ARCHAEA-ExPRESs targeting of alpha-tubulin 4 mRNA: a model for high-specificity trans-splicing.
Effectiveness of trans-splicing-mediated mRNA reprogramming depends on specificity and efficiency. We have previously developed a new strategy (ARCHAEA-ExPRESs) that uses a tRNA endonuclease derived from Archaea and its natural substrate, the bulge-helix-bulge (BHB) structure. ARCHAEA-ExPRESs provides increased specificity in functional targeting. In fact, this system is based on a double check, the base pairing and the formation of a BHB structure between the target mRNA and the targeting RNA. In this study, we demonstrate the high specificity of ARCHAEA-ExPRESs by tagging the endogenous alpha-tubulin 4 via trans-splicing. Alpha-tubulin 4 belongs to a gene family sharing high degree of nucleotide sequence homology. The formation of a perfect BHB structure between targeting RNAs and the isotype alpha-tubulin 4 enables selective trans-splicing. Most important, ARCHAEA-ExPRESs functionality is conserved in vivo following transient expression of archaeal tRNA endonuclease in mouse liver. Production of the recombinant protein is strictly dependent on the expression of the archaeal endonuclease, and the efficiency of the system depends on the relative amount of the target and targeting mRNAs. These data prove the effectiveness of ARCHAEA-ExPRESs in an endogenous highly demanding context and disclose the possibility to utilize this system in a variety of technological or therapeutic applications.
Deidda
Giancarlo
G
Istituto di Biologia Cellulare-CNR, Campus A. Buzzati-Traverso, Via E. Ramarini 32, I-00015 Monterondo Scalo, Rome, Italy. gdeidda@ibc.cnr.it
Rossi
Nicoletta
N
Putti
Sabrina
S
Tocchini-Valentini
Glauco P
GP
D000818:Animals; D001105:Archaea / Q000201:enzymology; D004720:Endonucleases; D005821:Genetic Techniques; D008099:Liver / Q000378:metabolism; D008722:Methods; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics*; D020040:Trans-Splicing; D014404:Tubulin / Q000235:genetics*
D012333:RNA, Messenger; D014404:Tubulin; C551900:alpha-tubulin 4, mouse; D004720:Endonucleases