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Questions and Answers
What is the primary purpose of RNA interference (RNAi) in organisms?
What is the primary purpose of RNA interference (RNAi) in organisms?
What role do microRNAs (miRNAs) play in gene regulation?
What role do microRNAs (miRNAs) play in gene regulation?
Which of the following proteins is known to suppress RNAi in certain viruses?
Which of the following proteins is known to suppress RNAi in certain viruses?
How do the miRNAs discovered in plants differ from those found in animals?
How do the miRNAs discovered in plants differ from those found in animals?
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What was a significant focus of Victor Ambros and Gary Ruvkun during their research?
What was a significant focus of Victor Ambros and Gary Ruvkun during their research?
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What is the primary outcome of post-transcriptional gene silencing (PTGS)?
What is the primary outcome of post-transcriptional gene silencing (PTGS)?
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How does antisense technology primarily function?
How does antisense technology primarily function?
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What was the original discovery of PTGS primarily associated with?
What was the original discovery of PTGS primarily associated with?
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Which component was used to introduce the DFR gene into petunia?
Which component was used to introduce the DFR gene into petunia?
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What determines the variability of the antisense effect in gene regulation?
What determines the variability of the antisense effect in gene regulation?
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Which of the following is a characteristic of RNA interference (RNAi) mechanisms?
Which of the following is a characteristic of RNA interference (RNAi) mechanisms?
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What is a key difference between plant and animal microRNAs?
What is a key difference between plant and animal microRNAs?
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In the context of gene regulation, what does co-suppression refer to?
In the context of gene regulation, what does co-suppression refer to?
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What is the main mechanism by which siRNAs mediate gene suppression?
What is the main mechanism by which siRNAs mediate gene suppression?
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What role does Dicer-like enzyme play in the miRNA biosynthesis process in plants?
What role does Dicer-like enzyme play in the miRNA biosynthesis process in plants?
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How do plant and animal miRNA systems differ in terms of miRNA binding sites in target genes?
How do plant and animal miRNA systems differ in terms of miRNA binding sites in target genes?
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What is a key function of regulatory genes targeted by miRNAs in both plants and animals?
What is a key function of regulatory genes targeted by miRNAs in both plants and animals?
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What is a common outcome of RNAi-mediated suppression of GLO1 in plants?
What is a common outcome of RNAi-mediated suppression of GLO1 in plants?
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What is the primary difference in miRNA biosynthesis between plants and animals?
What is the primary difference in miRNA biosynthesis between plants and animals?
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In terms of genetic location, where are miRNA genes predominantly found in plants?
In terms of genetic location, where are miRNA genes predominantly found in plants?
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How does the binding of miRNAs to their targets differ between plants and animals?
How does the binding of miRNAs to their targets differ between plants and animals?
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Study Notes
Molecular Biotechnology BE618 Lecture 7: Gene Silencing
- Gene silencing occurs at both post-transcriptional (PTGS) and transcriptional (TGS) levels.
- PTGS, also known as RNA interference (RNAi), downregulates gene expression after transcription.
- Examples of PTGS include transposons, retroviral genes, and heterochromatin.
- Antisense technology, used since ~1980, aims to repress specific genes by introducing an antisense gene.
- Antisense technology's effectiveness is variable, often mediated by RNAi rather than translation inhibition.
- Co-suppression, a form of PTGS, was observed when an introduced gene with homology to an endogenous gene in petunia led to suppression of both genes.
- Co-suppression results mainly from degradation of mRNAs from both the introduced and endogenous genes.
- Genes involved in the flavonoid/anthocyanin pathway, such as CHS (chalcone synthase) and DFR (dihydroflavonol reductase), are affected by this process.
- Introducing these genes into petunia with a strong viral promoter led to decreased mRNA and pigment levels in transgenics.
- The 2006 Nobel Prize for Physiology or Medicine was awarded for RNA interference discovery in C. elegans (Andrew Fire & Craig Mello).
- RNA interference (RNAi) was initially discovered in C. elegans while attempting to use antisense RNA in vivo.
- "Sense" RNAs, when contaminated with dsRNA, also demonstrated gene suppression.
- Double-stranded RNA (dsRNA) induces mRNA interference more effectively than antisense mRNA.
- Suppression signals can move between cells.
- RNAi occurs in various eukaryotes, including angiosperms, chlamydomonas (unicellular), mammals, C. elegans, Drosophila, and Neurospora, but not in yeast.
RNAi Pathway
- Long double-stranded RNA (dsRNA) is processed in the nucleus, creating shorter dsRNA.
- This shorter dsRNA is exported to the cytoplasm, binding to and being cleaved by dicer into small fragments (siRNAs or miRNAs).
- siRNAs or miRNAs are recruited by the RISC complex, unwinding and incorporating into a protein-RNA complex.
- The RISC-associated siRNA or miRNA binds to a complementary mRNA molecule.
- The mRNA is then cleaved and degraded, preventing protein synthesis of the target gene.
Mechanism of RNAi: Role of Dicer
- Cells undergoing RNAi contain small fragments of suppressed RNA.
- A nuclease called Dicer, purified from Drosophila embryos, is involved in processing these small RNA fragments.
- Mutating the Dicer gene inhibits RNAi.
- Dicer (an endonuclease) degrades dsRNA into 21-24 nt fragments.
- In higher eukaryotes, Dicer separates the fragments via intrinsic helicase activity.
Model for RNAi
- RNAi is a very efficient process, generating many small interfering RNAs (siRNAs) from a larger dsRNA.
- It uses Dicer or an ATP-dependent helicase to cleave and separate dsRNA strands.
- Cleaved RNA fragments form active siRNA complexes called RISC (RNA-induced silencing complex), which contain Argonaute instead of Dicer.
- siRNA complexes target cleavage of mRNA, inhibiting protein synthesis of the target gene.
RNAi Mechanism
- RNAi mechanism involves the use of RNA-dependent RNA polymerase (RDR).
- In plants, fungi, C. elegans, and Drosophila, RDR participates in RNA silencing initiation or amplification.
- Initiation or amplification of silencing requires RDR access to the RNA template.
- RDR access is sometimes blocked by proteins like CBP (cap-binding protein) and PABP (poly(A)-binding protein).
- The process can initiate or amplify RNA silencing by producing dsRNA, which then gets further processed into smaller siRNAs.
Why RNAi Silencing?
- RNA interference evolved to protect genomes from viruses and potentially transposons/mobile DNAs.
- Some viruses have proteins (e.g., HCPro, P19, Tat) that suppress silencing to facilitate viral replication.
MicroRNAs (miRNAs)
- Very small (micro) miRNAs have recently been discovered in plants and animals.
- They are similar to siRNAs, regulating gene expression through mRNA degradation or translational repression.
- miRNAs are used for more than just defense.
Discovery of microRNA in 1993 and Nobel Prize 2024
- Researchers, including Victor Ambros and Gary Ruvkun, discovered microRNAs (miRNAs) important in the development of the roundworm C. elegans.
- The lin-4 and lin-14 gene mutations in worms reveal the crucial role of miRNAs.
- miRNAs are encoded by their own genes, are processed differently and have similar function to siRNAs, but are endogenous (within the organism).
C. elegans and heterochronic gene lin-4
- C. elegans lin-4 gene encodes small RNAs with antisense complementarity to lin-14.
- These small RNAs play a critical role in regulating gene expression during development.
GLO1 role in pollination
- GLO1 is essential for successful pollination in plants.
- RNAi-mediated silencing of GLO1 in plants leads to reduced seed set and increased methylglyoxal levels.
- Transgenic GLO1 RNAi lines show reduced pollen attachment and germination.
Comparison of miRNA Biogenesis and Action
- The mechanisms of miRNA biogenesis and action differ slightly between plants and animals.
- Plants primarily use DCL1 (Dicer-like 1) for miRNA processing.
- Animals utilize Drosha and Dicer.
Summary of differences between plant and animal miRNA systems
- Plant and animal miRNA systems share some similarities, but also have key differences in miRNA gene number, location within the genome, miRNA biosynthesis mechanisms, site of miRNA targeting within a gene, and function of known target genes.
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