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What is the approximate length of a precursor miRNA (pre-miRNA)?
What is the approximate length of a precursor miRNA (pre-miRNA)?
MiRNAs are involved in regulating gene expression in prokaryotic cells.
MiRNAs are involved in regulating gene expression in prokaryotic cells.
False
What is the role of the RNase III enzyme Drosha in the context of miRNA production?
What is the role of the RNase III enzyme Drosha in the context of miRNA production?
Drosha cleaves the stem-loop structure of pri-miRNA to release pre-miRNA.
The pri-miRNA is processed in the nucleus by a complex that includes Drosha and the double-stranded RNA-binding protein _____ .
The pri-miRNA is processed in the nucleus by a complex that includes Drosha and the double-stranded RNA-binding protein _____ .
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Match the following components with their roles in miRNA processing:
Match the following components with their roles in miRNA processing:
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What is the primary mechanism by which miRNAs regulate gene expression?
What is the primary mechanism by which miRNAs regulate gene expression?
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The number of genes predicted to be regulated by miRNAs in the human genome is significant.
The number of genes predicted to be regulated by miRNAs in the human genome is significant.
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Describe one way RNAi mechanisms have evolved.
Describe one way RNAi mechanisms have evolved.
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What is the primary function of Drosha in the RNA interference (RNAi) pathway?
What is the primary function of Drosha in the RNA interference (RNAi) pathway?
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Dicer contains only one RNase III domain.
Dicer contains only one RNase III domain.
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Name the two primary reasons for the evolution of RNAi mechanisms.
Name the two primary reasons for the evolution of RNAi mechanisms.
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The enzyme that degrades RNA is called ____.
The enzyme that degrades RNA is called ____.
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Which structural class does Dicer belong to?
Which structural class does Dicer belong to?
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Match the following components of miRNA processing with their functions:
Match the following components of miRNA processing with their functions:
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Humans have multiple copies of both Dicer and Drosha.
Humans have multiple copies of both Dicer and Drosha.
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How can miRNAs be identified using sequencing?
How can miRNAs be identified using sequencing?
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What is the primary purpose of using PCR in the context of miRNA?
What is the primary purpose of using PCR in the context of miRNA?
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Over 60% of human genes are predicted to be regulated by microRNAs.
Over 60% of human genes are predicted to be regulated by microRNAs.
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What common diseases have been associated with mutations in microRNA sequences?
What common diseases have been associated with mutations in microRNA sequences?
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The human genome is thought to encode over _____ microRNAs.
The human genome is thought to encode over _____ microRNAs.
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Match the type of disease with its associated microRNA mutation:
Match the type of disease with its associated microRNA mutation:
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Which of the following best describes the function of miRNAs in biological processes?
Which of the following best describes the function of miRNAs in biological processes?
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MiRNAs are often found to be DOWNREGULATED in cancer.
MiRNAs are often found to be DOWNREGULATED in cancer.
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How has RNA interference (RNAi) been used in generating mutant phenotypes?
How has RNA interference (RNAi) been used in generating mutant phenotypes?
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What is the primary role of the dicer enzyme in miRNA processing?
What is the primary role of the dicer enzyme in miRNA processing?
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The mature RISC complex contains both the guide and passenger strands of the miRNA.
The mature RISC complex contains both the guide and passenger strands of the miRNA.
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Name one mechanism by which miRNA regulates gene expression.
Name one mechanism by which miRNA regulates gene expression.
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The guide strand typically binds to the _____ region of its target mRNA.
The guide strand typically binds to the _____ region of its target mRNA.
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Match the mechanism of miRNA regulation with its description:
Match the mechanism of miRNA regulation with its description:
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What typically happens to the mRNA after the binding of the miRNA in mammals?
What typically happens to the mRNA after the binding of the miRNA in mammals?
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MiRNA functions only at the transcriptional level in eukaryotic cells.
MiRNA functions only at the transcriptional level in eukaryotic cells.
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What specific type of RNA do RNase III family members target?
What specific type of RNA do RNase III family members target?
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What is the primary purpose of using RNAi in C.elegans research?
What is the primary purpose of using RNAi in C.elegans research?
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The sense strand and antisense strand are both produced from the gene insert in RNAi studies.
The sense strand and antisense strand are both produced from the gene insert in RNAi studies.
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What organism is commonly used in RNAi research for fat regulation and obesity gene identification?
What organism is commonly used in RNAi research for fat regulation and obesity gene identification?
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SiRNA stands for _____________________.
SiRNA stands for _____________________.
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Match the following RNAi delivery methods with their descriptions:
Match the following RNAi delivery methods with their descriptions:
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What is the length of small interfering RNA (siRNA)?
What is the length of small interfering RNA (siRNA)?
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C.elegans has been shown to have no fat regulatory genes previously implicated in fat storage.
C.elegans has been shown to have no fat regulatory genes previously implicated in fat storage.
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What is one way RNAi could be used therapeutically?
What is one way RNAi could be used therapeutically?
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Study Notes
Origin and Mechanism of MicroRNAs (miRNAs)
- MicroRNAs are small, non-coding RNA molecules produced from genomic DNA in eukaryotic cells.
- Primary miRNAs (pri-miRNAs) are regulated by transcription factors, often exceeding 1000 nucleotides in length, potentially producing multiple miRNAs.
- The microprocessor complex, comprising the RNase III enzyme Drosha and DGCR8, processes pri-miRNAs in the nucleus by cleaving their stem-loop structure to form precursor miRNAs (pre-miRNAs).
- Pre-miRNAs, approximately 60-70 nucleotides long, are exported to the cytoplasm by Exportin-5, utilizing GTP-Ran.
- Dicer cleaves the terminal loop of pre-miRNA, yielding miRNA:miRNA duplexes (21-25 nucleotides).
- The guide strand (antisense) is incorporated into the RISC (RNA-induced silencing complex) alongside Argonaute, while the passenger strand is discarded.
- Mature RISC modulates gene expression, primarily binding to the 3’ untranslated region (3’UTR) of target mRNAs, often with partial complementarity.
Regulation of Gene Expression by miRNAs
- RNA interference (RNAi) silences gene expression both transcriptionally and post-transcriptionally.
- Regulatory RNAs target mRNAs through complementary base pairing, leading to mRNA degradation or translation blockage.
- Some regulatory RNAs can repress gene transcription in the nucleus by altering chromatin structure.
- Mechanisms of miRNA-mediated regulation include:
- Endonucleolytic cleavage of target mRNA.
- Deadenylation (removal of polyA tail) leading to degradation.
- Inhibition of translation initiation by preventing ribosome binding.
- Inhibition after translation initiation resulting in loss of protein production.
Structure and Function of RNase III Family Members
- RNase III family members are nucleases specific to double-stranded RNA.
- Common structural motifs include:
- Class I (prokaryotic) with one RNase III domain and one double-stranded RNA-binding domain.
- Class II (eukaryotic, e.g., Drosha) with two RNase III domains and one dsRNA-binding domain, cleaving 11 bases from stem-loop junctions.
- Class III (eukaryotic, e.g., Dicer) features two RNase III domains, one dsRNA-binding domain, and a PAZ domain, crucial for processing pre-miRNA.
Evolution of RNAi Mechanisms
- RNAi mechanisms likely evolved to protect genomes against transposable elements, safeguarding gene function.
- They also offer defense against viral infections by silencing viral RNAs, evidenced by increased susceptibility of argonaute mutants in plants to viruses.
Identification of miRNAs
- MiRNA sequencing involves purifying miRNA from total RNA through size selection and attaching adaptors for reverse transcription to create complementary DNA.
- PCR amplifies the reverse transcription product, generating double-stranded DNA indicative of miRNA sequence.
- MiRNA arrays utilize synthetic oligonucleotides to detect mature miRNAs by hybridization and fluorescence quantification.
Expression and Regulation of miRNAs
- The human genome encodes over 2000 miRNAs, with at least 60% of genes predicted to be regulated by miRNAs.
- MiRNAs play a crucial role in multiple biological processes and are implicated in various diseases, including cancer.
Link Between miRNA Alterations and Human Diseases
- Changes in miRNA expression or mutations can lead to diseases such as hereditary progressive hearing loss (miR-96) and keratoconus (miR-184).
- Dysregulation of miRNAs is frequently observed in cancer, often resulting in downregulation.
Utilizing RNAi in C. elegans Research
- Researchers employ RNAi in C. elegans to silence individual genes systematically, elucidating gene functions related to processes like fat regulation.
- Methods include cloning genes into plasmids for transformation into E. coli or directly injecting double-stranded RNA into nematodes.
Therapeutic Applications of RNAi
- RNAi strategies can target disease-related genes (e.g., obesity) identified through research in model organisms like C. elegans.
- Therapeutic approaches include:
- Delivery of chemically synthesized small interfering RNAs (siRNAs) via various methods.
- Engineer viruses to transcribe siRNA in targeted cells.
- Nanoparticles can facilitate siRNA delivery, allowing entry into cells through endocytosis, where they bind to Argonaute to exert their effects.
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Description
This quiz explores the origins and mechanisms of micro RNA (miRNA) production in eukaryotic cells. It covers the regulatory roles of miRNAs in gene expression, the domain structure of RNase III family members, and methods for identifying miRNAs using arrays and sequencing. Test your understanding of these crucial molecular processes.
