Decrease in hnRNP A/B expression during erythropoiesis mediates a pre-mRNA splicing switch.
A physiologically important alternative pre-mRNA splicing switch, involving activation of protein 4.1R exon 16 (E16) splicing, is required for the establishment of proper mechanical integrity of the erythrocyte membrane during erythropoiesis. Here we identify a conserved exonic splicing silencer element (CE(16)) in E16 that interacts with hnRNP A/B proteins and plays a role in repression of E16 splicing during early erythropoiesis. Experiments with model pre-mRNAs showed that CE(16) can repress splicing of upstream introns, and that mutagenesis or replacement of CE(16) can relieve this inhibition. An affinity selection assay with biotinylated CE(16) RNA demonstrated specific binding of hnRNP A/B proteins. Depletion of hnRNP A/B proteins from nuclear extract significantly increased E16 inclusion, while repletion with recombinant hnRNP A/B restored E16 silencing. Most importantly, differentiating mouse erythroblasts exhibited a stage-specific activation of the E16 splicing switch in concert with a dramatic and specific down-regulation of hnRNP A/B protein expression. These findings demonstrate that natural developmental changes in hnRNP A/B proteins can effect physiologically important switches in pre-mRNA splicing.
Hou
Victor C
VC
Life Sciences Division, Lawrence Berkeley National Laboratory, University of California at Berkeley, Berkeley, CA 94720, USA.
Lersch
Robert
R
Gee
Sherry L
SL
Ponthier
Julie L
JL
Lo
Annie J
AJ
Wu
Michael
M
Turck
Chris W
CW
Koury
Mark
M
Krainer
Adrian R
AR
Mayeda
Akila
A
Conboy
John G
JG
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D016384:Consensus Sequence; D003598:Cytoskeletal Proteins; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000378:metabolism*; D004920:Erythropoiesis / Q000235:genetics*; D005091:Exons / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D020868:Gene Silencing; D006367:HeLa Cells; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007438:Introns / Q000235:genetics; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009479:Neuropeptides; D011485:Protein Binding; D011506:Proteins / Q000235:genetics*; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D014162:Transfection; D014714:Vertebrates / Q000235:genetics; D014982:Xenopus laevis
D003598:Cytoskeletal Proteins; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D008565:Membrane Proteins; D009479:Neuropeptides; D011506:Proteins; D012322:RNA Precursors; D016601:RNA-Binding Proteins; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1