RNA interference animation

Summary

The video script delves into RNA interference, a process popularized by experiments involving double-stranded RNA in worms. It explains how RNA polymerase generates pre-mRNAs, which are processed into mature mRNAs and translated in the cytoplasm. The script highlights the role of microRNAs and RNA silencing pathways in gene regulation, antiviral defense, and the creation of small interfering RNAs (siRNAs) by Dicer. It also discusses the RNA-induced silencing complex (RISC), which cleaves target mRNAs, and the amplification mechanisms involving RNA-dependent RNA polymerase (RdRP), crucial for antiviral responses and gene regulation in organisms like plants, fungi, and worms.

Takeaways

• 🧬 RNA interference is a process where double-stranded RNA (dsRNA) is used to silence genes in a sequence-specific manner.
• 🐛 The technique was popularized by injecting dsRNA into a worm's gonad, which is a common method for introducing transgenes in worms.
• 🛡️ dsRNA blocks the expression of endogenous genes, which is an important tool for studying gene function.
• 📝 Gene transcription by RNA polymerase II generates pre-mRNAs that are processed into mature mRNAs, which are then transported to the cytoplasm for translation.
• 🔄 RNA can regulate endogenous gene expression through mechanisms involving microRNAs and other short regulatory RNAs.
• 🌿 In plants and other organisms, RNA can be activated by endogenous transposition and plays a role in antiviral defense.
• 🔪 Dicer, a member of the RNase III family, cleaves dsRNA into small interfering RNAs (siRNAs) with two-nucleotide 3' overhangs.
• 🤝 siRNAs form a ribonucleoprotein complex called RISC, which includes an argonaute protein with an RNAse H-like domain.
• ⚔️ RISC mediates the unwinding of the siRNA duplex, allowing a single-stranded siRNA to bind to and cleave target mRNA in a sequence-specific manner.
• 🚫 Cleaved mRNA is recognized as aberrant and destroyed, preventing translation and silencing gene expression.
• 🌱 In plants, aberrant RNA from RISC-mediated cleavage can serve as a template for RNA-dependent RNA polymerase (RdRP), generating more siRNAs.
• 🔄 Some organisms have an amplification step involving single-stranded siRNAs binding to target mRNAs and serving as primers for RdRP to synthesize the antisense RNA strand.
• 🌱 RNA spreading between cells is suggested to underlie germline transmission of RNA in worms and has been described in plants.
• 🐇 RNA spreading has not been described in mammals, indicating differences in RNA interference mechanisms across species.

Q & A

What is RNA interference and how was it popularized?

RNA interference is a biological process where RNA molecules regulate gene expression. It was popularized by the work of researchers who injected long double-stranded RNAs into a worm's gonad, which blocked the expression of endogenous genes in a sequence-specific manner.

How do genes get transcribed in the cell?

Genes are transcribed by RNA polymerase II, which generates pre-mRNAs that are then processed to form mature mRNAs.

What is the role of mRNA in the cell?

mRNAs are transported from the nucleus to the cytoplasm where they serve as templates for protein synthesis through the process of translation.

What are microRNAs and how do they regulate gene expression?

MicroRNAs are genomically encoded short regulatory RNAs that can regulate endogenous gene expression in organisms such as algae, worms, and flies.

How does RNA play a role in antiviral defense?

In antiviral defense, double-stranded RNA from viruses is targeted for destruction by the RNA machinery, preventing viral replication and spread.

What is Dicer and how does it function in RNA interference?

Dicer is a member of the RNase III family of double-stranded RNA-specific endonucleases. It recognizes and cleaves long double-stranded RNAs into small interfering RNAs (siRNAs) with two-nucleotide long 3' overhangs.

What is the RNA-induced silencing complex (RISC) and what does it include?

The RNA-induced silencing complex (RISC) is a ribonucleoprotein complex that includes Slicer, an Argonaute protein with an RNA-binding domain. It mediates the unwinding and cleavage of target mRNAs.

How does the RISC complex mediate mRNA cleavage?

RISC first mediates the unwinding of the siRNA duplex, and then a single-stranded siRNA that is coupled to RISC binds to a target mRNA in a sequence-specific manner. This binding mediates target mRNA cleavage by the Slicer protein.

What happens to the cleaved mRNA and how does it lead to gene silencing?

The cleaved mRNA is recognized by the cell as aberrant and is destroyed, preventing translation from occurring and thus silencing the expression of the gene from which the mRNA was transcribed.

How does the RNA-dependent RNA polymerase (RdRP) contribute to RNA interference?

RdRP uses the aberrant RNA resulting from RISC-mediated cleavage as a template for unprimed RNA synthesis, generating more double-stranded RNA, which serves as a substrate for Dicer activity, producing more siRNAs.

What is the significance of RNA spreading in the context of RNA interference?

RNA spreading is a process that allows the RNA interference signal to move between cells, which is thought to underlie the germline transmission of RNA in worms and has also been described in plants.

Outlines

00:00

🧬 RNA Interference and Gene Regulation

This paragraph delves into the discovery and mechanism of RNA interference, a process that silences gene expression. It starts with the historical context of its discovery through the injection of double-stranded RNAs into a worm's gonad, which led to the blockage of endogenous gene expression. The paragraph explains the role of RNA polymerase II in generating pre-mRNAs, which are processed into mature mRNAs. It further discusses how microRNAs and other small regulatory RNAs can activate RNA interference. The central mechanism involves the cleavage of double-stranded RNAs by Dicer, an enzyme that generates small interfering RNAs (siRNAs) with two-nucleotide overhangs. These siRNAs form part of the RNA-induced silencing complex (RISC), which includes the 'slicer' protein. The RISC unwinds the siRNA duplex, and a single strand binds to a target mRNA, leading to its cleavage and subsequent destruction, thereby preventing translation and silencing the gene. The paragraph also touches on the role of RNA-dependent RNA polymerase (RdRP) in creating aberrant RNA from the cleaved mRNA, which can generate more siRNAs, and the amplification and spreading of RNA interference in organisms like plants and worms, but not in mammals.

Keywords

💡RNA interference

RNA interference, or RNAi, is a biological process in which RNA molecules regulate the activity of other genes. It is central to the video's theme as it discusses how this mechanism is triggered by double-stranded RNAs and leads to gene silencing. The script mentions that RNAi was popularized by experiments where long double-stranded RNAs were introduced into a worm's gonad, blocking gene expression in a sequence-specific manner.

💡Double-stranded RNAs (dsRNAs)

Double-stranded RNAs are RNA molecules composed of two complementary strands. In the context of the video, dsRNAs are used to initiate RNA interference. The script explains that when long dsRNAs are introduced into a cell, they are cleaved by Dicer, resulting in small interfering RNAs (siRNAs) that are key to the RNAi process.

💡Dicer

Dicer is an enzyme that plays a crucial role in RNA interference by cleaving long double-stranded RNAs into smaller pieces known as small interfering RNAs (siRNAs). The script describes Dicer as a member of the RNase III family of double-stranded RNA-specific endonucleases, which is essential for the formation of siRNAs that mediate gene silencing.

💡Small interfering RNAs (siRNAs)

siRNAs are short double-stranded RNAs that are generated from longer dsRNAs by the action of Dicer. They are characterized by two-nucleotide long 3' overhangs. The script highlights that siRNAs form a complex with proteins, known as the RNA-induced silencing complex (RISC), which is involved in the cleavage and subsequent degradation of target mRNAs.

💡RNA-induced silencing complex (RISC)

RISC is a ribonucleoprotein complex that includes proteins and siRNAs. It is pivotal to the RNAi mechanism as it mediates the unwinding of the siRNA duplex and the subsequent binding to target mRNAs. The script explains that RISC, with the help of the 'slicer' protein, cleaves the target mRNA in a sequence-specific manner, leading to gene silencing.

💡Slicer

Slicer is an argonaute protein with an RNA-binding domain that is part of the RISC complex. It is responsible for cleaving the target mRNA when bound to the siRNA. The script illustrates that the cleavage site is determined by the region of complementarity between the siRNA and the target mRNA, which is essential for the specificity of gene silencing.

💡Gene silencing

Gene silencing refers to the process by which the expression of a specific gene is prevented or reduced. In the video, gene silencing is achieved through RNA interference, where the cleavage of target mRNAs by RISC prevents their translation into proteins. The script provides the example of how aberrant mRNAs, resulting from RISC-mediated cleavage, are recognized and destroyed by the cell.

💡RNA-dependent RNA polymerase (RdRP)

RdRP is an enzyme that synthesizes RNA using an RNA template, a process that is crucial for the amplification of the RNAi response. The script describes how, in plants, the aberrant RNA resulting from RISC-mediated cleavage can serve as a template for RdRP, leading to the production of more dsRNA and, subsequently, more siRNAs through Dicer activity.

💡Amplification

Amplification in the context of RNAi refers to the process by which the initial dsRNA triggers the production of more siRNAs, thereby enhancing the silencing effect. The script explains that this is achieved through the action of RdRP and Dicer, which generate additional siRNAs from the aberrant RNA template.

💡RNA spreading

RNA spreading is the process by which the RNAi signal is transmitted between cells. The script mentions that this phenomenon has been described in plants and worms, suggesting a systemic response to the presence of dsRNAs. It is part of the broader theme of how RNAi can have widespread effects on gene expression within an organism.

💡Endogenous RNA mechanisms

Endogenous RNA mechanisms refer to the intrinsic RNA-based regulatory processes within an organism. The script points out that in some organisms, such as fungi, plants, worms, and mammals, RNA plays a role in regulating gene expression, including through processes like RNAi and the action of microRNAs.

Highlights

RNA interference was popularized by the injection of long double-stranded RNAs into a worm's gonad.

This method blocked the expression of endogenous genes in a sequence-specific manner.

Gene transcription by RNA polymerase II generates pre-mRNAs that are processed into mature mRNAs.

mRNAs are transported from the nucleus to the cytoplasm for translation.

RNA can regulate endogenous gene expression through microRNAs in various organisms.

RNA can be activated by endogenous transposition in plants and insect cells.

RNA plays a role in antiviral defense by targeting viral double-stranded RNA for destruction.

Long double-stranded RNAs are recognized and cleaved by Dicer, a member of the RNAse III family.

Cleavage by Dicer creates small interfering RNAs (siRNAs) with two-nucleotide long 3' overhangs.

siRNAs form the RISC (RNA silencing complex), which includes the Slicer protein.

RISC mediates the unwinding of the siRNA duplex and targets mRNA for cleavage.

The cleaved mRNA is recognized as aberrant and destroyed, preventing translation.

In plants, aberrant RNA from RISC-mediated cleavage serves as a template for RNA-dependent RNA polymerase (RdRP).

RdRP uses the aberrant RNA template for unprimed RNA synthesis, generating more siRNAs.

RNA interference involves an amplification step with single-stranded siRNAs priming RdRP.

The specificity of RNA interference is sensitive to natural sequence variation.

RNA spreading between cells is thought to underlie germline transmission of RNA in worms.

RNA spreading has been described in plants but not in mammals.

Transcripts

RNA interference was popularized by work

in

cagans when long double stranded rnas

were injected into a worm's gonad a

standard way of introducing trans genes

into worms they blocked the expression

of endogenous genes in a sequence

specific

manner in UK cariot most protein coating

genes are transcribed by RNA polymerase

2 which generates pre-mrnas that are

then processed to form mature

mrnas these mrnas are then transported

from the nucleus to the cytoplasm where

they are

translated RNA I can regulate endogenous

gene

expression

RNA can be set in motion by genomically

encoded short regulatory rnas known as

micro

rnas in algae worms and flies RNA can be

activated by endogenous

transposition in plants and cultured

insect cells RNA also has a role in

antiviral defense in which viral doubl

stranded RNA

are targeted for Destruction by the RNA

Machinery when long doubl stranded rnas

enter a cell they are recognized and

cleaved by daiser which is a member of

the rna's 3 family of double stranded

RNA specific

endonucleases cleavage by daer creates

short double stranded rnas that are

characterized by two nucleotide long

three prime

overhangs these are called small

interfering or si

rnas si rnas can form a

ribonucleoprotein complex called risk or

RNA silencing complex this complex

includes slicer an argonut protein with

an RNA H like domain called

peeee risk first mediates the unwinding

of the sna duplex a single stranded sna

that is coupled to risk then binds to a

Target mRNA in a sequent specific

manner The Binding mediates Target mRNA

cleavage by slicer the sight of the

cleavage falls in the middle of the

region of sirna

complementarity the cleaved mRNA can be

recognized by the cell as being aberant

and then destroyed this prevents

translation from occurring silencing the

expression of the gene from which which

the MRNA was

transcribed in Plants the aberant RNA

that results from the risk mediated

cleavage can also serve as a template

for RNA dependent RNA polymerase or

rdrp this process relies on unprimed RNA

synthesis in which the aberant RNA is

used as a

template the resulting double stranded

RNA is a substrate for dacer activity

which generates more si rnas in some

organisms with endogenous RNA mechanisms

for example fungi plants worms and

mammals RNA also involves another

amplification

step in this step singl stranded sirnas

not associated with risk bind to their

Target mrnas in a sequent specific way

and serve as a primer for rdrp to

polymerize the antisense RNA

strand such specificity is intrinsically

sensitive to Natural sequence

variation the double stranded RNA

molecule that is created serves as a

substrate for daiser which cuts it into

snas in turn these can either unwind and

Prime RNA dependent RNA

polymerization or together with risk

mediate the cleavage of Target

mrnas this amplification coupled with

RNA spreading between cells is thought

to underline germline transmission of

RNA in worms RNA spreading has also been

described in Plants but not in

mammals