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Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events
Published on: May 13, 2019
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Primary microRNA processing is functionally coupled to RNAP II transcription in vitro
Shanye Yin1, Yong Yu1, Robin Reed1
1Department of Cell Biology, Harvard Medical School, 240 Longwood Ave. Boston MA 02115.
Scientific Reports
|July 8, 2015
Summary
Transcription and microRNA processing are coupled, occurring simultaneously during gene expression. This study establishes a new in vivo model to demonstrate this functional coupling and co-transcriptional processing of pri-miRNA.
Area of Science:
- Molecular Biology
- Gene Expression
- RNA Processing
Background:
- Previous in vivo studies suggested a link between primary microRNA (pri-miRNA) processing and transcription by RNA polymerase II (RNAP II).
- Co-transcriptional processing of pri-miRNA has been hypothesized but lacked robust experimental models.
Purpose of the Study:
- To establish a reliable in vivo system for studying the functional coupling of transcription and pri-miRNA processing.
- To demonstrate co-transcriptional processing of pri-miRNA in a cell-free nuclear extract system.
- To investigate the impact of transcription on the kinetics and efficiency of pri-miRNA maturation.
Main Methods:
- Development of a robust in vivo system using HeLa cell nuclear extracts.
- Transcription of pri-miRNA by RNAP II within the established system.
- Analysis of pri-miRNA processing into pre-miRNA in the presence of transcription.
Main Results:
- Pri-miRNA transcription by RNAP II and subsequent processing to pre-miRNA were successfully achieved in HeLa cell nuclear extracts.
- The processing of pri-miRNA was significantly enhanced in kinetics and efficiency compared to naked pri-miRNA.
- This enhancement effect was observed across multiple different pri-miRNAs, indicating generality.
- Nascent pri-miRNA was efficiently processed while still associated with the DNA template.
Conclusions:
- Directly demonstrates the functional coupling between transcription and pri-miRNA processing.
- Establishes the first in vivo model systems for studying transcription-pri-miRNA processing coupling.
- Provides strong evidence for co-transcriptional processing of pri-miRNA as a biologically relevant mechanism.
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