A comparative analysis of RNA targeting strategies in the thymosin beta 4 gene.
The thymosin beta 4 (Tbeta4) gene is of biological and pharmaceutical relevance because of its anti-inflammatory and wound-healing properties. As such, it is an example of a gene that may be targeted in immunotherapy regimens. Therefore, we have used the Tbeta4 gene to compare alternative strategies for RNA targeting, namely short hairpin (sh) RNAi versus external guide sequence (EGS)-mediated RNase P cleavage. Tbeta4 has two transcripts (UTbeta4 and LTbeta4) formed by alternative splicing that differ in both expression levels and the biological activity of their encoded products. Thus, we were able to compare the capacity of shRNAi/EGS mini-genes to target molecules of high and low abundance; to specifically target alternatively spliced mRNAs; and to discriminate between very closely related alleles encoding for identical proteins. Finally, we compared transient gene knockdown in tissue culture with results in stable systems in vitro and in vivo. The data demonstrate that shRNAi and EGS can both target the Tbeta4 gene, but that the extent of RNA reduction with shRNAi ( approximately 90%) is greater. RNAi targeting shows varying efficacy against two overlapping RNAs, is largely but not completely splice form-specific, and preferentially, but not exclusively, targets a perfect-sequence match. Very high targeting achieved with an shRNAi expressed from an RNA polymerase III promoter in transient transfection was not maintained in stably transfected clones and was not efficiently transmitted through the mouse germline. These results demonstrate the versatility and the limitations of RNA targeting strategies, and suggest that particular biological and clinical needs may be best met by varying the strategy.
Gibbons
Deena L
DL
Peter Gorer Department of Immunobiology, Guy's King's St Thomas' School of Medicine, Guy's Hospital, London SE1 9RT, UK.
Shashikant
Coodivaalli
C
Hayday
Adrian C
AC
D017398:Alternative Splicing; D001483:Base Sequence; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism*; D013947:Thymosin / Q000235:genetics* / Q000378:metabolism; D000099201:Thymosin beta(4)
D012333:RNA, Messenger; D013947:Thymosin; D000099201:Thymosin beta(4)