HnRNP L-mediated regulation of mammalian alternative splicing by interference with splice site recognition.
Heterogeneous nuclear ribonucleoprotein (hnRNP) L can regulate alternative mRNA splicing in diverse ways, binding to exonic or intronic sites and acting as either an activator or repressor. To investigate the mechanistic basis of hnRNP L-regulated alternative splicing, we focus here on two specific cases of hnRNP L-dependent splice site recognition. First, in the case of TJP1 our microarray data had suggested that exon 20 inclusion is regulated by hnRNP L as a repressor. Here we demonstrate by mutational analysis that exon skipping is mediated by a short silencer sequence consisting of three hnRNP L high-score binding motifs located upstream of the 3' splice site of the regulated exon. UV crosslinking and immunoprecipitation experiments showed that hnRNP L binding interferes with 3' splice site recognition by U2AF65. Second, SLC2A2 contains a CA-repeat sequence close to the 5' splice site of the regulated exon 4. Using psoralen crosslinking, we demonstrate that hnRNP L represses splicing by preventing 5' splice site recognition of the U1 snRNP. In sum, our data provide new insights into the mechanisms of how hnRNP L-bound to intronic sites-regulates exon recognition.
Heiner
Monika
M
Institute of Biochemistry, Justus Liebig University of Giessen, Giessen, Germany.
Hui
Jingyi
J
Schreiner
Silke
S
Hung
Lee-Hsueh
LH
Bindereif
Albrecht
A
D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D005091:Exons / Q000235:genetics; D051273:Glucose Transporter Type 2 / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D034664:Heterogeneous-Nuclear Ribonucleoprotein L / Q000378:metabolism*; D006801:Humans; D007438:Introns / Q000235:genetics; D008322:Mammals / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012322:RNA Precursors / Q000378:metabolism; D022821:RNA Splice Sites / Q000235:genetics*; D012261:Ribonucleoproteins / Q000378:metabolism; D037941:Silencer Elements, Transcriptional / Q000235:genetics; D000072668:Splicing Factor U2AF; D062826:Zonula Occludens-1 Protein
D051273:Glucose Transporter Type 2; D034664:Heterogeneous-Nuclear Ribonucleoprotein L; D008565:Membrane Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012322:RNA Precursors; D022821:RNA Splice Sites; D012261:Ribonucleoproteins; C495680:SLC2A2 protein, human; D000072668:Splicing Factor U2AF; C569042:TJP1 protein, human; C000607494:U2AF2 protein, human; D062826:Zonula Occludens-1 Protein