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RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA
Published on: April 10, 2018
Natural antisense RNA promotes 3' end processing and maturation of MALAT1 lncRNA
Xinying Zong1, Shinichi Nakagawa2, Susan M Freier3
1Department of Cell and Developmental Biology, University of Illinois Urbana, IL 61801, USA.
Abstract:
The RNase P-mediated endonucleolytic cleavage plays a crucial role in the 3' end processing and cellular accumulation of MALAT1, a nuclear-retained long noncoding RNA that promotes malignancy. The regulation of this cleavage event is largely undetermined. Here we characterize a broadly expressed natural antisense transcript at the MALAT1 locus, designated as TALAM1, that positively regulates MALAT1 levels by promoting the 3' end cleavage and maturation of MALAT1 RNA. TALAM1 RNA preferentially localizes at the site of transcription, and also interacts with MALAT1 RNA. Depletion of TALAM1 leads to defects in the 3' end cleavage reaction and compromises cellular accumulation of MALAT1. Conversely, overexpression of TALAM1 facilitates the cleavage reaction in trans Interestingly, TALAM1 is also positively regulated by MALAT1 at the level of both transcription and RNA stability. Together, our data demonstrate a novel feed-forward positive regulatory loop that is established to maintain the high cellular levels of MALAT1, and also unravel the existence of sense-antisense mediated regulatory mechanism for cellular lncRNAs that display RNase P-mediated 3' end processing.
Insights
A novel antisense transcript, TALAM1, promotes the processing and accumulation of MALAT1 long noncoding RNA. This discovery reveals a feed-forward regulatory loop crucial for maintaining MALAT1 levels in cells.
Area of Science:
- Molecular Biology
- RNA Biology
- Gene Regulation
Background:
- MALAT1 is a nuclear-retained long noncoding RNA critical for malignancy.
- RNase P-mediated endonucleolytic cleavage is essential for MALAT1 3' end processing and accumulation.
- Regulatory mechanisms governing MALAT1 processing remain largely unknown.
Purpose of the Study:
- To identify and characterize regulatory factors involved in MALAT1 3' end processing.
- To elucidate the role of natural antisense transcripts in regulating MALAT1 levels.
- To investigate the interplay between MALAT1 and its antisense transcript.
Main Methods:
- Characterization of a novel antisense transcript (TALAM1) at the MALAT1 locus.
- RNA localization studies (preferential localization at the transcription site).
- RNA-protein interaction assays.
- Depletion and overexpression studies of TALAM1.
- Analysis of MALAT1 transcription and RNA stability.
Main Results:
- TALAM1 positively regulates MALAT1 levels by promoting its 3' end cleavage and maturation.
- TALAM1 interacts with MALAT1 RNA and localizes to the site of transcription.
- TALAM1 depletion impairs MALAT1 processing and cellular accumulation.
- MALAT1 positively regulates TALAM1 transcription and RNA stability, forming a feed-forward loop.
Conclusions:
- A novel feed-forward positive regulatory loop between MALAT1 and its antisense transcript TALAM1 maintains high MALAT1 cellular levels.
- This study reveals a sense-antisense mediated regulatory mechanism for lncRNAs processed by RNase P.
- TALAM1 is a key regulator of MALAT1 processing and accumulation.
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