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The response of A549 transcriptome to RNA Pol II slowdown with alpha-amanitin and DRB

ABSTRACT: The response of A549 transcriptome to RNA Pol II slowdown with alpha-amanitin and DRB

PROVIDER: PRJNA642131 | ENA |

REPOSITORIES: ENA

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The response of A549 transcriptome to RNA Pol II slowdown with alpha-amanitin and DRB

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HEK293T cells were transfected with the Rbp1-amr or slow (R729H-amr) α-amanitin resistant subunit of RNA Pol II and selected with α-amanitin 24 hours after transfection for additional 24 hours

Project description:HEK293T cells were transfected with the Rbp1-amr or slow (R729H-amr) α-amanitin resistant subunit of RNA Pol II and selected with α-amanitin 24 hours after transfection for additional 24 hours. Total RNA was extracted and global changes in gene expression were determined using microarray chips. MiRNAs are transcribed by RNA pol II but the transcriptional features influencing their synthesis are poorly defined. Here we report that a TATA-box in miRNA and a subset of protein-coding genes is associated with increased sensitivity to a slow rate of transcription elongation. We also show that promoters driven by TATA-box or NF-κB elicit high transcription re-initiation rate, but paradoxically lower levels of miRNA. Interestingly, miRNA synthesis was converted to a more productive mode by decreasing initiation rate, but less productive when the re-initiation rate increased. This phenomenon was found to be associated with a delay in miR-146a induction by NF-κB. We also demonstrate that miRNAs are remarkably strong pause sites. Our findings suggest that lower efficiency of miRNA synthesis directed by the TATA-box or NF-κB is a consequence of frequent transcription initiation that lead to Pol II crowding at pause sites, thereby increasing the chance of collision and premature termination. These findings highlight the importance of the transcription initiation mechanism for miRNA synthesis, and have implications for TATA-box promoters in general.

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Project description:Transcription and pre-mRNA alternative splicing was analyzed by in isogenic human HEK293 cell lines that inducibly express a-amanitin resistant mutants of the RNA polymerase II large subunit with slow and fast elongation rates. Alternative splicing was analyzed by, RNA-seq and RASL-seq of polyA+ RNA from a-amanitin treated cells; transcription elongation rate was analyzed by BrUTP-labelled GRO-seq of a-amanitin treated cells at time points after release from a DRB (5,6-dichloro-1-bold beta-D-ribofuranosylbenzimidazole) block.

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