Identification of antisense RNA stem-loops that inhibit RNA-protein interactions using a bacterial reporter system.
A bacterial reporter system for the identification of antisense RNA stem-loops that inhibit RNA-protein interactions was constructed. RNA stem-loops were chosen since they have been shown to act as triggers in a variety of natural antisense systems. An RNA stem-loop library was screened for sequences that bind to the U1 hpII and inhibit U1A protein binding using a bacterial system based on phage lambda N mediated antitermination. As a result, antisense RNA stem-loops that appeared to be binding to the boxA region, which is important for the formation of functional antitermination complex, and not to the hpII loop were identified. In vivo and in vitro mutagenic analysis showed that the inhibitor RNA stem-loops were indeed binding to the boxA region through Watson-Crick base pairing and disrupting binding to host factors NusG, NusB and S10. The results show that the bacterial reporter system may be of general use for the identification of antisense RNA stem-loops that disrupt RNA-protein interactions.
Yano
Akiko
A
Department of Life Sciences, Tokyo Gakugei University, 4-1-1 Nukuikita-machi, Koganei, Tokyo 184-8501, Japan.
Kunimoto
Mayumi
M
Minami
Takako
T
Harada
Kazuo
K
D001419:Bacteria / Q000235:genetics; D001665:Binding Sites; D017930:Genes, Reporter; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D016372:RNA, Antisense / Q000737:chemistry*; D012342:RNA, Small Nuclear / Q000737:chemistry; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000037:antagonists & inhibitors; D013728:Terminator Regions, Genetic; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics
C059417:N protein, Bacteriophage lambda; D016372:RNA, Antisense; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C405483:U1 small nuclear RNA; C075339:U1A protein; D054334:Viral Regulatory and Accessory Proteins; D001616:beta-Galactosidase