Intronic polyadenylation in the human glycinamide ribonucleotide formyltransferase gene

Abstract

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.

No Evidence for Mutations that Deregulate GARS–AIRS–GART Protein Levels in Children with Down Syndrome

“…The exons coding for ATP-binding and active-site residues of GARS (exons 5, 6, 9) were coamplified with the exon 15 encoding for the 3rd glycinerich loop of AIRS [15] and a poly-A signal sequence within Intron 11 (Intron-11A) [8] in Multiplex Set-I reactions along with singleplex controls (Fig. 2a).…”

“…2a). Exons 13,18,19 that include the 2nd glycine rich loop of AIRS and the substrate binding loop of GART, respectively, [15] were co-amplified with the Poly-(A) signal sequence located within Intron 11B [8] in Multiplex Set-II reactions (Fig. 2b).…”

“…Primers were designed with Primer3 software (http://hpc. ilri.cgiar.org/cgi-bin/primer3_www.cgi); forward primers for intron 11A and 11B were as reported previously [8]. Primer sequences and amplification conditions are detailed in Table 1 and amplification procedures were standardized as described previously [21,22].…”

“…GARS-AIRS-GART spans *38 kb with 22 exons [6] and the cDNA has been cloned by functional complementation in Escherichia coli [7]. The gene harbors two polyadenylation signal sequences in intron 11 which participate in alternative splicing leading to the generation of monofunctional GARS and tri-functional GARS-AIRS-GART transcripts [8]. Purines play vital roles in embryonic development [9,10] and nervous system function [11].…”

“…1, the inheritance of single/multiple copies of a mutation gives rise to a dosage effect; however, a combination of mutations in different regions of the GARS-AIRS-GART gene that encodes distinct enzymatic functions may disrupt epistatic interactions and account for the range of phenotypic severity of DS-related MR. Interestingly, the inheritance of 1-4 copies of mutations in the a-globin chain disrupts interactions in the a 2 b 2 complex of haemoglobin leading to the observed range of severity of b-thalassemia [14]. Accordingly, GARS-AIRS-GART regions previously identified as being crucial for alternative splicing [8], structure-function or regulation of gene expression [15] were prioritized for screening of mutations that could potentially alter protein levels in DS patients. We used multiplex/ singleplex PCR-based target amplification followed by amplicon size determination and fingerprinting.…”