A versatile ribosomal protein promoter-based reporter system for selective assessment of RNA stability and post-transcriptional control.
Assessment of post-transcriptional control relies on use of transcriptional inhibitors and is masked by copious and cryptic transcriptional induction. We screened several cellular promoters that are constitutively active yet noninducible to external stimuli. The ribosomal protein RPS30 promoter was chosen; its TATA signal and sp1 site location were optimized. The modified promoter (RPS30M) is selective to post-transcriptional effects of AU-rich elements (ARE) in the 3'UTR, while it is not transcriptionally responsive to a wide variety of agents including pro-inflammatory cytokines and RNA-binding proteins. Specific cis-acting elements can be appended to RPS30M by a cloning-free approach to allow coupled transcriptional/post-transcriptional assessment, as demonstrated with NF-kappaB and beta-catenin/wnt signaling experiments. Moreover, efficient tetracycline-regulated RPS30M was created for quantitative assessment of the half-lives of mRNAs containing AREs. The described approach provides enhanced versatility and suitability for selective post-transcriptional assessment with or without transcriptional induction.
Hitti
Edward
E
Program in BioMolecular Research, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia 11211.
Al-Yahya
Suhad
S
Al-Saif
Maher
M
Mohideen
Peer
P
Mahmoud
Linah
L
Polyak
Stephen J
SJ
Khabar
Khalid S A
KS
D020413:3' Untranslated Regions; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D006801:Humans; D016328:NF-kappa B / Q000235:genetics; D011401:Promoter Regions, Genetic; D012323:RNA Processing, Post-Transcriptional; D020871:RNA Stability / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D012269:Ribosomal Proteins / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D014158:Transcription, Genetic
D020413:3' Untranslated Regions; D016328:NF-kappa B; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C076845:FAU protein, human