A new twist in trypanosome RNA metabolism: cis-splicing of pre-mRNA

It has been known for almost a decade and a half that in trypanosomes all mRNAs are trans-spliced by addition to the 5' end of the spliced leader (SL) sequence. During the same time period the conviction developed that classical cis-splicing introns are not present in the trypanosome genome and that the trypanosome gene arrangement is highly compact with small intergenic regions separating one gene from the next. We have now discovered that these tenets are no longer true. Poly(A) polymerase (PAP) genes in Trypanosoma brucei and Trypanosoma cruzi are split by intervening sequences of 653 and 302 nt, respectively. The intervening sequences occur at identical positions in both organisms and obey the GT/AG rule of cis-splicing introns. PAP mRNAs are trans-spliced at the very 5' end as well as internally at the 3' splice site of the intervening sequence. Interestingly, 11 nucleotide positions past the actual 5' splice site are conserved between the T. bruceiand T. cruzi introns. Point mutations in these conserved positions, as well as in the AG dinucleotide of the 3' splice site, abolish intron removal in vivo. Our results, together with the recent discovery of cis-splicing introns in Euglena gracilis, suggest that both trans- and cis-splicing are ancient acquisitions of the eukaryotic cell.     

Multiple introns in a conjugation-specific gene from Tetrahymena thermophila

Multiple introns have been found in a gene from a ciliated protozoan. This Tetrahymena thermophila gene (cnjB) is large (7.5 kb mRNA) and active only during conjugation, the organism's sexual cycle. Six introns ranging in size from 62 bp to 676 bp were found when we sequenced a 3.1 kb segment of the cnjB gene together with its corresponding cDNA. We estimate, by extrapolation of our current data, a total of approximately 30 introns in this gene with a total gene size (introns plus exons) of 15 kb or more. The number of introns is surprising given the scarcity of introns in ciliate genes examined to date. Our findings constitute the first example of multiple introns in a ciliate gene. Having the sequence of several introns has allowed us to construct consensus sequences for T. thermophila mRNA introns. The 5' and 3' intron junctions resemble those of general nuclear mRNA (GT/AG rule is followed) but differences are seen. In particular, stretches of 10 or more adenines and thymines are found adjacent to the conserved GT and AGs at the junctions. Unusual aspects of the coding region of this gene are discussed.     

The sedlin gene for spondyloepiphyseal dysplasia tarda escapes X-inactivation and contains a non-canonical splice site

Mutations in the sedlin gene cause spondyloepiphyseal dysplasia tarda (SEDT), a rare X-linked chondrodysplasia. Affected males suffer short stature, deformation of the spine and hips, and deterioration of intervertebral discs with characteristic radiographic changes in the vertebrae. We have sequenced two full-length cDNA clones corresponding to the human sedlin gene. The longest cDNA is 2836 bp, containing a 218 bp 5' untranslated region, a 423 bp coding region, and a 2195 bp 3' untranslated region. The second cDNA does not contain exon 2, suggesting alternative splicing. Sedlin was finely mapped in Xp22.2 by Southern blot analysis on a yeast artificial chromosome/bacterial artificial chromosome map. Comparison of the cDNA sequence and genomic sequence identified six sedlin exons of 67, 142, 112, 147, 84, and 2259 bp. The corresponding introns vary in size from 339 to 14,061 bp. Splice site sequences for four of the five introns conform to the GT/AG consensus sequences, however, the splice site between exons 4 and 5 displays a rare non-canonical splice site sequence, AT/AC. Northern blot analysis showed expression of the sedlin gene in all human adult and fetal tissues tested, with the highest levels in kidney, heart, skeletal muscle, liver, and placenta. Four mRNA sizes were detected with the major band being 3 kb and minor bands of 5, 1.6, and 0.9 kb (the smallest product may reflect a sedlin pseudogene). Sedlin is expressed from both the active and the inactive human X chromosomes helping to explain the recessive nature and consistent presentation of the disease. Human sedlin shows homology to a yeast gene, which conditions endoplasmic reticulum/golgi transport. Characterization of the human sedlin cDNA and determination of the sedlin gene structure enable functional studies of sedlin and elucidation of the pathogenesis of SEDT.     

In vitro splicing of mutually exclusive exons from the chicken beta-tropomyosin gene: role of the branch point location and very long pyrimidine stretch.

The chicken beta-tropomyosin gene contains 11 exons, two of which are spliced into mRNA only in skeletal muscle. One pair of alternative exons, 6A and 6B, is found in the middle of the gene; they are spliced in a mutually exclusive manner. The non-muscle splice 6A-7 is by far the predominant in vitro reaction in a HeLa cell nuclear extract. A minor product is the 6A-6B splice, which is excluded in all tissues. This minor product results from the use of a branch point located 105 nt upstream of the 3' end of the intron separating exons 6A and 6B. The region between the branch point sequence and the final AG contains a stretch of approximately 80 pyrimidines. We have examined the role of the distance of the branchpoint to the 3' splice site and of the sequences between these two elements. Our results suggest that at least two cis-acting elements contribute to the mutual exclusivity of exons 6A and 6B. The intron between exons 6A and 6B is intrinsically poorly 'spliceable' both because the branch point is too far upstream of the 3' end of the intron to give efficient splicing and because of the particular sequence lying between this branch point and the 3' splice site.     

Cryptic intron activation within the large exon of the mouse polymeric immunoglobulin receptor gene: cryptic splice sites correspond to protein domain boundaries.

The fourth exon of the mouse polymeric immuno-globulin receptor (pIgR) is 654 nt long and, despite being surrounded by large introns, is constitutively spliced into the mRNA. Deletion of an 84 nt sequence from this exon strongly activated both cryptic 5' and 3' splice sites surrounding a 78 nt cryptic intron. The 84 nt deletion is just upstream of the cryptic 3' splice site; the cryptic 3' splice site was likely activated because the deletion created a better 3' splice site. However, the cryptic 5' splice site was also required to activate the cryptic splice reaction; point mutations in either of the cryptic splice sites that decreased their match to the consensus splice site sequence inactivated the cryptic splice reaction. The activation and inactivation of these cryptic splice sites as a pair suggests that they are being co-recognized by the splicing machinery. Interestingly, the large fourth exon of the pIgR gene encodes two immunoglobulin-like extracellular protein domains; the cryptic 3' splice site coincides with the junction between these protein domains. The cryptic 5' splice site is located between protein subdomains where an intron is found in another gene of the immunoglobulin superfamily.     

Nucleotide sequence of the gene for human prothrombin

A human genomic DNA library was screened for the gene coding for human prothrombin with a cDNA coding for the human protein. Eighty-one positive lambda phage were identified, and three were chosen for further characterization. These three phage hybridized with 5' and/or 3' probes prepared from the prothrombin cDNA. The complete DNA sequence of 21 kilobases of the human prothrombin gene was determined and included a 4.9-kilobase region that was previously sequenced. The gene for human prothrombin contains 14 exons separated by 13 intervening sequences. The exons range in size from 25 to 315 base pairs, while the introns range from 84 to 9447 base pairs. Ninety percent of the gene is composed of intervening sequence. All the intron splice junctions are consistent with sequences found in other eukaryotic genes, except for the presence of GC rather than GT on the 5' end of intervening sequence L. Thirty copies of Alu repetitive DNA and two copies of partial KpnI repeats were identified in clusters within several of the intervening sequences, and these repeats represent 40% of the DNA sequence of the gene. The size, distribution, and sequence homology of the introns within the gene were then compared to those of the genes for the other vitamin K dependent proteins and several other serine proteases.     

Intron definition in splicing of small Drosophila introns.

Approximately half of the introns in Drosophila melanogaster are too small to function in a vertebrate and often lack the pyrimidine tract associated with vertebrate 3' splice sites. Here, we report the splicing and spliceosome assembly properties of two such introns: one with a pyrimidine-poor 3' splice site and one with a pyrimidine-rich 3' splice site. The pyrimidine-poor intron was absolutely dependent on its small size for in vivo and in vitro splicing and assembly. As such, it had properties reminiscent of those of yeast introns. The pyrimidine-rich intron had properties intermediate between those of yeasts and vertebrates. This 3' splice site directed assembly of ATP-dependent complexes when present as either an intron or exon and supported low levels of in vivo splicing of a moderate-length intron. We propose that splice sites can be recognized as pairs across either exons or introns, depending on which distance is shorter, and that a pyrimidine-rich region upstream of the 3' splice site facilitates the exon mode.     

Requirements for mini-exon inclusion in potato invertase mRNAs provides evidence for exon-scanning interactions in plants

Invertases are responsible for the breakdown of sucrose to fructose and glucose. In all but one plant invertase gene, the second exon is only 9 nt in length and encodes three amino acids of a five-amino-acid sequence that is highly conserved in all invertases of plant origin. Sequences responsible for normal splicing (inclusion) of exon 2 have been investigated in vivo using the potato invertase, invGF gene. The upstream intron 1 is required for inclusion whereas the downstream intron 2 is not. Mutations within intron 1 have identified two sequence elements that are needed for inclusion: a putative branchpoint sequence and an adjacent U-rich region. Both are recognized plant intron splicing signals. The branchpoint sequence lies further upstream from the 3' splice site of intron 1 than is normally seen in plant introns. All dicotyledonous plant invertase genes contain this arrangement of sequence elements: a distal branchpoint sequence and adjacent, downstream U-rich region. Intron 1 sequences upstream of the branchpoint and sequences in exons 1, 2, or 3 do not determine inclusion, suggesting that intron or exon splicing enhancer elements seen in vertebrate mini-exon systems are absent. In addition, mutation of the 3' and 5' splice sites flanking the mini-exon cause skipping of the mini-exon, suggesting that both splice sites are required. The branchpoint/U-rich sequence is able to promote splicing of mini-exons of 6, 3, and 1 nt in length and of a chicken cTNT mini-exon of 6 nt. These sequence elements therefore act as a splicing enhancer and appear to function via interactions between factors bound at the branchpoint/U-rich region and at the 5' splice site of intron 2, activating removal of this intron followed by removal of intron 1. This first example of splicing of a plant mini-exon to be analyzed demonstrates that particular arrangement of standard plant intron splicing signals can drive constitutive splicing of a mini-exon.