Analysis of inhibitory action of modified U1 snRNAs on target gene expression: discrimination of two RNA targets differing by a 1 bp mismatch.
The modified U1 snRNA gene can suppress expression of a target transgene. In the present study, its potential utility to inhibit a dominant negative/gain of function mutation is explored. Using a green fluorescent protein (GFP) target gene, inhibition was achieved in all cells transduced with U1antiGFP directed at multiple sites within GFP. Using a chloramphenicol acetyltransferase (CAT) target gene, inhibition was not increased by increasing the hybridization domain from 10 to 16 bp or when a site in an upstream exon or intron was targeted. To determine if a U1 anti-target design could discriminate between two transcripts that differ by a 1-2 bp mismatch, GFPtpz and GFPsaph were chosen as targets because they share sequence homology except for three regions where a 1, 2 or 3 bp mismatch exists. The results demonstrated that U1antiGFP correctly reduced its cognate GFP expression by >90% and therefore U1 anti-target constructs are able to discriminate a 1 or 2 bp mismatch in their target mRNA. Thus, these U1 anti-target constructs may be effective in a strategy of somatic gene therapy for a dominant negative/gain of function mutation due to the discreteness of its discrimination. It may complement other anti-target strategies to reduce the cellular load of a mutant transcript.
Liu
Peng
P
Department of Genetics and Developmental Biology, Mail Code 3301, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA.
Gucwa
Amy
A
Stover
Mary Louise
ML
Buck
Emily
E
Lichtler
Alexander
A
Rowe
David
D
D016475:3T3 Cells; D000818:Animals; D020137:Base Pair Mismatch / Q000235:genetics*; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D005091:Exons / Q000235:genetics; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D007438:Introns / Q000235:genetics; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D011061:Poly A / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection; D019076:Transgenes / Q000235:genetics
D012333:RNA, Messenger; D012342:RNA, Small Nuclear; C405483:U1 small nuclear RNA; D011061:Poly A; D015500:Chloramphenicol O-Acetyltransferase