Promoter-associated RNA is required for RNA-directed transcriptional gene silencing in human cells - PubMed (original) (raw)

Detection and characterization of a EF1a promoter-associated RNA variant. (A) 5′ biotin-linked antisense RNAs targeted to the EF1a promoter elute with a promoter-associated RNA. Cultures were treated with either EF52 siRNA containing a 5′ biotin-linked sense annealed to the antisense (Control) or 5′ biotin-linked antisense RNA alone (EF52) (100 nM Lipofectamine 2000). Samples from the preelution step (Input) are shown along with the post-biotin/avidin elution (Elution). The elutes were collected 24 h after transfection, DNase treated, subject to RT with either a random hexamer primer (dTT) (BioRad; iScript), or EF1a-specific primer (EF1a) that spans the EF1a promoter/exon 1 junction (

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, no. 804) and PCR amplified with EF1a-specific primers (

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, 803/804). (B) The 5′ biotin tagged antisense RNA eluted EF1a promoter-associated RNA is sensitive to RNase A and resistant to RNase H treatment. Sample lanes represent elutes from cultures treated with (1) EF52 sense RNA + 5′ biotin linker, (2) EF52 antisense RNA + 5′ biotin linker, and (3) EF52 sense RNA plus 5′ biotin linker annealed to the EF52 antisense plus 5′ biotin linker. The resultant elutes underwent RT (BioRad; iScript) followed by PCR (

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, EF1a-specific primers 803/804) after the various RNase treatments. (C) Characterization of the EF1a promoter-associated RNA as determined by bidirectional RT-PCR. RT was performed on DNase treated 293T cell RNA with various primers either sense (1) or antisense (2) (

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, 803 or 805, respectively) followed by PCR with both primers. (D) RT with primer 2 followed by PCR with primers 1 and 2 generates a ≈156-bp band corresponding to the EF1a promoter whereas RT with primer 1 followed by PCR with primers 1 and 2 does not. The results from three independent cultures are shown. (E) 5′ and 3′ RACE analysis of the EF1a promoter-associated RNA was performed on 293T cell RNA with EF1a primers specific for the promoter-associated RNA. The 5′ RACE analysis was performed with primer 5 (

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, EFpRNARev), whereas the 3′ RACE analysis was performed with the 3′RACE F primer (

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). The resulting RACE products were cloned, sequenced, and shown to be spliced and processed accordingly and to contain a 5′ UTR which overlaps ≈230 bp of the EF1a promoter.