader sequence upstream of an ORF encoding either a wildtype or methotrexate resistant dihydrofolate reductase (DHFR) reduces DHFR translation up to 10-fold in a monocistronic DHFR expression vector. However, insertion of another ORF upstream of the EMC leader to produce a dicistronic mRNA does not further reduce DHFR translation. In the presence of the EMC virus leader, DHFR translation is not dependent on sequences inserted into the 5' end of the mRNA. We demonstrate that stable high level expression of inserted cDNAs may be rapidly achieved by selection for methotrexate resistance in DHFR deficient as well as DHFR containing cells. In contrast to previously described dicistronic expression vectors, these new vectors do not undergo rearrangement or deletion upon selection for amplification by propagation in increasing concentrations of methotrexate. The explanation may be either that the EMC virus leader sequence allows internal initiation of translation or that cryptic splice sites in the EMC virus sequence mediate production of monocistronic mRNAs. These vectors may be generally useful to rapidly obtain high level expression of cDNA genes in mammalian cells.