Polyadenylation site-specific differences in the activity of the neuronal βCstF-64 protein in PC-12 cells.
Recent genome-wide analyses have implicated alternative polyadenylation - the process of regulated mRNA 3' end formation - as a critical mechanism that influences multiple steps of mRNA metabolism in addition to increasing the protein-coding capacity of the genome. Although the functional consequences of alternative polyadenylation are well known, protein factors that regulate this process are poorly characterized. Previously, we described an evolutionarily conserved family of neuronal splice variants of the CstF-64 mRNA, βCstF-64, that we hypothesized to function in alternative polyadenylation in the nervous system. In the present study, we show that βCstF-64 mRNA and protein expression increase in response to nerve growth factor (NGF), concomitant with differentiation of adrenal PC-12 cells into a neuronal phenotype, suggesting a role for βCstF-64 in neuronal gene expression. Using PC-12 cells as model, we show that βCstF-64 is a bona fide polyadenylation protein, as evidenced by its association with the CstF complex, and by its ability to stimulate polyadenylation of luciferase reporter mRNA. Using luciferase assays, we show that βCstF-64 stimulates polyadenylation equivalently at the two weak poly(A) sites of the β-adducin mRNA. Notably, we demonstrate that the activity of βCstF-64 is less than CstF-64 on a strong polyadenylation signal, suggesting polyadenylation site-specific differences in the activity of the βCstF-64 protein. Our data address the polyadenylation functions of βCstF-64 for the first time, and provide initial insights into the mechanism of alternative poly(A) site selection in the nervous system.
Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430-6540, USA.
Ganesh S
GS
Shankarling
Clinton C
CC
MacDonald
D000818:Animals; D001921:Brain / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D039241:Cleavage Stimulation Factor / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D020932:Nerve Growth Factor / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D016716:PC12 Cells; D026723:Polyadenylation; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D014158:Transcription, Genetic
D039241:Cleavage Stimulation Factor; C492659:Cstf1 protein, rat; D020033:Protein Isoforms; D012333:RNA, Messenger; D020932:Nerve Growth Factor
3′ RACE; 3′ rapid amplification of cDNA ends; Add2; Alternative polyadenylation; Alternative splicing; CSTF2; CSTF2T; CstF; CstF-64; CstF-77; Cstf2; DMEM; DSE; Dulbecco's Minimal Eagle Media; N-CAM; NF1; NGF; NGSP; Neuronal gene expression; RLU; RT-PCR; SLAP; brain-specific splice variant of CstF-64; cleavage stimulation factor; cleavage stimulation factor, 64,000 M(r) subunit; cleavage stimulation factor, 77,000 M(r) subunit; downstream sequence element; human gene encoding CstF-64; human gene encoding τCstF-64; mouse gene encoding β-adducin; mouse or rat gene encoding CstF-64; nerve growth factor; nested gene-specific primer; neural cell adhesion molecule; neurofibromatosis type 1; relative luciferase unit; reverse transcriptase-mediated polymerase chain reaction; stem-loop luciferase assay for polyadenylation; β-adducin; βCstF-64