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Updated: Jun 29, 2026
06:48
A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells
Published on: June 16, 2022
Two cap-binding proteins CBP20 and CBP80 are involved in processing primary MicroRNAs
Sanghee Kim1, Jun-Yi Yang, Jun Xu
1Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065-6399, USA.
Plant & Cell Physiology
|October 3, 2008
Summary
Cap-binding proteins (CBPs) 20 and 80 are essential for plant microRNA (miRNA) biogenesis and ABA response. Mutants show reduced miRNA levels, impacting development and ABA sensitivity.
Area of Science:
- Plant molecular biology
- Gene regulation
- Biochemistry
Background:
- MicroRNAs (miRNAs) are crucial regulators of plant development, mediating gene expression post-transcriptionally.
- Mutations affecting miRNA biogenesis lead to pleiotropic developmental defects, including serrated leaves and abscisic acid (ABA) hypersensitivity.
- miRNA genes are transcribed by RNA polymerase II, producing capped primary miRNA (pri-miRNA) transcripts.
Purpose of the Study:
- To investigate the role of cap-binding proteins (CBP) 20 and 80 in miRNA biogenesis and processing in Arabidopsis.
- To determine if CBP20 and CBP80 interact with pri-miRNAs and influence their stability.
- To elucidate the involvement of CBP20 and CBP80 in ABA-mediated regulation of miRNA levels and downstream gene expression.
Main Methods:
- Analysis of Arabidopsis mutants (cbp20, cbp80) with altered miRNA and pri-miRNA levels.
- Co-immunoprecipitation assays to detect interactions between pri-miRNAs and CBP proteins.
- ABA treatment experiments to assess protein stability and miRNA induction.
- Quantitative analysis of MYB transcript levels in response to altered miRNA accumulation.
Main Results:
- cbp20 and cbp80 mutants exhibit reduced mature miRNA levels and elevated pri-miRNA levels.
- CBP20 and CBP80 physically associate with multiple pri-miRNAs (159, 166, 168, 172).
- CBP20 and CBP80 protein levels are stabilized by ABA via post-translational modification, crucial for miR159 induction during germination.
- Lack of miR159 in cbp20/80 mutants leads to increased MYB33/MYB101 transcripts, causing ABA hypersensitivity.
Conclusions:
- CBP20 and CBP80 are novel components involved in the processing and biogenesis of plant miRNAs.
- These proteins play a critical role in ABA signaling pathways by regulating specific miRNA accumulation.
- CBP20 and CBP80 function in a pathway overlapping with known miRNA biogenesis factors like SE and HYL1.
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01:22MicroRNAs
MicroRNA (miRNA) are short, regulatory RNA transcribed from introns—non-coding regions of a gene—or intergenic regions—stretches of DNA present between genes. Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA ends...
01:22MicroRNAs
MicroRNA (miRNA) are short, regulatory RNA transcribed from introns (non-coding regions of a gene) or intergenic regions (stretches of DNA present between genes). Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself, forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA...
01:22MicroRNAs
MicroRNA (miRNA) are short, regulatory RNA transcribed from introns—non-coding regions of a gene—or intergenic regions—stretches of DNA present between genes. Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA ends...
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In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA to distinguish it from mature mRNA.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it (7-Methyl guanosine). This 5’ cap helps the...
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it (7-Methyl guanosine). This 5’ cap helps the...
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In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA to distinguish it from mature mRNA.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a cap to the 5' end of the growing transcript. In this process, a 5' phosphate is replaced by modified guanosine that has a methyl group attached (7-methyl guanosine). This 5' cap helps the cell...
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a cap to the 5' end of the growing transcript. In this process, a 5' phosphate is replaced by modified guanosine that has a methyl group attached (7-methyl guanosine). This 5' cap helps the cell...