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Published on: August 21, 2019
Mammalian 5'-capped microRNA precursors that generate a single microRNA
Mingyi Xie1, Mingfeng Li2, Anna Vilborg1
1Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Boyer Center for Molecular Medicine, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT 06536, USA.
Abstract:
MicroRNAs (miRNAs) are short RNA gene regulators typically produced from primary transcripts that are cleaved by the nuclear microprocessor complex, with the resulting precursor miRNA hairpins exported by exportin 5 and processed by cytoplasmic Dicer to yield two (5p and 3p) miRNAs. Here, we document microprocessor-independent 7-methylguanosine (m(7)G)-capped pre-miRNAs, whose 5' ends coincide with transcription start sites and 3' ends are most likely generated by transcription termination. By establishing a small RNA Cap-seq method that employs the cap-binding protein eIF4E, we identified a group of murine m(7)G-capped pre-miRNAs genome wide. The m(7)G-capped pre-miRNAs are exported via the PHAX-exportin 1 pathway. After Dicer cleavage, only the 3p-miRNA is efficiently loaded onto Argonaute to form a functional microRNP. This unusual miRNA biogenesis pathway, which differs in pre-miRNA synthesis, nuclear-cytoplasmic transport, and guide strand selection, enables the development of shRNA expression constructs that produce a single 3p-siRNA.
Insights
Researchers discovered a new pathway for microRNA (miRNA) biogenesis. This pathway bypasses typical processing, yielding specific 3p-siRNAs for gene silencing applications.
Area of Science:
- Molecular Biology
- Genetics
- RNA Biology
Background:
- MicroRNAs (miRNAs) are key gene regulators processed through a canonical pathway involving the microprocessor complex and Dicer.
- This pathway generates mature 5p and 3p miRNA duplexes.
Purpose of the Study:
- To identify and characterize an alternative miRNA biogenesis pathway.
- To investigate the mechanism of microprocessor-independent pre-miRNA formation and processing.
- To explore the potential of this pathway for novel gene silencing tools.
Main Methods:
- Development of a small RNA Cap-seq method utilizing the cap-binding protein eIF4E.
- Genome-wide identification of 7-methylguanosine (m(7)G)-capped pre-miRNAs in murine cells.
- Analysis of pre-miRNA nuclear-cytoplasmic export and Dicer processing.
Main Results:
- Discovery of microprocessor-independent, m(7)G-capped pre-miRNAs originating from transcription start and termination sites.
- Identification of PHAX-exportin 1 as the export pathway for these capped pre-miRNAs.
- Demonstration that only the 3p-miRNA is efficiently loaded into Argonaute after Dicer cleavage, forming a functional microRNP.
Conclusions:
- A novel miRNA biogenesis pathway exists, distinct in pre-miRNA synthesis, transport, and guide strand selection.
- This pathway allows for the generation of single 3p-siRNAs, distinct from canonical miRNAs.
- This finding enables the design of shRNA expression constructs for targeted gene silencing via single 3p-siRNA production.
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