Small RNAs
Small RNAs
Definition and Overview
Small RNAs are approximately 20-30 nucleotides (nt) in length.
Known as small non-coding RNAs or small regulatory RNAs.
They play a critical role in inhibiting gene expression.
Regulatory mechanisms mediated by small RNAs are referred to as RNA silencing or RNA interference (RNAi).
Categories of Endogenous Small RNAs
Belong to three major categories:
miRNAs (microRNAs)
Found in animals, plants, and fungi.
siRNAs (short interfering RNAs)
Found in animals, plants, and fungi.
piRNAs (piwi-interacting RNAs)
Found in animal germlines.
miRNAs
Biogenesis of miRNAs
miRNAs are encoded by the genome and are often found in clusters.
They are transcribed as primary-miRNA (pri-miRNAs) with tissue-specific expression patterns.
In the nucleus:
Pri-miRNA is cleaved by the Drosha enzyme from the microprocessor complex to form pre-miRNA.
Pre-miRNA is exported to the cytoplasm and processed further by Dicer to form mature double-stranded miRNAs (~22 bp).
Both Drosha and Dicer are ribonucleases (RNases).
miRBase: miRNA Database
miRBase contains entries from 271 organisms.
As of release v22, the human genome has:
1917 annotated hairpin precursors
2654 mature sequences
Over 500 are annotated with high confidence.
Methods to Detect and Measure miRNA
Microarray-based profiling
High throughput, semi-quantitative.
Next Generation Sequencing (miRNA sequencing)
High throughput, quantitative.
RT-PCR
Sensitive, quantitative, but not high-throughput.
Loading of Mature miRNA onto Argonaut Protein
Mature miRNA is double-stranded.
One strand (the guide strand) associates stably with Ago (a key component of RISC).
The other strand (the passenger strand, denoted miRNA*) is discarded.
Guide strand selection is dictated by the relative thermodynamic stabilities of the duplex ends.
The sequence of the miRNA provided in databases is that of the guide strand, while the passenger strand is marked with an asterisk (*).
Targeting Mechanism of miRNAs
The miRNA guide strand binds to the Ago protein in RISC.
RISC: RNA-induced silencing complex that contains the guide strand, Ago protein, and other accessory proteins.
Target mRNAs are recognized through partial base-pairing with the miRNA sequence.
miRNAs silence gene expression by inhibiting translation and/or promoting mRNA degradation.
miRNA Target Recognition
miRNA target sites are typically located in the 3'UTR of mRNAs.
The crucial base-pairing region between miRNA and target mRNA is the 6mer seed found in the 5' region of the miRNA.
6mer seed sites exhibit limited efficacy.
7mer and 8mer sites are stronger and more likely to produce silencing effects.
Rare sites may include 3' supplementary pairing, slightly improving effectiveness, and 3'-compensatory sites, which can compensate for mismatches in the seed region.
Predicting miRNA Target Sites
Evolutionary conservation of target binding sites is not considered in all prediction models (e.g., miRDB).
TargetScan gives more weight to conserved target sites in evolutionary terms.
Challenges in Target Site Prediction
For metazoan miRNAs, a challenge is to capture most target sites without too many false predictions.
Target Regulation Statistics
Each miRNA can have many target mRNAs, with mammalian miRNAs averaging around 300 conserved targets each.
Conversely, each mRNA may be regulated by many miRNAs.
Searching for Functionally Relevant miRNA Targets
Computational predictions are used to identify genes potentially targeted based on sequence complementarity to the miRNA.
Experimental examination includes:
Checking miRNA and target mRNA expression in specific cells/tissues (high or moderate expression indicates likely functional impact).
Overexpressing miRNA (miRNA mimics) to see if mRNA targets are downregulated.
Inhibiting miRNA (antisense oligos, miRNA sponges) to check for mRNA upregulation.
Removing target sites from mRNA to see if regulation is retained.
Using reporters with miRNA target sites in the 3'UTR.
Reagents for Studying miRNA Function
miRNA mimics: Synthetic oligonucleotides with sequences matching mature miRNA; induce miRNA effects when introduced into cells.
Antisense oligonucleotides: Bind to the miRNA guide strand, inhibiting miRNA function.
miRNA sponges: Contain multiple tandem binding sites for a miRNA, blocking its function.
miRNA and Gene Expression
The human genome has over 2,000 mature miRNA sequences.
More than 60% of human protein-coding genes harbor predicted miRNA target sites.
Deletion of Dicer and Drosha is embryonically lethal in mice.
miRNA function deregulation is associated with various diseases, particularly cancer, with overall downregulation observed in most cancers.
Pleiotropic Effects of miRNA Regulation
A single miRNA can target many genes, some with opposing effects on the same cellular process. Their effect is contextual, based on cellular conditions.
miRNAs often exert only mild effects on gene expression but may lead to repression of multiple main targets, thereby regulating various facets of cellular processes.
Role in Cancer
miRNAs can act as oncogenes (oncomirs) or tumor suppressors.
Oncogenes: Encourage tumorigenesis.
Tumor suppressor genes: Inhibit cell proliferation and tumor progression.
siRNAs
Sources of siRNAs
Initially, natural siRNAs were believed to be exogenous (from viral RNA), functioning primarily to combat environmental threats.
Advancements in sequencing have identified endogenous sources that produce siRNAs, though their biological roles remain not fully understood.
siRNA Induction into RISC
siRNAs are generated from Dicer cleavage.
The guide strand associates with RISC and checks with Argonaut protein, while the passenger strand is degraded.
Mechanism of siRNA-Induced Silencing
With perfect complementarity between the siRNA guide strand and target mRNA, cleavage occurs by the Ago protein within the duplex, followed by degradation of the resultant mRNA fragments, regenerating RISC. This method is referred to as canonical siRNA-induced silencing.
Mismatches and Off-target Effects
If there are mismatches between the guide strand and target mRNA, siRNA may act similarly to miRNA, suppressing gene function via translational inhibition and transcript degradation.
One siRNA can inhibit multiple mRNAs with partial sequence complementarity to its seed region, leading to potential off-target effects of siRNA interference.
Comparative Gene Silencing Mechanisms
Both siRNAs and miRNAs are specificity factors that recognize target mRNA through base-pairing, facilitated by the Argonaute protein, which induces silencing:
Extensive sequence complementarity (siRNA) can cause mRNA cleavage.
Sequencing complementarity in the seed region may lead to mRNA degradation and translational repression.
Applications of RNAi in Research
RNA interference technology facilitates manipulation of gene expression in genetically challenging systems for modifying DNA.
siRNA Reagents for Gene Knockdown
Commercially available siRNAs are chemically synthesized RNA duplexes targeting specific mRNA sequences, typically around 21 nt in length.
They are introduced into cultured cells through a process called transfection.
Gene silencing via siRNAs is transient, often restricted to 2-4 days post-transfection, and can prompt off-target effects, silencing genes with partial complementarity.
Mitigating Off-target Effects
Methods to increase targeting specificity and reduce off-target effects include:
Careful siRNA design to avoid high-frequency miRNA seed sequences.
Pooling multiple siRNAs targeting the same gene to reduce individual siRNA concentrations.
Chemical modifications.
Chemical Modifications for Increased Stability
Common oligonucleotide modifications include:
Locked Nucleic Acid (LNA): Enhances hybridization affinity.
Phosphorothioate modifications: Increase stability.
2'-O-Methyl modifications.
Long-term Gene Silencing with shRNAs
shRNAs (small hairpin RNAs) induce sustained gene silencing by:
Introducing shRNA genes into cells via viral vectors for stable genome integration.
Transcription of shRNA genes to produce siRNAs through miRNA processing machinery.
siRNA vs. shRNA
siRNA (short-term):
Transiently present in cytoplasm.
Suitable for quick and short-term gene knockdown.
Cannot offer permanent silencing of essential genes.
shRNA (long-term):
Stably integrated into the genome, offering long-term effects.
Overcomes transfection challenges in hard-to-transfect cell types.
Expression controlled through inducible promoters.
Typically more complex and time-consuming to work with.
High-throughput Cell-based RNAi Screening
Use of plate-based siRNA or shRNA libraries to screen for targeting effects.
Each well contains siRNA or shRNA aimed at individual genes.
Genome sequences have enabled construction of extensive RNAi reagent libraries for high-throughput screening.
Screening Validation Steps:
Confirm gene knockdown.
Utilize varying siRNAs to check reproducibility of phenotypes.
Employ functional complementation assays to verify gene effects (resistant versions of genes).
Conduct other pathway-specific assays.
dsRNA-Mediated Interference Across Cellular Boundaries
Injection studies showed silencing effects not only in injected organisms but also in progeny, suggesting induction reaching germ cells and cross-cellular interference.
siRNA Amplification
RNA-dependent RNA polymerase (RdRP): Converts target mRNAs into new dsRNAs, amplifying initial siRNAs and facilitating sustained silencing that may transfer to progeny.
Observed generally in eukaryotes apart from mammals and insects.
Therapeutic Applications of RNAi
RNA-induced gene silencing has promising therapeutic implications, surpassing conventional drugs in flexibility.
Challenges in therapeutic RNAi include appropriate delivery systems, stability enhancement via chemical modifications, and reduced off-target effects:
miRNA-based drugs currently lack successful Phase II trial outcomes; three are in Phase I trials, often terminated due to adverse effects and inefficacy.
siRNA-based drugs: By 2023, the FDA has approved three siRNA-based medications (patisiran, givosiran, and lumasiran) for treating non-cancerous conditions after positive Phase III studies with minimal side effects. Eight additional drugs are in Phase III, and sixteen in Phase II trials.