Newly established in vitro system with fluorescent proteins shows that abnormal expression of downstream prion protein-like protein in mice is probably due to functional disconnection between splicing and 3' formation of prion protein pre-mRNA.
We and others previously showed that, in some lines of prion protein (PrP)-knockout mice, the downstream PrP-like protein (PrPLP/Dpl) was abnormally expressed in brains partly due to impaired cleavage/polyadenylation of the residual PrP promoter-driven pre-mRNA despite the presence of a poly(A) signal. In this study, we newly established an in vitro transient transfection system in which abnormal expression of PrPLP/Dpl can be visualized by expression of the green fluorescence protein, EGFP, in cultured cells. No EGFP was detected in cells transfected by a vector carrying a PrP genomic fragment including the region targeted in the knockout mice intact upstream of the PrPLP/Dpl gene. In contrast, deletion of the targeted region from the vector caused expression of EGFP. By employing this system with other vectors carrying various deletions or point mutations in the targeted region, we identified that disruption of the splicing elements in the PrP terminal intron caused the expression of EGFP. Recent lines of evidence indicate that terminal intron splicing and cleavage/polyadenylation of pre-mRNA are functionally linked to each other. Taken together, our newly established system shows that the abnormal expression of PrPLP/Dpl in PrP-knockout mice caused by the impaired cleavage/polyadenylation of the PrP promoter-driven pre-mRNA is due to the functional dissociation between the pre-mRNA machineries, in particular those of cleavage/polyadenylation and splicing. Our newly established in vitro system, in which the functional dissociation between the pre-mRNA machineries can be visualized by EGFP green fluorescence, may be useful for studies of the functional connection of pre-mRNA machineries.
Yoshikawa
Daisuke
D
Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto 1-12-4, Nagasaki 852-8523, Japan.
Kopacek
Juraj
J
Yamaguchi
Naohiro
N
Ishibashi
Daisuke
D
Yamanaka
Hitoki
H
Yamaguchi
Yoshitaka
Y
Katamine
Shigeru
S
Sakaguchi
Suehiro
S
D020413:3' Untranslated Regions / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D045744:Cell Line, Tumor; D058851:GPI-Linked Proteins; D005786:Gene Expression Regulation / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics*; D051379:Mice; D018345:Mice, Knockout; D026723:Polyadenylation / Q000235:genetics; D011328:Prions / Q000096:biosynthesis / Q000235:genetics*; D012322:RNA Precursors / Q000096:biosynthesis* / Q000378:metabolism*; D012326:RNA Splicing / Q000502:physiology*
D020413:3' Untranslated Regions; D058851:GPI-Linked Proteins; D011328:Prions; C122036:Prnd protein, mouse; D012322:RNA Precursors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins