Sequence of human adenosine deaminase cDNA including the coding region and a small intron

The nucleotide sequence for an unusual, cloned human adenosine deaminase cDNA has been determined. Contained within a sequence of 1535 nucleotides is a coding sequence of 1089 nucleotides that encodes a protein of 40,762 daltons. The coding sequence is interrupted by a non-coding region containing 76 nucleotides. Both the 3' and 5' ends of this region have consensus sequences generally associated with splice sites. The 3' untranslated sequence contained 308 nucleotides, including a polyadenylation signal sequence 20 nucleotides from the end. The cloned cDNA appears to correspond to a nuclear mRNA precursor which contains a small intron.     

Deletion analysis of a unique 3'' splice site indicates that alternating guanine and thymine residues represent an efficient splicing signal.

The 3' splice site of the second intron (I2) of the human apolipoprotein-AII gene, (GT)16GGGCAG, is unique in that, although fully functional, a stretch of alternating guanine and thymine residues replaces the polypyrimidine tract usually associated with 3' splice junctions. The transient expression of successive 5' deletion mutants has defined the minimum number of nucleotides at the 3' end of apo-AII I2 that are required to direct efficient splicing. Processing in two cell-types, representing apo-AII producing and non-producing tissue was identical; in both, only by removing all the GT repeats did the 3' splice site of apo-AII I2 become completely non-functional. Similar deletion analyses of "classic" 3' splice sites, which conform to the consensus sequence (Y)nNYAG, have indicated that a minimum of 14 nucleotides of the polypyrimidine tract are required for detectable levels of processing to take place. Here we report that the six nucleotides (GT)2GG, which directly replace this tract in a deletion mutant of the 3' splice site of apo-AII I2 are sufficient to direct the splicing process efficiently and correctly.     

5' exon requirement for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA and identification of a cryptic 5' splice site in the 3' exon

The intervening sequence (IVS) of the Tetrahymena thermophila ribosomal RNA precursor undergoes accurate self-splicing in vitro. The work presented here examines the requirement for Tetrahymena rRNA sequences in the 5' exon for the accuracy and efficiency of splicing. Three plasmids were constructed with nine, four and two nucleotides of the natural 5' exon sequence, followed by the IVS and 26 nucleotides of the Tetrahymena 3' exon. RNA was transcribed from these plasmids in vitro and tested for self-splicing activity. The efficiency of splicing, as measured by the production of ligated exons, is reduced as the natural 5' exon sequence is replaced with plasmid sequences. Accurate splicing persists even when only four nucleotides of the natural 5' exon sequence remain. When only two nucleotides of the natural exon remain, no ligated exons are observed. As the efficiency of the normal reaction diminishes, novel RNA species are produced in increasing amounts. The novel RNA species were examined and found to be products of aberrant reactions of the precursor RNA. Two of these aberrant reactions involve auto-addition of GTP to sites six nucleotides and 52 nucleotides downstream from the 3' splice site. The former site occurs just after the sequence GGU, and may indicate the existence of a GGU-binding site within the IVS RNA. The latter site follows the sequence CUCU, which is identical with the four nucleotides preceding the 5' splice site. This observation led to a model where where the CUCU sequence in the 3' exon acts as a cryptic 5' splice site. The model predicted the existence of a circular RNA containing the first 52 nucleotides of the 3' exon. A small circular RNA was isolated and partially sequenced and found to support the model. So, a cryptic 5' splice site can function even if it is located downstream from the 3' splice site. Precursor RNA labeled at its 5' end, presumably by a GTP exchange reaction mediated by the IVS, is also described.     

Identification and characterization by antisense oligonucleotides of exon and intron sequences required for splicing.

Certain thalassemic human beta-globin pre-mRNAs carry mutations that generate aberrant splice sites and/or activate cryptic splice sites, providing a convenient and clinically relevant system to study splice site selection. Antisense 2'-O-methyl oligoribonucleotides were used to block a number of sequences in these pre-mRNAs and were tested for their ability to inhibit splicing in vitro or to affect the ratio between aberrantly and correctly spliced products. By this approach, it was found that (i) up to 19 nucleotides upstream from the branch point adenosine are involved in proper recognition and functioning of the branch point sequence; (ii) whereas at least 25 nucleotides of exon sequences at both 3' and 5' ends are required for splicing, this requirement does not extend past the 5' splice site sequence of the intron; and (iii) improving the 5' splice site of the internal exon to match the consensus sequence strongly decreases the accessibility of the upstream 3' splice site to antisense 2'-O-methyl oligoribonucleotides. This result most likely reflects changes in the strength of interactions near the 3' splice site in response to improvement of the 5' splice site and further supports the existence of communication between these sites across the exon.     

Branch point selection in alternative splicing of tropomyosin pre-mRNAs.

The rat tropomyosin 1 gene gives rise to two mRNAs encoding rat fibroblast TM-1 and skeletal muscle beta-tropomyosin via an alternative splicing mechanism. The gene is comprised of 11 exons. Exons 1 through 5 and exons 8 and 9 are common to all mRNAs expressed from this gene. Exons 6 and 11 are used in fibroblasts as well as smooth muscle whereas exons 7 and 10 are used exclusively in skeletal muscle. In the present studies we have focused on the mutually exclusive internal alternative splice choice involving exon 6 (fibroblast-type splice) and exon 7 (skeletal muscle-type splice). To study the mechanism and regulation of alternative splice site selection we have characterized the branch points used in processing of the tropomyosin pre-mRNAs in vitro using nuclear extracts obtained from HeLa cells. Splicing of exon 5 to exon 6 (fibroblast-type splice) involves the use of three branch points located 25, 29, and 36 nucleotides upstream of the 3' splice site of exon 6. Splicing of exon 6 (fibroblast-type splice) or exon 7 (skeletal muscle type-splice) to exon 8 involves the use of the same branch point located 24 nucleotides upstream of this shared 3' splice site. In contrast, the splicing of exon 5 to exon 7 (skeletal muscle-type splice) involves the use of three branch sites located 144, 147 and 153 nucleotides, upstream of the 3' splice site of exon 7. In addition, the pyrimidine content of the region between these unusual branch points and the 3' splice site of exon 7 was found to be greater than 80%. These studies raise the possibility that the use of branch points located a long distance from a 3' splice site may be an essential feature of some alternatively spliced exons. The possible significance of these unusual branch points as well as a role for the polypyrimidine stretch in intron 6 in splice site selection are discussed.     

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.     

Tri-partite assay for studying exon ligation by the ai5gamma group II intron

Group II introns self-splice via a two-step mechanism: cleavage at the 5' splice site followed by exon ligation at the 3' splice site. The second step has been difficult to study in vitro because it is generally faster than the first. Herein we describe development and partial kinetic characterization of a novel assay for studying the second step in isolation. In this system, a truncated linear intron (nucleotides 1-881) mediates exon ligation between two oligonucleotide substrates: a 19 nt 5' exon and a 3' substrate consisting of the last 6 nucleotides of the intron plus a 6 nucleotide 3' exon. We found that neither the exact structure of domain 6 nor the identity of nucleotides flanking the 3' splice site is critical for accurate 3' splice site choice by the ai5gamma group II intron. The multiple turnover k(cat) (0.14 min(-)(1)) is slower than the single turnover k(obs) (0.6-0.7 min(-)(1)), consistent with rate-limiting product release under steady-state conditions. Decreased single turnover rates at lower pHs were more consistent with loss of catalytic activity than with rate-limiting chemistry. Binding of the 3' substrate (K(m) = 2.6 microM) could be improved by changing a long-range A:U base pair involving the last intronic nucleotide (the gamma-gamma' interaction) to G:C (K(m(3)(')(substrate)) = 1 microM).     

Role of intron-contained sequences in formation of moloney murine leukemia virus env mRNA.

Formation of the Moloney murine leukemia virus envelope mRNA involves the removal of a 5,185-base pair-long intron. Deletion analysis of two Moloney murine leukemia virus-derived expression vectors revealed the existence of two short regions within the viral intron which are required for the efficient formation of the spliced RNA species. One region was present upstream from the 3' splice junction, extended at least 85 nucleotides beyond the splice site, and was not more than 165 nucleotides long. As yeast polymerase II introns, the Moloney murine leukemia virus intron contains the sequence 5'-TACTAAC-3' 15 nucleotides upstream from the 3' splice site. A second region located in the middle of the intron, within a 560-nucleotide-long sequence, was also essential for formation of the spliced RNA species. The efficient splicing of the env mRNA in the absence of expression of viral genes raises the possibility that similar mechanisms are used to remove introns of (some) cellular genes.     

A mutational analysis of U12-dependent splice site dinucleotides

Introns spliced by the U12-dependent minor spliceosome are divided into two classes based on their splice site dinucleotides. The /AU-AC/ class accounts for about one-third of U12-dependent introns in humans, while the /GU-AG/ class accounts for the other two-thirds. We have investigated the in vivo and in vitro splicing phenotypes of mutations in these dinucleotide sequences. A 5' A residue can splice to any 3' residue, although C is preferred. A 5' G residue can splice to 3' G or U residues with a preference for G. Little or no splicing was observed to 3' A or C residues. A 5' U or C residue is highly deleterious for U12-dependent splicing, although some combinations, notably 5' U to 3' U produced detectable spliced products. The dependence of 3' splice site activity on the identity of the 5' residue provides evidence for communication between the first and last nucleotides of the intron. Most mutants in the second position of the 5' splice site and the next to last position of the 3' splice site were defective for splicing. Double mutants of these residues showed no evidence of communication between these nucleotides. Varying the distance between the branch site and the 3' splice site dinucleotide in the /GU-AG/ class showed that a somewhat larger range of distances was functional than for the /AU-AC/ class. The optimum branch site to 3' splice site distance of 11-12 nucleotides appears to be the same for both classes.     

The human immunodeficiency virus type 1 Rev protein and the Rev-responsive element counteract the effect of an inhibitory 5' splice site in a 3' untranslated region.

A 5' splice site located in a 3' untranslated region (3'UTR) has been shown previously to inhibit gene expression. Natural examples of inhibitory 5' splice sites have been identified in the late 3'UTRs of papillomaviruses and are thought to inhibit viral late gene expression at early stages of the viral life cycle. In this study, we demonstrate that the interaction of the human immunodeficiency virus type 1 Rev protein with the Rev-responsive element (RRE) overcomes the inhibitory effects of a 5' splice site located within a 3'UTR. This was studied by using both a bovine papillomavirus type 1 L1 cDNA expression vector and a chloramphenicol acetyltransferase expression vector containing a 5' splice site in the 3'UTR. In both systems, coexpression of Rev enhanced cytoplasmic expression from vectors containing the RRE even when the RRE and the inhibitory 5' splice site were separated by up to 1,000 nucleotides. In addition, multiple copies of a 5' splice site in a 3'UTR were shown to act synergistically, and this effect could also be moderated by the interaction of Rev and the RRE. These studies provide additional evidence that at least one mechanism of Rev action is through interactions with the splicing machinery. We have previously shown that base pairing between the U1 small nuclear RNA and a 3'UTR 5' splice site is required for inhibition of gene expression. However, experiments by J. Kjems and P. A. Sharp (J. Virol. 67:4769-4776, 1993) have suggested that Rev acts on spliceosome assembly at a stage after binding of the U1 small nuclear ribonucleoprotein to the 5' splice site. This finding suggests that binding of additional small nuclear ribonucleoproteins, as well as other splicing factors, may be necessary for the inhibitory action of a 3'UTR 5' splice site. These data also suggest that expression of the papillomavirus late genes in terminally differentiated keratinocytes can be regulated by a viral or cellular Rev-like activity.     

Relationship Between Moloney Murine Leukemia and Sarcoma Virus RNAs: Purification and Hybridization Map of Complementary DNAs from Defined Regions of Moloney Murine Sarcoma Virus 124

Complementary DNAs (cDNA's) specific for various regions of the Moloney murine sarcoma virus (MSV) 124 RNA genome were prepared by cross-hybridization techniques. A cDNA specific for the first 1,000 nucleotides adjacent to the RNA 3' end (cDNA 3') was prepared and shown to also be complementary to the 3'-terminal 1,000 nucleotides of a related Moloney murine leukemia virus (MLV) genome. A cDNA complementary to the "MSV-specific" portion of the MSV 124 genome was prepared. This cDNA was shown not to anneal to Moloney MLV RNA and to anneal to a portion of the viral RNA of about 1,500 to 1,800 nucleotides in length, located 1,000 nucleotides from the 3' end of MSV RNA. A cDNA common to the genome of MSV and MLV was also obtained and shown to anneal to the 5'-terminal two-thirds, as well as to the 3'-terminal 1,000 nucleotides, of the MSV RNA genome. This cDNA also annealed to the RNA from MLV and mainly to the 5'-terminal half of the MLV genome. It is concluded that the 6-kilobase Moloney MSV 124 RNA genome has a sequence arrangement that includes (i) a 3' portion of about 1,000 nucleotides, which is also present at the 3' terminus of MLV; (ii) an MSV-specific region, not shared with MLV, which extends between 1,000 and 2,500 nucleotides from the 3' terminus; and (iii) a second "common" region, again shared with MLV, which extends from 2,500 nucleotides to the 5' terminus. This second common region appears to be located in the 5' half of the 10-kilobase MLV genome as well. Experiments in which a large excess of cold MLV cDNA was annealed to (3)H-labeled polyadenylic acid-containing fragments of MSV RNA gave results consistent with this arrangement of the MSV genome.     

Deletion mutants that alter differential RNA processing in the E3 complex transcription unit of adenovirus

Region E3 of the adenovirus encodes about ten overlapping mRNAs (a to j) with different splicing patterns and with two RNA 3' end sites termed E3A and E3B. We have examined how deletions in 12 viable virus mutants affect differential RNA processing in E3. We assayed E3 mRNAs by the nuclease-gel and RNA blot procedures. Some deletions had no effect whereas others (e.g. deletion of a 3' splice or the E3A 3' end signal) had the anticipated effects on RNA processing. However, deletions in two regions had surprising effects. Deletions in one region (nucleotides 1691 to 2044) enhanced splicing at the upstream 951 5' splice site and the downstream 2157 and/or 2880 3' splice sites. Some of these deletions prevented RNA 3' end formation at the downstream E3A site. Deletion in the other region (nucleotides 2173 to 2237) enhanced an upstream splice site (951 to 2157) such that almost all pre-mRNA was processed into mRNA f. We suggest that these two regions contain cis-acting signals that regulate differential RNA processing. We discuss the results in terms of RNA folding and scanning models for splicing, as well as models for differential RNA 3' end formation at the E3A versus the E3B site.     

The role of branchpoint-3'' splice site spacing and interaction between intron terminal nucleotides in 3'' splice site selection in Saccharomyces cerevisiae.

A conserved 3' splice site YAG is essential for the second step of pre-mRNA splicing but no trans-acting factor recognizing this sequence has been found. A direct, non-Watson-Crick interaction between the intron terminal nucleotides was suggested to affect YAG selection. The mechanism of YAG recognition was proposed to involve 5' to 3' scanning originating from the branchpoint or the polypyrimidine tract. We have constructed a yeast intron harbouring two closely spaced 3' splice sites. Preferential selection of a wild-type site over mutant ones indicated that the two sites are competing. For two identical sequences, the proximal site is selected. As previously observed, an A at the first intron nucleotide spliced most efficiently with a 3' splice site UAC. In this context, UAA or UAU were also more efficient 3' splice sites than UAG and competed more efficiently than the wild-type sequence with a 3' splice site UAC. We observed that a U at the first intron nucleotide is used for splicing in combination with 3' splice sites UAG, UAA or UAU. Our data indicate that the 3' splice site is not primarily selected through an interaction with the first intron nucleotide. Selection of the 3' splice site depends critically on its distance from the branchpoint but does not occur by a simple leaky scanning mechanism.     

A single nucleotide difference at the 3'' end of an intron causes differential splicing of two histocompatibility genes.

The murine histocompatibility class I genes, H-2 Kb and Kk, display considerable homology at their 3' ends. In fact, from exon 5 to the termination codon, only two nucleotides differ between the two genes, one at the 5' end and the other at the 3' end of intron 7. Despite this similarity, the gene products have distinctly different mol. wts as determined by SDS-PAGE. By constructing two hybrid genes, pC2 and pC4, we demonstrated that it is the cytoplasmic parts of the antigens (encoded by exons 6-8) which are responsible for the major difference in mol. wt. We have used site-directed mutagenesis to change the two nucleotides in intron 7 of the H-2 Kk gene to those present in the H-2 Kb gene. S1 nuclease mapping has been used to identify the actual splice site of the authentic Kb and Kk genes, the hybrid genes and the mutagenized genes. We have shown that it is the 3' nucleotide difference, nine nucleotides upstream of the 3' splice site, which causes the different excision of intron 7 of the Kb gene. The 5' nucleotide difference does not alter the splicing. The choice of branch points and 3' splice signals for intron 7 of five H-2 class I genes, is discussed.