Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Intron A of the human growth hormone gene does not contain an A residue within 56 nucleotides preceding the 3' splice site. The analysis of the excised intron lariat revealed a C residue 28 nucleotides upstream from the 3' splice site as the major branch acceptor nucleotide. Two additional minor branched nucleotides were identified as U residues at positions -22 and -36. An adenosine substitution at position -22 results in lariat formation solely to this nucleotide. Therefore, C and U residues can function efficiently as natural branch acceptors, but an A residue is preferred if available in the proper region. In addition, the data strongly reinforce the importance of the distance constraint for lariat formation. To explain selection of the branch acceptor nucleotide, potential base-pairing interactions of branch point sequences with the U2 RNA are discussed.
Mol Cell Biol 1988 May
PMID:Unusual branch point selection in processing of human growth hormone pre-mRNA. 296 99

The nucleotide sequence of the small-subunit rRNA (18S rRNA) coding gene in the higher fungus Protomyces inouyei contains two group I introns. This is the first report of two group I introns in the 18S rRNA coding region. Based on the comparison of the two introns of Protomyces inouyei with those of the green alga Ankistrodesmus stipitatus, and the other two higher fungi Pneumocystis carinii and Ustilago maydis, the Protomyces introns are group I introns containing the highly conserved sequence elements P, Q, R, and S. Intron A of Protomyces inouyei is located in the same position as in Pneumocystis carinii while intron B shares the location with that in Ustilago maydis. The phylogenetic relationships strongly support horizontal transfer of these group I introns.
J Mol Evol 1993 Jul
PMID:The higher fungus Protomyces inouyei has two group I introns in the 18S rRNA gene. 836 Sep 15

The rat GnRH gene consists of four short exons (denoted 1, 2, 3, and 4) and three introns (A, B, and C). All three introns are spliced from the primary transcript, resulting in a mature mRNA. Northern blot and RT-PCR analyses showed that the GnRH primary transcript and its splicing intermediates are more prevalent than the mature GnRH mRNA in a variety of non-GnRH-producing tissues. To delineate the possible splicing mechanism of introns, an in vitro HeLa splicing system was used. Introns B and C were efficiently spliced, while intron A spanning between exon 1 and exon 2 was not. The retention of intron A was relieved when the 5'- and/or 3'-splice sites of intron A were point mutated based on the consensus sequence. The splicing activity was even more strengthened when a putative branchpoint site was moved to the upstream region of the pyrimidine tract of intron A. Intron A could be partially spliced when whole exons (2, 3, and 4) were linked up with intron A. There are two putative exonic splicing enhancers (ESEs) in exon 3 and exon 4. The ESE on exon 4 (ESE4) is much stronger than that on exon 3. The closer the ESE4 to the 3'-splice site of intron A, the better the splicing activity became. However, in the presence of the nuclear extract from GnRH neurons, there was an enhancement in the splicing activity notwithstanding the distance between ESE4 and 3'-splice site of intron A. These results suggest that the ESE4 functions as both the constitutive and regulated enhancer. Collectively, our study provides evidence that enhanced splicing of intron A by putative GnRH neuron-specific splicing factor(s) interacting with the ESEs is a prerequisite for mature GnRH synthesis.
Mol Endocrinol 1999 Nov
PMID:Enhanced splicing of the first intron from the gonadotropin-releasing hormone (GnRH) primary transcript is a prerequisite for mature GnRH messenger RNA: presence of GnRH neuron-specific splicing factors. 1055 82

In an earlier study, we found that excision of the first intron (intron A) from the rat GnRH primary transcript is attenuated in non-GnRH-producing cells. This attenuation can be partially relieved by exonic splicing enhancers (ESEs) located in GnRH exons 3 and 4. In the present study, we confirmed that intron A of the mouse GnRH pre-mRNA was not excised in a HeLa nuclear extract (NE) in vitro or in COS-7 cells in vivo. Intron A could, however, be partially removed when exon 3 and/or 4 were linked to exon 2. In the presence of an ESE in exon 4 (ESE4), an addition of GT1 NE further increased the excision rate of intron A, whereas the addition of KK1 (a non-GnRH-producing cell) NE decreased it. To define the GnRH neuron-specific splicing activity, GT1 NE was fractionated by ultracentrifugation and ammonium sulfate precipitation. A 50-90% ammonium sulfate pellet (ASP50-90) fraction was further precipitated with 20 mM MgCl(2) to isolate a serine/arginine-rich (SR) protein fraction. Among the ASP fractions, ASP40-50 significantly increased the excision rate of intron A in the presence of HeLa NE or SR protein-rich fraction. However, the ASP40-50 fraction alone could not remove intron A. This result suggests the presence of a cofactor protein(s) in the ASP40-50 fraction that may mediate the interaction between a 3' spliceosome complex and the ESE4-SR protein complex. UV cross-linking and gel mobility shift analysis revealed that Tra2alpha but not other SR proteins tested, specifically binds to ESE4. Moreover, Tra2alpha stimulated intron A excision in a dose-dependent manner. These results imply that Tra2alpha and a cofactor protein in the ASP40-50 fraction are involved in mediating the GnRH neuron-specific excision of intron A from the GnRH primary transcript.
Mol Endocrinol 2002 Nov
PMID:Exonic splicing enhancer-dependent splicing of the gonadotropin-releasing hormone premessenger ribonucleic acid is mediated by tra2alpha, a 40-kilodalton serine/arginine-rich protein. 1240 32

Recombinant factor VIII is one of the most complex mammalian proteins and a biotechnology venture required for the treatment of hemophilia A. The complexity of the protein, post-translational modifications and limitations of expression elements make the production of active recombinant FVIII a challenge. Here we report the production of biologically active Factor VIII in two different cell lines, CHO and HepG2, by transient transfection. Two expression vectors based on the CMV promoter were used: one harboring CMV Intron A (InA) and the other without it. To bypass difficulties in secretion, we also studied the influence of co-expression of the human splice isoform of the XBP1 gene. We report the production of recombinant FVIII possessing bioengineered FVIII heavy and light chains, linked by a minimal B domain. In our study, HepG2, a human hepatocyte cell line, expressed Factor VIII ten-fold more than a CHO cell line, and in HepG2 cells, the expression of XBP1 improved Factor VIII activity. For CHO cells, expression was improved by the presence of InA, but no further improvement was noted with XBP1 co-expression. These data suggest that the minimal B domain rFVIII preserves Factor VIII biological activity and that different expression elements can be used to improve its production.
Mol Biotechnol 2008 Jun
PMID:Production of recombinant human factor VIII in different cell lines and the effect of human XBP1 co-expression. 1832 49