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5BBG0205 Molecular Basis of Gene Expression > L5: RNA mediated regulation of gene expression (RNAi) > Flashcards

L5: RNA mediated regulation of gene expression (RNAi) Flashcards

1
Q

What are microRNAs (miRNAs) and how do they function?

A
  • miRNAs are small non-coding RNA molecules (~22 nucleotides) that regulate gene expression.
  • Produced from primary miRNA (pri-miRNA) transcripts.
  • Regulate endogenous gene expression
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2
Q

Where are microRNA genes found and transcribed?

A
  • Found in various genomes: plants, C. elegans, Drosophila, humans.
  • Genes located in intergenic regions with their own promoters
  • Can be in anti-sense orientation within genes
  • Some miRNAs transcribed from introns of genes in same sense orientation
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3
Q

What is the evolutionary significance of miRNAs?

A
  • miRNAs considered a common evolutionary strategy for gene regulation.
  • Many miRNAs in C. elegans have homologues in mammals.
  • miRNAs are present in diverse organisms
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4
Q

How many microRNAs are present in the human genome and how do they regulate genes?

A
  • human genome encodes nearly 6000 microRNAs
  • at least 60% of human genes regulated by microRNAs
  • some miRNAs target different sites on same mRNA with similar nucleotide sequences
  • some miRNAs target multiple mRNAs with similar nucleotide sequences
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5
Q

How are primary miRNAs (pri-miRNAs) produced and regulated?

A
  • Pri-miRNAs transcribed in nucleus from genomic DNA.
  • Transcribed by RNA polymerase II (Pol II) with their own promoters.
  • TFs regulate the production of pri-miRNAs
  • Pri-miRNAs can be quite long and can yield multiple miRNAs
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6
Q

Describe the pathway of mammalian miRNA biogenesis and RNAi silencing

A
  • pri-miRNA transcribed in the nucleus
  • microprocessor complex (Drosha & DGCR8) cleaves a hairpin loop from pri-miRNA, forming pre-miRNA.
  • exportin 5 transports pre-miRNA from nucleus to cytoplasm using GTP energy.
  • in cytoplasm, Dicer cleaves terminal loop of pre-miRNA, yielding a miRNA duplex.
  • dicer associates with Argonaute protein, inducing RNA-induced silencing complex (RISC) formation.
  • Guide (antisense) strand is selected and loaded into Argonaute while passenger (sense) strand is discarded.
  • Mature RISC targets mRNAs in partially complementary manner, regulating gene expression
  • Mechanisms of target regulation include deadenylation, degradation, inhibition of translation initiation, & more
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7
Q

What are the key steps in the miRNA biogenesis pathway?

A
  1. Transcription of pri-miRNA in the nucleus.
  2. Cleavage of hairpin loop from pri-miRNA by microprocessor complex (Drosha and DGCR8).
  3. Export of pre-miRNA to the cytoplasm via Exportin 5
  4. Dicer cleavage of pre-miRNA’s terminal loop, forming miRNA duplex
  5. Formation of RISC through Dicer and Argonaute association
  6. Selection of guide strand, loading into Argonaute, and degradation of passenger strand
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8
Q

How does the mature RISC regulate gene expression?

A
  • Mature RISC targets mRNAs partially complementary manner
  • can induce deadenylation, leading to mRNA degradation.
  • Inhibition of translation initiation can also occur.
  • Guide strand, loaded into Argonaute, binds to 3’ untranslated region (3’ UTR) of mRNA.
  • guide strand’s partial complementarity to its target mRNA enables regulation
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9
Q

What is the role of the RNase III family in miRNA biogenesis?

A
  • RNase III family includes nucleases with specificity for double-stranded RNA (dsRNA).
  • Found across various organisms from bacteria to animals.
  • 3 structural classes: Class I (e.g., E. coli RNase III), Class II (e.g., Drosha), Class III (e.g., Dicer).
  • Drosha, part of the RNase III family, cleaves pri-miRNA in the nucleus
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10
Q

How is asymmetric cleavage achieved during miRNA biogenesis?

A

.- Drosha (an RNase III enzyme) cleaves the lower stem of pri-miRNA.
- this cleavage happens ~11 bp upstream from the junction of flanking sequences and the lower stem-loop.
- the microprocessor complex’s asymmetric cleavage leaves 2 unpaired nucleotides at the 3’OH end of the sense strand of pre-miRNA

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11
Q

What is the significance of the microRNA pathway in gene regulation?

A
  • miRNAs = crucial regulators of gene expression
  • play a role in post-transcriptional gene silencing
  • pathway involves multiple steps, from transcription to target regulation, to ensure precise gene regulation.
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12
Q

How does Dicer process pre-miRNA in the cytoplasm?

A
  • PAZ domain of Dicer binds to both ends of pre-miRNA hairpin
  • Two RNase III domains cut the dsRNA stem, removing the terminal loop.
  • produces a miRNA duplex, usually 21-25 nucleotides long.
  • structure of the PAZ domain, RNase III domains & the miRNA duplex govern its length
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13
Q

What is the significance of the miRNA duplex structure?

A
  • miRNA duplex length (21-25 nucleotides) is specific to each type of Dicer & position of pre-miRNA
  • Variants in Dicer & Argonaute protein families across organisms lead to RNAi mechanism variations
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14
Q

How are miRNAs identified and characterized?

A
  • miRNA sequencing involves:
    1. Purification of miRNA from total RNA using size selection.
    2. Ligating adapters to miRNA ends.
    3. Creating cDNA using reverse transcription.
    4. Amplifying cDNA through PCR.
    5. Sequencing PCR products to identify miRNA sequences
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15
Q

How are mRNA arrays used to study miRNA expression profiles?

A
  • Synthetic oligonucleotides complementary to mature miRNAs spotted on slides
  • Labeled miRNA from different tissues hybridizes to oligonucleotides on slides.
  • Fluorescence measurement identifies present miRNAs and their expression levels
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16
Q

How are miRNAs linked to human diseases?

A
  • Changes in miRNA expression & mutations within miRNAs relate to human diseases
  • Examples include miR-96 mutation causing hereditary hearing loss, and miR-184 mutation linked to hereditary keratoconus and cataract.
  • miRNAs have roles in development, cancer, heart disease, dementia, etc
17
Q

What are the applications of RNA interference (RNAi)?

A
  • RNAi downregulates gene expression.
    RNAi is used to:
  • Study gene function.
  • Develop anti-viral drugs.
    Create gene therapies.
    Design anti-cancer treatments
18
Q

How is RNAi used for studying novel phenotypes?

A
  • RNAi can induce gene downregulation in C. elegans by introducing dsRNA.
  • Techniques include injecting dsRNA into nematodes or feeding them bacteria expressing dsRNA.
  • RNAi helps identify genes contributing to specific phenotypes
19
Q

What is RNAi-based therapy, and how is it delivered?

A
  • RNAi therapy treats diseases using siRNA.
    Delivery methods include:
  • Lipid nanoparticles (LNPs).
  • Sugar molecule-siRNA conjugates.
  • Viral vectors.
20
Q

How is RNAi used in clinical trials?

A
  • RNAi-based therapeutics are being tested in clinical trials.
  • Examples include Bevasiranib, an siRNA that downregulates VegF mRNA for treating age-related macular degeneration
21
Q

What is the process of RNA-induced silencing in therapeutic applications?

A
  • Target mRNA is transcribed from a dsDNA fragment
  • Target mRNA binds to RISC with delivered siRNA.
  • Antisense strand of siRNA is loaded into RISC.
  • Antisense strand guides cleavage of target mRNA in RISC, causing its degradation
22
Q

How does RNA interference (RNAi) work against viral RNA?

A
  • Viral RNA enters cytoplasm via endocytosis.
  • Viral RNA transforms into dsRNA.
  • Dicer processes dsRNA into siRNA.
  • siRNA enters RISC, binds target mRNA, leading to mRNA cleavage and degradation
23
Q

How does RNAi silence transposons?

A
  • Transposons create dsRNA in the nucleus.
  • dsRNA exits nucleus, binds Dicer, forming siRNA.
  • siRNA enters RISC, binds transposon mRNA, causing mRNA cleavage and degradation
24
Q

What is the role of RNAi in microRNA gene regulation?

A
  • RNA polymerase synthesizes pri-miRNA in the nucleus.
  • Pri-miRNA exits nucleus, binds Dicer, producing miRNA.
  • miRNA enters RISC, binds target mRNA, suppressing protein synthesis
25
Q

How does RNAi work against exogenous RNA (siRNA)?

A
  • Exogenous RNA enters cytoplasm via endocytosis.
  • Exogenous RNA becomes siRNA.
  • siRNA enters RISC, binds target mRNA, leading to mRNA cleavage and degradation.
26
Q

How does RNAi inhibit RNA synthesis?

A
  • RISC complex with miRNA/siRNA and target mRNA causes chromatin condensation.
  • Chromatin condensation inhibits RNA synthesis by RNA polymerase

5BBG0205 Molecular Basis of Gene Expression (9 decks)

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