Exonic splicing enhancer-dependent splicing of the gonadotropin-releasing hormone premessenger ribonucleic acid is mediated by tra2alpha, a 40-kilodalton serine/arginine-rich protein.
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.
Seong
Jae Young
JY
Hormone Research Center, Chonnam National University, Kwangju 500-757, Korea.
Han
Jin
J
Park
Sungjin
S
Wuttke
Wolfgang
W
Jarry
Hubertus
H
Kim
Kyungjin
K
D000818:Animals; D001120:Arginine; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D005091:Exons; D007987:Gonadotropin-Releasing Hormone / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012694:Serine; D014158:Transcription, Genetic; D014162:Transfection
D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C474542:Tra2alpha protein, mouse; D007987:Gonadotropin-Releasing Hormone; D012694:Serine; D001120:Arginine