Pre-mRNA splicing of IgM exons M1 and M2 is directed by a juxtaposed splicing enhancer and inhibitor.

Splicing of certain pre-mRNA introns is dependent on an enhancer element, which is typically purine-rich. It is generally thought that enhancers increase the use of suboptimal splicing signals, and one specific proposal is that enhancers stabilize binding of U2AF65 to weak polypyrimidine (Py) tracts. Here, we test this model using an IgM pre-mRNA substrate, which contains a well-characterized enhancer. Although the enhancer was required for in vitro splicing, we found it had no effect on U2AF65 binding. Unexpectedly, replacement of the natural IgM Py tract, branchpoint, and 5' splice site with consensus splicing signals did not circumvent the enhancer requirement. These observations led us to identify a novel regulatory element within the IgM M2 exon that acts as a splicing inhibitor; removal of the inhibitor enabled splicing to occur in the absence of the enhancer. The IgM M2 splicing inhibitor is evolutionarily conserved, can inhibit the activity of an unrelated, constitutively spliced pre-mRNA, and acts by repressing splicing complex assembly. Interestingly, the inhibitor itself forms an ATP-dependent complex that contains U2 snRNP. We conclude that splicing of IgM exons M1 and M2 is directed by two juxtaposed regulatory elements-an enhancer and an inhibitor-and that a primary function of the enhancer is to counteract the inhibitor.

Intronic polyadenylation in the human glycinamide ribonucleotide formyltransferase gene.

The mouse glycinamide ribonucleotide formyltransferase (GART) locus is known to produce two functional proteins, one by recognition and use of an intronic polyadenylation site and the other by downstream splicing. We now report a similar intronic polyadenylation mechanism for the human GART locus. The human GART gene has two potential polyadenylation signals within the identically located intron as that involved in intronic polyadenylation in the mouse gene. Each of the potential polyadenylation signals in the human gene was followed by an extensive polyT rich tract, but only the downstream signal was preceded by a GT tract. Only the downstream signal was utilized. The polyT rich tract which followed the functional polyadenylation site in the human GART gene was virtually identical in sequence to a similarly placed region in the mouse gene. An exact inverted complement to the polyT rich stretch following the active polyadenylation signal was found in the upstream intron of the human gene, suggesting that a hairpin loop may be involved in this intronic polyadenylation.

Analysis of a mouse gene encoding three steps of purine synthesis reveals use of an intronic polyadenylation signal without alternative exon usage.

A single mouse genomic locus encodes proteins catalyzing three steps of purine synthesis, glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS), and glycinamide ribonucleotide formyltransferase (GART). This gene has 22 exons and spans 28 kilobases. The existence of a second genetic locus and closely related pseudogenes was ruled out by Southern analysis. Mouse tissues express two related classes of messages encoded by this single locus: a trifunctional GARS-AIRS-GART mRNA and a monofunctional GARS mRNA. These transcripts used the same set of multiple transcriptional start sites, and both used the same first 10 exons. CCAAT and TATA elements were not found for this locus. Exon 11, which represented the last coding sequence of the GARS domain, was differentially utilized for the two messages. The trifunctional mRNA was generated by splicing exon 11 to exon 12, the first coding sequence for the AIRS domain with subsequent use of a polyadenylation signal at the end of exon 22. Genomic sequence corresponding to the 3'-UTR of the monofunctional GARS mRNA was contiguous with exon 11, so that the smaller message arose from the recognition of one of the multiple polyadenylation signals present within the intron between exons 11 and 12. Hence, polyadenylation of the primary transcript at a position corresponding to an intron of the genomic locus was responsible for the generation of the monofunctional GARS class of mRNAs. This utilization of an intronic polyadenylation site without alternative exon usage is comparable to the mechanism whereby both secreted and membrane-bound forms of the immunoglobulin mu heavy chain are made from a single genetic locus.

Mouse cDNAs encoding a trifunctional protein of de novo purine synthesis and a related single-domain glycinamide ribonucleotide synthetase.

Three of the enzymatic activities of de novo purine synthesis, glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS) and glycinamide ribonucleotide formyltransferase (GART), can be catalyzed by a single 110-kDa protein in mouse cells. Western blots using a polyclonal antibody (Ab) to this protein identified two species, the trifunctional 110-kDa protein and a 50-kDa cytosolic protein with GARS, but not GART activity. We used Ab and, subsequently, oligodeoxyribonucleotide screens to isolate cDNAs corresponding to these two proteins from mouse T-cell cDNA expression libraries. The sequence of one class of these cDNAs and the partial sequence of a corresponding genomic clone defined an open reading frame (ORF) encoding a 1010-amino-acid (aa) protein, individual domains of which showed high homology to each of the monofunctional bacterial GARS, AIRS and GART proteins, and to each domain of chicken and human trifunctional GARS-AIRS-GARTs. cDNAs corresponding to the smaller protein contained a 1.3-kb ORF with complete identity to the GARS domain of, but with a 3' untranslated region different from, the trifunctional cDNAs. Hence, both cDNAs appear to derive from the same gene due to either differential splicing or use of an intronic polyadenylation signal. The functional requirement for the expression of both trifunctional protein with GARS activity and monofunctional, catalytically active GARS is unknown.

Recognition of unusual DNA structures by human DNA (cytosine-5)methyltransferase.

The symmetry of the responses of the human DNA (cytosine-5)methyltransferase to alternative placements of 5-methylcytosine in model oligodeoxynucleotide duplexes containing unusual structures has been examined. The results of these experiments more clearly define the DNA recognition specificity of the enzyme. A simple three-nucleotide recognition motif within the CG dinucleotide pair can be identified in each enzymatically methylated duplex. The data can be summarized by numbering the four nucleotides in the dinucleotide pair thus: 1 4/2 3. With reference to this numbering scheme, position 1 can be occupied by cytosine or 5-methylcytosine; position 2 can be occupied by guanosine or inosine; position 3, the site of enzymatic methylation, can be occupied only by cytosine; and position 4 can be occupied by guanosine, inosine, O6-methylguanosine, cytosine, adenosine, an abasic site, or the 3' hydroxyl group at the end of a gapped molecule. Replacing the guanosine normally found at position 4 with any of the moieties introduces unusual (non-Watson-Crick) pairing at position 3 and generally enhances methylation of the cytosine at that site. The exceptional facility of the enzyme in actively methylating unusual DNA structures suggests that the evolution of the DNA methyltransferase, and perhaps DNA methylation itself, may be linked to the biological occurrence of unusual DNA structures.