Binding of hnRNP H and U2AF65 to respective G-codes and a poly-uridine tract collaborate in the N50-5'ss selection of the REST N exon in H69 cells.
The splicing of the N exon in the pre-mRNA coding for the RE1-silencing transcription factor (REST) results in a truncated protein that modifies the expression pattern of some of its target genes. A weak 3'ss, three alternative 5'ss (N4-, N50-, and N62-5'ss) and a variety of putative target sites for splicing regulatory proteins are found around the N exon; two GGGG codes (G2-G3) and a poly-Uridine tract (N-PU) are found in front of the N50-5'ss. In this work we analyzed some of the regulatory factors and elements involved in the preferred selection of the N50-5'ss (N50 activation) in the small cell lung cancer cell line H69. Wild type and mutant N exon/β-globin minigenes recapitulated N50 exon splicing in H69 cells, and showed that the N-PU and the G2-G3 elements are required for N50 exon splicing. Biochemical and knockdown experiments identified these elements as U2AF65 and hnRNP H targets, respectively, and that they are also required for N50 exon activation. Compared to normal MRC5 cells, and in keeping with N50 exon activation, U2AF65, hnRNP H and other splicing factors were highly expressed in H69 cells. CLIP experiments revealed that hnRNP H RNA-binding occurs first and is a prerequisite for U2AF65 RNA binding, and EMSA and CLIP experiments suggest that U2AF65-RNA recognition displaces hnRNP H and helps to recruit other splicing factors (at least U1 70K) to the N50-5'ss. Our results evidenced novel hnRNP H and U2AF65 functions: respectively, U2AF65-recruiting to a 5'ss in humans and the hnRNP H-displacing function from two juxtaposed GGGG codes.
Departamento de Bioquímica, Centro de Investigación y de Estudios Avanzados del I.P.N., México DF, México.
Carlos
C
Ortuño-Pineda
José Manuel
JM
Galindo-Rosales
José Victor
JV
Calderón-Salinas
Nicolás
N
Villegas-Sepúlveda
Odila
O
Saucedo-Cárdenas
Mónica
M
De Nova-Ocampo
Jesús
J
Valdés
D017398:Alternative Splicing; D001483:Base Sequence; D002460:Cell Line; D005091:Exons; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011072:Poly U / Q000235:genetics*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D022821:RNA Splice Sites; D034741:RNA, Small Interfering / Q000235:genetics; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000072668:Splicing Factor U2AF; D000099229:RE1-Silencing Transcription Factor
D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H; D009687:Nuclear Proteins; D011072:Poly U; D020033:Protein Isoforms; D012322:RNA Precursors; D022821:RNA Splice Sites; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; D000099229:RE1-Silencing Transcription Factor; C000607494:U2AF2 protein, human