title: The germ cell nuclear proteins hnRNP G-T and RBMY activate a testis-specific exon.
abstract: The human testis has almost as high a frequency of alternative splicing events as brain. While not as extensively studied as brain, a few candidate testis-specific splicing regulator proteins have been identified, including the nuclear RNA binding proteins RBMY and hnRNP G-T, which are germ cell-specific versions of the somatically expressed hnRNP G protein and are highly conserved in mammals. The splicing activator protein Tra2beta is also highly expressed in the testis and physically interacts with these hnRNP G family proteins. In this study, we identified a novel testis-specific cassette exon TLE4-T within intron 6 of the human transducing-like enhancer of split 4 (TLE4) gene which makes a more transcriptionally repressive TLE4 protein isoform. TLE4-T splicing is normally repressed in somatic cells because of a weak 5' splice site and surrounding splicing-repressive intronic regions. TLE4-T RNA pulls down Tra2beta and hnRNP G proteins which activate its inclusion. The germ cell-specific RBMY and hnRNP G-T proteins were more efficient in stimulating TLE4-T incorporation than somatically expressed hnRNP G protein. Tra2b bound moderately to TLE4-T RNA, but more strongly to upstream sites to potently activate an alternative 3' splice site normally weakly selected in the testis. Co-expression of Tra2beta with either hnRNP G-T or RBMY re-established the normal testis physiological splicing pattern of this exon. Although they can directly bind pre-mRNA sequences around the TLE4-T exon, RBMY and hnRNP G-T function as efficient germ cell-specific splicing co-activators of TLE4-T. Our study indicates a delicate balance between the activity of positive and negative splicing regulators combinatorially controls physiological splicing inclusion of exon TLE4-T and leads to modulation of signalling pathways in the testis. In addition, we identified a high-affinity binding site for hnRNP G-T protein, showing it is also a sequence-specific RNA binding protein.
authors.affiliation: Institute of Human Genetics, Newcastle University, Newcastle upon Tyne, United Kingdom.
authors.forename: Yilei
authors.initials: Y
authors.lastname: Liu
authors.forename: Cyril F
authors.initials: CF
authors.lastname: Bourgeois
authors.forename: Shaochen
authors.initials: S
authors.lastname: Pang
authors.forename: Marek
authors.initials: M
authors.lastname: Kudla
authors.forename: Natacha
authors.initials: N
authors.lastname: Dreumont
authors.forename: Liliane
authors.initials: L
authors.lastname: Kister
authors.forename: Yong-Hua
authors.initials: YH
authors.lastname: Sun
authors.forename: James
authors.initials: J
authors.lastname: Stevenin
authors.forename: David J
authors.initials: DJ
authors.lastname: Elliott
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005091:Exons; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000378:metabolism*; D006801:Humans; D007438:Introns; D008297:Male; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013094:Spermatozoa / Q000378:metabolism*; D013737:Testis / Q000378:metabolism*; D015027:Zebrafish / Q000196:embryology / Q000235:genetics / Q000378:metabolism
chemical_list: D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009687:Nuclear Proteins; D020033:Protein Isoforms; C415905:RBMXL2 protein, human; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; C465817:TLE4 protein, human