7.5 Post-transcriptional regulation: RNA interference and microRNAs
3 min read•august 16, 2024
and microRNAs are powerful gene regulators. They use small RNA molecules to silence specific genes by targeting mRNAs. This process involves complex pathways and enzymes like and RISC.
These mechanisms play crucial roles in gene expression control. They're involved in development, disease, and cellular processes. Understanding them is key to grasping how cells fine-tune their genetic programs.
RNA Interference: Mechanisms and Gene Silencing
RNAi Pathway Components and Process
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- RNA interference (RNAi) uses double-stranded RNA (dsRNA) to target and degrade specific mRNA molecules
- Enzyme Dicer cleaves long dsRNA into short interfering RNAs (siRNAs) of 21-23 nucleotides
- siRNAs incorporate into RNA-induced silencing complex (RISC) guiding it to complementary mRNA sequences
- Argonaute proteins, particularly Argonaute 2 (), provide catalytic activity for target mRNA cleavage in RISC
- RNAi initiates from endogenous dsRNA or exogenous sources (viral infections, artificially introduced dsRNA)
Gene Silencing Mechanisms
- Transcriptional silencing occurs through chromatin modification
- Post-transcriptional silencing happens via mRNA degradation or
- strength varies based on complementarity between and target mRNA
- RNAi can spread systemically in some organisms (plants, C. elegans) through RNA-dependent RNA polymerases
- Off-target effects may occur when siRNAs bind to partially complementary sequences
MicroRNAs: Biogenesis and Function
miRNA Biogenesis
- MicroRNAs (miRNAs) regulate gene expression post-transcriptionally as small, non-coding RNAs of ~22 nucleotides
- RNA polymerase II transcribes genes into primary miRNAs (pri-miRNAs)
- processes pri-miRNAs in the nucleus into precursor miRNAs (pre-miRNAs)
- transports pre-miRNAs to the cytoplasm
- Dicer further processes pre-miRNAs into
- loads one strand of mature miRNA for target mRNA guidance
miRNA Function and Target Recognition
- miRNAs typically bind to (UTR) of target mRNAs
- Binding leads to translational repression or mRNA degradation
- (nucleotides 2-8 of miRNA) determines target recognition specificity
- Single miRNA can regulate multiple target genes (miR-155 regulates over 100 genes)
- Individual mRNA can be targeted by multiple miRNAs (p21 mRNA targeted by miR-17, miR-20a, miR-106b)
- Complex regulatory networks form through miRNA-mRNA interactions
- miRNA function influenced by cellular context and target mRNA abundance
Applications of RNAi and MicroRNAs
Research Applications
- studies use RNAi for targeted gene knockdown experiments
- High-throughput RNAi screens identify genes involved in specific cellular processes (cell cycle regulation, apoptosis)
- miRNA profiling serves as diagnostic and prognostic tool in diseases (miR-21 overexpression in various cancers)
- CRISPR-Cas9 technology edits miRNA genes or target sites for functional studies
Therapeutic Applications
- siRNA-based therapeutics target disease-causing genes (patisiran for hereditary transthyretin amyloidosis)
- miRNA mimics restore function of tumor-suppressor miRNAs (miR-34a mimic for cancer treatment)
- (anti-miRNAs) inhibit oncogenic miRNAs (anti-miR-122 for hepatitis C treatment)
- Delivery systems optimize cellular uptake and reduce off-target effects
- Lipid nanoparticles
- Viral vectors
- Conjugated delivery systems (N-acetylgalactosamine-siRNA conjugates)
- Combination therapies with traditional drugs enhance therapeutic efficacy
MicroRNA Mutations: Effects on Gene Expression
Types of miRNA Mutations
- Single nucleotide polymorphisms (SNPs) in miRNA genes alter expression or target specificity
- Seed sequence mutations change target mRNA recognition patterns
- Regulatory region mutations affect miRNA expression levels
- miRNA gene amplifications or deletions lead to over- or under-expression
- Epigenetic modifications alter miRNA expression patterns
- DNA methylation
- Histone modifications
Consequences of miRNA Mutations
- Biogenesis disruptions affect mature miRNA production (mutations in pri-miRNA processing sites)
- Target recognition alterations lead to dysregulation of multiple genes
- Expression level changes impact entire regulatory networks
- Aberrant gene expression contributes to disease development (miR-15a/16-1 cluster deletion in chronic lymphocytic leukemia)
- Mutations in miRNA target sites within mRNAs disrupt regulation (KRAS 3' UTR mutation in lung cancer)
Key Terms to Review (27)
3' untranslated region: The 3' untranslated region (3' UTR) is a segment of messenger RNA (mRNA) that follows the coding sequence and precedes the polyadenylation signal. This region plays a crucial role in post-transcriptional regulation by influencing mRNA stability, localization, and translation efficiency. Additionally, the 3' UTR is often where regulatory elements, such as microRNA binding sites, are found, making it vital for gene expression control.
Ago2: Ago2 is a protein that plays a crucial role in the RNA interference (RNAi) pathway by serving as a component of the RNA-induced silencing complex (RISC). This protein is involved in the loading of small interfering RNAs (siRNAs) and microRNAs (miRNAs) onto RISC, facilitating the regulation of gene expression through mRNA degradation or translational repression. Ago2’s ability to interact with these small RNA molecules is essential for the post-transcriptional regulation of gene expression, influencing various biological processes.
Antagomirs: Antagomirs are chemically modified oligonucleotides designed to specifically inhibit microRNAs (miRNAs) by binding to their target sequences. This interaction prevents the miRNAs from carrying out their regulatory functions on messenger RNAs (mRNAs), effectively reducing the expression of specific genes. Antagomirs play a crucial role in post-transcriptional regulation by modulating the activity of miRNAs, which are key players in RNA interference and gene silencing.
Dicer: Dicer is an endonuclease enzyme that plays a crucial role in the RNA interference (RNAi) pathway by processing long double-stranded RNA (dsRNA) and precursor microRNAs (pre-miRNAs) into shorter, functional RNA fragments. These small RNA molecules, typically 20-25 nucleotides long, are essential for gene silencing and regulation of gene expression through the action of the RNA-induced silencing complex (RISC). Dicer's ability to generate these small interfering RNAs (siRNAs) and microRNAs (miRNAs) is a key step in post-transcriptional regulation.
Dicer enzyme: The Dicer enzyme is a crucial ribonuclease that processes long double-stranded RNA (dsRNA) and precursor microRNAs (pre-miRNAs) into small interfering RNAs (siRNAs) and microRNAs (miRNAs). It plays a vital role in post-transcriptional regulation by facilitating RNA interference, which controls gene expression and helps maintain cellular homeostasis.
Drosha-dgcr8 complex: The drosha-dgcr8 complex is a crucial RNA processing complex that plays a key role in the biogenesis of microRNAs (miRNAs) by initiating the first step of miRNA maturation. This complex functions within the nucleus, where it recognizes and cleaves primary miRNA transcripts (pri-miRNAs) into shorter precursor forms called pre-miRNAs. The activity of this complex is essential for post-transcriptional regulation, influencing gene expression through RNA interference and the subsequent action of mature miRNAs.
Exportin-5: Exportin-5 is a transport protein that plays a critical role in the export of specific RNA molecules from the nucleus to the cytoplasm, particularly in the context of microRNA and small interfering RNA pathways. It recognizes and binds to double-stranded RNA structures, facilitating their transport through the nuclear pore complex, which is essential for post-transcriptional regulation mechanisms involving RNA interference.
Functional genomics: Functional genomics is the study of gene functions and interactions using high-throughput methods to analyze the expression and regulation of genes within a genome. This area of research aims to understand how genes contribute to biological processes by examining their roles in cellular functions, pathways, and responses to environmental changes. It involves integrating data from various techniques such as transcriptomics, proteomics, and metabolomics to provide a comprehensive view of gene activity and regulation.
Gene silencing: Gene silencing is a biological process that reduces or eliminates the expression of a specific gene, often through mechanisms like RNA interference (RNAi) and the action of microRNAs. This phenomenon plays a crucial role in regulating gene activity, ensuring that genes are expressed only when needed, and maintaining cellular homeostasis. By controlling gene expression, gene silencing influences various cellular processes including development, differentiation, and response to environmental signals.
Gene therapy: Gene therapy is a medical technique that aims to treat or prevent diseases by introducing, removing, or altering genetic material within a patient's cells. This innovative approach can address genetic disorders at their root cause, offering potential cures for inherited diseases and improving the effectiveness of existing treatments.
Mature miRNA duplexes: Mature miRNA duplexes are the final, functional forms of microRNAs that result from the processing of primary miRNA transcripts. These duplexes consist of two strands: a guide strand that is incorporated into the RNA-induced silencing complex (RISC) and a passenger strand that is typically degraded. These duplexes play a crucial role in post-transcriptional regulation by targeting messenger RNAs (mRNAs) for degradation or translational repression, thereby influencing gene expression.
Microrna: Microrna (miRNA) is a small, non-coding RNA molecule that plays a crucial role in the regulation of gene expression at the post-transcriptional level. These molecules typically consist of about 21 to 25 nucleotides and function by binding to complementary sequences on target messenger RNAs (mRNAs), leading to their degradation or inhibition of translation. This regulatory mechanism is essential for various biological processes, including development, differentiation, and cellular responses to environmental changes.
Microrna biogenesis pathway: The microrna biogenesis pathway is a cellular process that generates microRNAs (miRNAs) from primary transcripts, leading to the regulation of gene expression at the post-transcriptional level. This pathway involves several key steps including transcription, processing by specific enzymes, and incorporation into the RNA-induced silencing complex (RISC), which ultimately leads to target mRNA degradation or repression.
MiRNA: miRNA, or microRNA, is a small, non-coding RNA molecule that plays a crucial role in the regulation of gene expression at the post-transcriptional level. By binding to complementary sequences on target messenger RNAs (mRNAs), miRNAs can inhibit translation or lead to mRNA degradation, effectively fine-tuning the protein output of genes and influencing various cellular processes.
Northern blotting: Northern blotting is a laboratory technique used to detect specific RNA molecules within a complex mixture. This method allows researchers to analyze RNA expression levels, identify RNA size, and assess RNA processing events, connecting it to post-transcriptional regulation mechanisms like RNA interference and microRNAs as well as various types of RNA in biological systems.
Post-transcriptional modification: Post-transcriptional modification refers to the various biochemical processes that occur on RNA molecules after transcription, ultimately influencing gene expression and RNA stability. These modifications include capping, polyadenylation, and splicing, which help mature the precursor mRNA into functional mRNA that can be translated into proteins. Additionally, this term is closely associated with regulatory mechanisms such as RNA interference and the role of microRNAs in controlling gene expression.
Pre-miRNA: Pre-miRNA, or precursor microRNA, is a short, hairpin-shaped RNA molecule that serves as an intermediate form in the biogenesis of microRNAs. It is synthesized in the nucleus from primary miRNA (pri-miRNA) transcripts and is essential for the subsequent processing into mature miRNA, which plays a key role in post-transcriptional regulation of gene expression.
RISC Complex: The RISC complex, or RNA-induced silencing complex, is a multi-protein complex that plays a crucial role in the gene regulation process known as RNA interference (RNAi). This complex is responsible for guiding small interfering RNAs (siRNAs) and microRNAs (miRNAs) to their target mRNA molecules, leading to post-transcriptional gene silencing by either degrading the mRNA or inhibiting its translation. The RISC complex is essential for regulating gene expression, maintaining cellular homeostasis, and defending against viral infections.
RNA interference: RNA interference (RNAi) is a biological process in which small RNA molecules inhibit gene expression or translation by targeting specific mRNA molecules for degradation. This mechanism is crucial for regulating gene expression after transcription, impacting various cellular processes and playing a significant role in development, defense against viruses, and maintaining genomic stability.
Rna stability: RNA stability refers to the lifespan and degradation rate of RNA molecules within a cell, which significantly affects gene expression and regulation. The stability of RNA is crucial because it determines how long an RNA molecule can function before being degraded, influencing the amount of protein produced. Factors that affect RNA stability include sequences in the RNA itself, interactions with proteins, and mechanisms like RNA interference and the action of microRNAs.
Rt-qpcr: rt-qpcr, or reverse transcription quantitative polymerase chain reaction, is a laboratory technique used to measure the amount of specific RNA in a sample by first converting it into complementary DNA (cDNA) through reverse transcription and then amplifying that cDNA to quantify the RNA levels. This method is particularly significant for studying gene expression and the roles of RNA in various biological processes, including post-transcriptional regulation through mechanisms such as RNA interference and microRNAs.
Seed sequence: A seed sequence refers to a short, conserved nucleotide sequence that is crucial for the functionality of microRNAs and their role in post-transcriptional regulation. These sequences typically bind to complementary sites in target messenger RNAs (mRNAs), leading to mRNA degradation or inhibition of translation, thereby controlling gene expression. The precise pairing between the seed sequence and its target is fundamental for the specificity of microRNA action in various biological processes.
SiRNA: siRNA, or small interfering RNA, is a class of double-stranded RNA molecules that play a crucial role in the process of RNA interference (RNAi). These molecules are typically 20-25 nucleotides long and function by specifically targeting and degrading messenger RNA (mRNA) transcripts, leading to a decrease in the expression of certain genes. siRNA is essential for post-transcriptional regulation, as it helps control gene expression and can be used for gene silencing in various biological processes.
SiRNA pathway: The siRNA pathway is a biological process involving small interfering RNAs (siRNAs) that play a crucial role in the regulation of gene expression and RNA interference (RNAi). This pathway is essential for the degradation of messenger RNA (mRNA) and is activated by double-stranded RNA (dsRNA), leading to the silencing of specific genes. It serves as a vital mechanism in post-transcriptional regulation, allowing cells to respond to viral infections and regulate cellular processes by controlling which genes are expressed.
Small interfering RNA: Small interfering RNA (siRNA) is a class of double-stranded RNA molecules, typically 20-25 nucleotides long, that play a crucial role in the regulation of gene expression through a process called RNA interference (RNAi). siRNAs are involved in post-transcriptional regulation by targeting specific messenger RNAs (mRNAs) for degradation, thereby preventing the translation of those mRNAs into proteins. This mechanism is essential for controlling gene expression, maintaining cellular processes, and defending against viral infections.
Target mRNA degradation: Target mRNA degradation refers to the process by which specific messenger RNA (mRNA) molecules are selectively degraded to regulate gene expression. This mechanism is crucial in controlling the levels of proteins synthesized in cells, ensuring that only the necessary proteins are produced at the right time. It plays a significant role in post-transcriptional regulation, particularly through processes such as RNA interference and the action of microRNAs.
Translational repression: Translational repression is a regulatory mechanism that prevents the synthesis of proteins by inhibiting the translation of messenger RNA (mRNA) into proteins. This process plays a crucial role in controlling gene expression, ensuring that proteins are produced only when necessary, and is significantly influenced by factors like RNA interference and microRNAs, which can bind to mRNA and block its translation or degrade it.