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MicroRNAs01:22

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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...
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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...
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Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
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The pentose sugar in DNA is deoxyribose, while in RNA the pentose sugar is ribose. The difference between the sugars is the presence of the hydroxyl group on the ribose's second carbon and a hydrogen on the deoxyribose's second carbon. The phosphate residue attaches to the hydroxyl group of the 5′ carbon of one sugar and the hydroxyl group of the 3′ carbon of the sugar of the next nucleotide, which forms  a 5′ to 3′ phosphodiester linkage.
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The Upf proteins that carry out nonsense-mediated decay (NMD) are found in all eukaryotic organisms, including humans. Each protein has an individual role, but they need to work in collaboration. Upf1 is an ATP-dependent RNA helicase that unwinds the RNA helix. Because Upf1 can unwind any RNA, Upf2 and Upf3 are required to help Upf1 discriminate between nonsense and normal mRNAs.
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A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells
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Mismatched and wobble base pairs govern primary microRNA processing by human Microprocessor.

Shaohua Li1, Trung Duc Nguyen1, Thuy Linh Nguyen1

  • 1Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, China.

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Microprocessor enzyme activity is key for microRNA (miRNA) production. Mismatches and wobble base pairs in pri-miRNAs affect processing efficiency, explaining alternative cleavage and differential miRNA expression due to RNA editing or SNPs.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • MicroRNAs (miRNAs) are crucial regulators of gene expression.
  • The Microprocessor complex is essential for processing primary miRNAs (pri-miRNAs) into mature miRNAs.
  • Understanding Microprocessor's catalytic mechanism is vital for comprehending miRNA biogenesis and function.

Purpose of the Study:

  • To investigate the catalytic mechanism of Microprocessor in cleaving pri-miRNAs.
  • To identify RNA structural elements within pri-miRNAs that influence Microprocessor processing.
  • To explore how RNA modifications and genetic variations affect miRNA production.

Main Methods:

  • High-throughput enzymatic assays were employed to study Microprocessor activity.
  • Randomized pri-miRNAs were used to probe the enzyme's catalytic mechanism.
  • Analysis of RNA structural elements, including mismatches and wobble base pairs, in pri-miRNA substrates.

Main Results:

  • Multiple mismatches and wobble base pairs in the upper stem of pri-miRNAs were identified as key determinants of processing efficiency and accuracy.
  • These RNA elements explain alternative cleavage patterns observed for some human pri-miRNAs.
  • RNA-editing events and single nucleotide polymorphisms (SNPs) were shown to alter pri-miRNA processing by modifying these RNA elements.

Conclusions:

  • The study elucidates the role of specific RNA structural features in pri-miRNA processing by Microprocessor.
  • Findings provide a mechanistic basis for understanding how RNA modifications and genetic variations lead to differential miRNA expression.
  • This work enhances the understanding of miRNA biogenesis regulation and its implications in disease.