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Questions and Answers
What is the primary role of tmRNA in bacterial cells?
What is the primary role of tmRNA in bacterial cells?
Which of the following is a characteristic function of riboswitches?
Which of the following is a characteristic function of riboswitches?
Long non-coding RNAs (lncRNAs) have what kind of informational status?
Long non-coding RNAs (lncRNAs) have what kind of informational status?
Which class of small RNA is responsible for gene silencing in eukaryotes?
Which class of small RNA is responsible for gene silencing in eukaryotes?
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What common theme surrounds the discovery of eukaryotic small RNAs?
What common theme surrounds the discovery of eukaryotic small RNAs?
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What does the Hammerhead Ribozyme primarily perform?
What does the Hammerhead Ribozyme primarily perform?
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Which of the following is NOT considered a class of eukaryotic small RNAs?
Which of the following is NOT considered a class of eukaryotic small RNAs?
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What is a common function of bacterial small RNAs?
What is a common function of bacterial small RNAs?
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What is the primary role of Dicer in the siRNA processing pathway?
What is the primary role of Dicer in the siRNA processing pathway?
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How do miRNAs typically achieve gene regulation?
How do miRNAs typically achieve gene regulation?
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What characterizes the interaction between siRNAs and their target mRNAs?
What characterizes the interaction between siRNAs and their target mRNAs?
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What structural features do PAZ and PIWI domains contribute to in Argonaute proteins?
What structural features do PAZ and PIWI domains contribute to in Argonaute proteins?
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What is the role of Dicer in the miRNA processing pathway?
What is the role of Dicer in the miRNA processing pathway?
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What is a key difference in how miRNAs and siRNAs functionally interact with their targets?
What is a key difference in how miRNAs and siRNAs functionally interact with their targets?
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What does the PAZ domain in RNAse III enzymes do?
What does the PAZ domain in RNAse III enzymes do?
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What determines the sorting of duplex RNAs on Argonaute proteins during RISC assembly?
What determines the sorting of duplex RNAs on Argonaute proteins during RISC assembly?
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In mammalian systems, which molecules are identified as being essential for RNA interference?
In mammalian systems, which molecules are identified as being essential for RNA interference?
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What is the significance of the 'guide strand' in the miRNA processing outcome?
What is the significance of the 'guide strand' in the miRNA processing outcome?
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In which organism was the recapitulation of RNAi in vitro first successfully demonstrated?
In which organism was the recapitulation of RNAi in vitro first successfully demonstrated?
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Which statement about the processing of exogenously introduced RNA and endogenous dsRNA is accurate?
Which statement about the processing of exogenously introduced RNA and endogenous dsRNA is accurate?
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What is the primary function of small RNAs like miRNAs and siRNAs?
What is the primary function of small RNAs like miRNAs and siRNAs?
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What does co-suppression refer to in plants?
What does co-suppression refer to in plants?
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Which feature is commonly found in pri-miRNAs?
Which feature is commonly found in pri-miRNAs?
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What was discovered about sense and antisense RNA in relation to gene silencing?
What was discovered about sense and antisense RNA in relation to gene silencing?
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Study Notes
Noncoding/Regulatory RNAs
- Regulatory RNAs play various roles throughout biology.
- Their discovery and analysis are important areas of study.
- These RNAs regulate gene expression in typical ways.
- Specific examples of regulatory RNAs include tmRNA, ribozymes, RyhB, miRNAs, siRNAs, piRNAs, and tsRNAs.
Small (and Long) RNAs in Biology
- Bacteria have diverse non-coding RNAs that regulate gene expression.
- These are typically expressed through standard cellular pathways like transcription and processing.
- Examples include tmRNA, ribozymes, and RyhB.
- Eukaryotes possess various short (20-30 nucleotides) RNAs, playing diverse roles, discovered later than those in bacteria.
- Examples include miRNAs, siRNAs, piRNAs, and tsRNAs.
- Eukaryotes also have many longer non-coding RNAs, with less understood functions.
- Xist is an example of a long non-coding RNA.
Highly Specialized Function of tmRNA
- tmRNA performs the function of tRNA and mRNA to rescue ribosomes stalled during protein synthesis.
- It often corrects damaged messenger RNAs, thereby impacting protein synthesis.
- Research involving tmRNA includes analyses of protein tagging and degradation systems.
Typical Action by Bacterial Small RNA
- Bacterial small RNAs, like RyhB, regulate gene expression in response to changes in iron levels.
- High iron levels cause obstruction of ribosome binding to mRNA.
- Low levels enable recruitment of mRNA targeting for cleavage.
Riboswitches
- Riboswitches bind small molecules to regulate downstream processes.
- Their binding, or lack thereof, influences gene expression through changes in mRNA structure.
- The B12 riboswitch is one example in which a metabolite-binding mRNA regulates gene expression.
RNA as Enzymes: Hammerhead Ribozyme
- RNA can act as enzymes, exemplified by the hammerhead ribozyme, capable of self-cleavage.
- This self-cleavage is an instance of an endo-nucleolytic reaction involving a hydroxyl group.
Common Themes for Eukaryotic Small RNAs
- Discovery of eukaryotic small RNAs involved diverse methods and early clues.
- Their origins and subsequent processing into functioning forms, including loading into Argonaute family proteins, are relevant aspects in understanding their action.
- Understanding their functions in gene regulation is another key topic.
Discovery of miRNAs
- Lin-14 and Lin-14 mutants exhibited similar phenotypes in development, indicating a shared regulatory role.
- Lin-14 shows variable protein expression during development, high in L1 and low in L2/L3.
- Lin-4 encodes a small discrete RNA that downregulates Lin-14 expression, acting as a negative regulator.
lin4 Encodes a Small Discrete RNA
- lin-4 RNA is expressed at specific times during development.
- The RNA sequence of lin-4 could complement the lin-14 3' UTR, illustrating a potential regulatory mechanism.
Small RNAs Conserved from Worms to Humans
- Small RNAs, specifically RNA species like let-7, are conserved from worms, like C. elegans, to humans, illustrating an evolutionary connection.
- Conservation of sequence and expression patterns signifies fundamental roles in biological processes.
How Many miRNAs Are There?
- miRNAs are encoded as genes.
- They form hairpin structures to carry out their function.
- They are highly conserved meaning similar elements are likely found across a number of organisms.
- miRNAs are involved with a myriad of gene expression control.
What do Precursors Look Like?
- miRNAs are transcribed from genes as larger RNA precursors.
- These precursors can contain introns and are often clustered.
- They are processed, first in the nucleus and then cytoplasm, through enzymes like Drosha and Dicer.
- After processing, the resulting miRNA is often a duplex with a small 2 nucleotide overhang at the 3’ end.
RNAse III Enzymes
- RNAse III enzymes play a significant role in RNA processing.
- These enzymes function as "molecular rulers" defining precise RNA lengths, crucial for Dicer function.
- Enzymes like Drosha interact with DGCR8 (containing PAZ domains) in early miRNA processing.
- Dicer has its own PAZ domain for later processing.
miRNA Processing Steps
- miRNA duplexes are loaded into Argonautes to execute gene regulation.
- The guide strand stays bound while other strands (the passenger) is released.
- These strands are marked with 5' and 3' designations to distinguish them.
Discovery of RNAi (and siRNAs)
- RNA interference (RNAi) is a mechanism of gene silencing involving small RNAs, like siRNAs.
- Co-suppression in plants and quelling in Neurospora are examples of RNAi phenomena.
An Important Observation
- Sense and antisense RNA molecules have equivalent silencing effects, emphasizing the role of dsRNA as a silencing trigger.
Potent and Specific Genetic Interference
- Double-stranded RNA (dsRNA) can potently and specifically downregulate gene expression (RNA interference or RNAi).
- Ancestral pathways play a protective role against viruses and transposable elements (transposons) which could adversely affect gene expression.
Recapitulation of RNAi
- RNAi can be recapitulated in vitro using Drosophila embryos, and cell culture models such as Drosophila S2 cells and HeLa cells, which shows target RNA is susceptible to degradation.
dsRNA is Processed into 21-mers
- Double-stranded RNA (dsRNA) is processed into 21-nucleotide fragments for silencing through enzymes such as Dicer.
- These processes and types of RNA degradation or silencing are well documented in plants and cellular models.
Sounds Like a Job for Dicer
- Dicer processes duplexes into siRNAs, which are a type of silencing RNA, in both exogenous and endogenous dsRNA contexts.
- SiRNAs can be transient or expressed stably in cells .
- All forms result in the production of siRNAs which are not considered genes but rather as a form of functional silencing RNA.
What are the Effector Molecules?
- miRNAs and siRNAs regulate gene expression via different mechanisms, including translational repression, and target cleavage.
- Their functions are influenced by the degree and type of complementary base pairing with target molecules.
Purification of RISC
- Mass spectrometry identifies Argonaute 2 (Ago2) as a key component of the RNA-induced silencing complex (RISC).
- RISC is important in RNAi and influences processes by which genes are silenced/expressed.
What do PAZ and PIWI Domains Do?
- PAZ domains are essential for binding the 3' end of RNAs.
- PIWI domains are strikingly similar to RNaseH in their structure, and function in mediating cleavage.
X-Ray Structures of Ago Architecture
- Structures reveal multiple domains, including PAZ and PIWI, within the Argonaute protein structure, which gives a mechanistic insight for how they function.
How do Duplex RNAs Get Sorted?
- Asymmetric loading of duplex RNAs into Argonaute proteins is crucial for determining which strand will act as the guide strand and which will be the passenger strand.
- This can involve helicase enzymes and or specific interactions between the protein domain responsible for loading.
RISC Assembly
- RISC assembly from RNA duplexes involves the joining of specific RNA strands and components mediating gene silencing;
- Specific interactions between the component parts of the protein complex are crucial, and often involve structural features such as base pairing and or spatial positioning.
What do Targets Look Like?
- miRNAs can bind imperfectly to target mRNAs and mediate translational repression and or cleavage.
- siRNAs bind perfectly to target mRNAs and induce cleavage of the target mRNA.
miRNAs Use "Seed" Pairing to Identify Targets
- miRNAs utilize seed sequences (nucleotides 2-8) for target recognition and regulate gene expression via either cleavage or translational repression.
- Often there are small regulatory regions that contribute to the overall regulatory complexity.
Challenges in miRNA Target Identification
- Imperfect base pairing between miRNA and target mRNAs complicates target prediction, yet allows for broader targeting specificity
How do Mammalian miRNAs Silence Gene Expression?
- Mammalian miRNAs primarily mediate gene silencing via mRNA cleavage and or translational repression, achieved through interactions with proteins and/or RNA complexes.
What Drives miRNA Turnover?
- miRNA stability varies across different types and cellular contexts, with elements like Tudor-SN potentially involved in regulating their turnover.
- miRNA degradation occurs through processes mediated or influenced by different proteins and/or pathways involving a process of target-directed miRNA degradation (TDMD)
Diverse miRNA: Target RNA Interactions
- miRNAs can interact with target RNAs through diverse mechanisms involving seed pairing, full pairing, or extended pairing.
How do siRNAs Silence Gene Expression?
- siRNAs cause mRNA cleavage followed by standard RNA degradation pathways, with precise cleavage sites located between nucleotides 10-11.
Specialization of Small RNA Pathways
- Dcr-2/Ago2 is associated with RNA interference (RNAi), whereas Dcr-1/Ago1 is involved in miRNA function.
Circular RNAs
- Circular RNAs (circRNAs): are a form of regulatory RNA that can act like miRNA sponges to influence miRNA potency and target access for regulation.
tRNA-derived small RNAs
- tRNA fragments and halves are a considerable source of regulatory small RNAs.
Guardians of the Genome: piRNAs
- piRNAs are long non-coding RNAs derived from transposon-rich loci within DNA.
- They regulate gene expression by interfering with target transcripts (often through mechanisms of secondary piRNAs)
What do we know about piRNAs (PIWI-interacting)?
- piRNAs are a class of long single-stranded RNAs transcribed from specific chromosomal regions and are often associated with processing that results in secondary piRNAs.
What do the piRNAs Do?
- piRNAs regulate gene expression during early development, predominantly silencing transcripts from repetitive genomic regions, which could be problematic.
What do we know about hcRNAs (heterochromatic)?
- hcRNAs is a type of RNA that primarily maintains genome integrity near centromeres.
- They promote efficient epigenetic modification primarily at various specific and overlapping chromosomal regions
Evolutionary Origins of These Pathways
- RNA interference (RNAi) machinery appears to have originated early in eukaryotic evolution.
- This RNAi machinery plays a crucial role in antiviral defense.
CRISPR: in other lectures
- CRISPR is a bacterial-adaptive immune system.
- CRISPR systems have three primary stages (insertion, transcription, and targeting).
- Different subtypes of the CRISPR system exist, like Cas9 (type II) or Cascade (type I).
- Cas9 systems have been widely used to edit genomes in cellular or model organisms.
Genome engineering with CRISPR
- CRISPR is increasingly utilized for efficient genome engineering, such as base editing and DNA repair.
- Methods to utilize crispr for precise genome engineering are frequently developed and refined.
Long (intergenic) non-coding RNAs (I(i)ncRNAs)
- Long non-coding RNAs (lncRNAs) like Xist perform significant regulatory functions in various aspects of cellular biology, especially in the regulation of gene expression
Take home messages
- There are more functional noncoding RNAs (ncRNAs) and genes than previously thought.
- ncRNAs play diverse regulatory roles across organisms.
- Mechanisms for RNA regulation are similar to those of protein binding.
- Evolution of specific regulatory RNAs may be simple given the role of complementarity.
Additional Resources
- Resources for further study on RNAs.
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