Generation and action of siRNAs and miRNAs

Summary

This script delves into the intricate world of small regulatory RNAs, focusing on microRNAs (miRNAs) and small interfering RNAs (siRNAs). It explains their origin, processing, and function in gene regulation. miRNAs originate from genes, undergo nuclear processing by Drosha and Dicer, and form the RISC complex to silence target mRNAs. siRNAs, derived from double-stranded RNA, follow a similar path but differ in their perfect complementarity and cleavage mechanism. Both types of small RNAs play crucial roles in controlling gene expression and are essential for understanding post-transcriptional regulation.

Takeaways

• 🌟 MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are two types of small regulatory RNAs that play crucial roles in gene regulation by affecting mRNA stability and translation.
• 🌿 miRNAs originate from actual genes found in the genomes of multicellular animals and plants, while siRNAs are derived from double-stranded RNA from various sources, including endogenous and exogenous origins.
• 📜 Primary miRNA transcripts, known as pri-miRNAs, are transcribed by RNA Polymerase II and fold into a stem-loop structure that is processed into a precursor miRNA (pre-miRNA) by the Drosha-DGCR8 microprocessor complex.
• 🚀 The pre-miRNA is exported to the cytoplasm where it undergoes further processing by Dicer, an RNase III enzyme, to generate a miRNA duplex with overhangs and monophosphates.
• 🔄 The miRNA duplex is loaded into the Argonaute protein, where one strand, the guide strand, is retained and the other, the passenger strand, is removed through a sorting process that is not fully understood.
• 🎯 The Argonaute-miRNA complex, known as the RISC (RNA-induced silencing complex), targets mRNAs with imperfect complementarity, particularly in the 3' untranslated regions (UTRs), guided by the 'seed' sequence of the miRNA.
• 🛡 miRNAs typically do not cleave their target mRNAs but instead recruit factors like TRBP (TNRC6 in humans) to repress translation and destabilize the mRNA, without relying on the slicer activity of Argonaute.
• 🧬 In contrast to miRNAs, siRNAs are fully complementary duplexes that, after being processed by Dicer, are loaded into Argonaute proteins where the passenger strand is cleaved by the endonuclease activity of the protein's PAZ domain.
• 🔍 siRNA-Argonaute complexes scan for and bind to fully complementary target RNAs, leading to the activation of the PAZ domain's cleavage activity and slicing of the target mRNA.
• ♻️ The products of slicing are thought to be immediately targeted for degradation by the exosome and other RNA decay pathways, thereby silencing the gene expression.
• 🔗 Both miRNAs and siRNAs associate with Argonaute proteins to mediate gene silencing, but they differ in their origin, processing pathway, targets, and mechanism of action.

Q & A

What are the two types of small regulatory RNAs mentioned in the script?

The two types of small regulatory RNAs mentioned are microRNAs (miRNAs) and small interfering RNAs (siRNAs).

What is the role of Argonaut proteins in relation to small RNAs?

Argonaut proteins are associated with both miRNAs and siRNAs and play a crucial role in mediating their regulatory functions, such as gene silencing.

How do miRNAs originate in the cell?

miRNAs originate from actual genes found in the genomes of multicellular animals and plants. They begin as primary transcripts or pri-miRNAs, which are transcribed by RNA polymerase II.

What is the function of the microprocessor complex in miRNA processing?

The microprocessor complex, consisting of Drosha and DGCR8, performs a cleavage reaction that removes the five prime and three prime extensions from the pri-miRNA, producing a precursor miRNA (pre-miRNA).

How is the pre-miRNA transported to the cytoplasm for further processing?

The pre-miRNA is recognized by Exportin 5, a nuclear export factor, which transports it to the cytoplasm for subsequent processing.

What enzyme is responsible for the second cleavage reaction of pre-miRNAs in the cytoplasm?

The second cleavage reaction, referred to as dicing, is catalyzed by Dicer, an RNase III enzyme.

What are the two strands of the miRNA duplex called after the second cleavage reaction?

The two strands of the miRNA duplex are called the guide strand and the passenger strand.

How does the miRNA guide strand determine which target RNAs will be silenced?

The miRNA guide strand, once loaded into the Argonaut protein, identifies target RNAs with imperfect complementarity, particularly in the 3' UTRs of mRNAs, through a region known as the seed sequence.

What is the difference between miRNAs and siRNAs in terms of their origin?

miRNAs are encoded in the genome as specific genes, while siRNAs are derived from double-stranded RNA that can come from various sources, including endogenous duplex RNA or exogenous sources like viral RNAs.

How does the mechanism of gene silencing differ between miRNAs and siRNAs?

miRNAs typically promote gene silencing through translational repression and mRNA destabilization without cleaving the target RNA, whereas siRNAs lead to the cleavage of fully complementary target RNAs, a process known as slicing.

What is the role of the TRBP protein in miRNA-mediated gene silencing?

The TRBP protein, which contains multiple glycine-tryptophan repeats, is thought to be involved in repressing translation and destabilizing mRNA, although the exact mechanisms are not fully understood.

Outlines

00:00

🧬 MicroRNA Biogenesis and Function

This paragraph delves into the intricate world of microRNAs (miRNAs), a class of small regulatory RNAs that play a pivotal role in gene expression regulation. Originating from actual genes in multicellular animals and plants, miRNAs begin as primary transcripts, transcribed by RNA polymerase II. They undergo a series of processing steps, starting with the microprocessor complex, which includes Drosha and DGCR8, to form a pre-miRNA. This is then exported to the cytoplasm and further processed by Dicer, resulting in a miRNA duplex. The guide strand of this duplex is selected for loading into the Argonaute protein, forming the RISC complex, which is responsible for gene silencing by binding to target mRNAs, typically in their 3' UTRs, through imperfect complementarity, especially within the 'seed' sequence. The mechanism of gene silencing involves the recruitment of additional factors like TRBP, which may repress translation and destabilize mRNA, although the exact mechanisms are not fully understood.

05:01

🧬 siRNA Processing and Gene Silencing

The second paragraph focuses on small interfering RNAs (siRNAs), another class of small regulatory RNAs that differ from miRNAs in their origin and processing. siRNAs are derived from double-stranded RNA, which can be of endogenous or exogenous origin, such as from viruses or experimental introduction. Unlike miRNAs, siRNAs require only the Dicer enzyme for processing in the cytoplasm, which cleaves the duplex RNA into short, fully base-paired duplexes. These siRNA duplexes are then loaded into the Argonaute protein, leading to the selective removal of the passenger strand by the endonuclease activity of the Ago protein's PIWI domain. The resulting single-stranded siRNA-Ago complexes scan for fully complementary target RNAs, leading to cleavage, or 'slicing,' of the target mRNA. This cleavage is facilitated by conformational changes in the RNA's H-PIWI domain upon binding, activating its cleavage activity. The products of this slicing reaction are thought to be rapidly degraded by cellular RNA decay pathways, such as the exosome.

Keywords

💡MicroRNAs (miRNAs)

MicroRNAs, or miRNAs, are a class of small, non-coding RNAs that play a crucial role in the regulation of gene expression. They are derived from actual genes found in the genomes of multicellular animals and plants. In the video, miRNAs are described as starting as primary transcripts, which are processed into mature miRNAs through several steps involving specific enzymes and proteins. They are essential for understanding the theme of gene regulation as they influence mRNA stability and translation.

💡Small Interfering RNAs (siRNAs)

Small interfering RNAs, or siRNAs, are another class of small regulatory RNAs that are involved in the RNA interference (RNAi) pathway. Unlike miRNAs, siRNAs are not encoded as specific genes but are derived from double-stranded RNA of various origins, including exogenous sources like viral RNA. The script explains that siRNAs are processed by the enzyme Dicer, leading to the formation of duplexes that are fully complementary. They are central to the video's theme as they illustrate an alternative mechanism of gene regulation through RNAi.

💡Argonaute proteins

Argonaute proteins are a family of proteins that associate with both miRNAs and siRNAs. They play a key role in the RNA silencing complex by binding to the small RNAs and guiding the complex to its target mRNA. The script mentions that miRNAs and siRNAs differ in their origin and processing but associate with Argonaute proteins, which is critical for understanding the mechanisms of gene regulation discussed in the video.

💡Drosha

Drosha is a nuclear RNAse III enzyme that is part of the 'microprocessor complex' involved in the initial processing of primary miRNA transcripts. It works in conjunction with the DGCR8 protein to cleave the transcript, generating a precursor miRNA. The script describes Drosha's role in the biogenesis of miRNAs, which is essential for understanding the processing pathway of miRNAs.

💡DGCR8

DGCR8, or DiGeorge syndrome critical region gene 8, is an RNA-binding protein that interacts with Drosha to form the microprocessor complex. It is essential for the first step of miRNA processing, where it helps Drosha to cleave the primary miRNA transcript. The script uses DGCR8 as an example of the specialized proteins required for miRNA maturation, highlighting its importance in gene regulation.

💡Exportin-5

Exportin-5 is a nuclear export factor responsible for transporting pre-miRNAs from the nucleus to the cytoplasm. The script mentions Exportin-5 in the context of trafficking pre-miRNAs for further processing, which is a critical step in miRNA biogenesis and an important aspect of the video's theme on gene regulation.

💡Dicer

Dicer is an RNase III endonuclease enzyme that processes both pre-miRNAs and long duplex RNAs into mature miRNAs and siRNAs, respectively. The script describes Dicer's role in the 'dicing' reaction, which is the second cleavage step for miRNAs and the sole processing step for siRNAs, emphasizing its central role in small RNA pathways.

💡TRBP2

TRBP2, or TAR RNA-binding protein 2, is a double-stranded RNA binding protein that forms a complex with Dicer. It is involved in the second cleavage reaction that generates the miRNA duplex. The script explains TRBP2's function in the miRNA processing pathway, which is integral to the video's discussion on miRNA biogenesis.

💡Seed sequence

The seed sequence is a term used to describe the 2-8 nucleotide region at the 5' end of a miRNA, which is crucial for target recognition and binding. The script explains that Argonaute proteins bound to miRNAs typically identify sequences with imperfect complementarity in the 3' UTRs of mRNAs, with the seed sequence being the most important pairing region. This concept is fundamental to understanding how miRNAs regulate gene expression.

💡Gene silencing

Gene silencing refers to the process by which the expression of a gene is suppressed, either at the transcriptional or post-transcriptional level. In the context of the video, gene silencing is achieved through the action of miRNAs and siRNAs, which bind to their target mRNAs and inhibit their translation or promote their degradation. This concept is central to the video's theme of RNA-based regulation of gene expression.

💡RNAi

RNA interference, or RNAi, is a biological process in which small RNA molecules, such as siRNAs, regulate gene expression by silencing specific genes. The script discusses RNAi in the context of siRNAs, which are part of this pathway. Understanding RNAi is key to grasping the mechanisms of post-transcriptional gene regulation highlighted in the video.

Highlights

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are two families of small regulatory RNAs that regulate mRNA stability and translation in eukaryotes.

Both miRNAs and siRNAs associate with Argonaut proteins but differ in their origin, processing pathway, targets, and mechanism of action.

miRNAs originate from actual genes found in the genomes of multicellular animals and plants.

Primary miRNA transcripts are transcribed by RNA polymerase II and fold into a stem-loop structure.

The microprocessor complex, composed of Drosha and DGCR8, processes the primary miRNA transcript into a precursor miRNA.

Precursor miRNAs are exported to the cytoplasm and undergo further processing by Dicer.

Dicer, in complex with TRBP2, generates the miRNA duplex with 5' monophosphates and 3' overhangs.

The miRNA duplex is loaded into the Argonaut protein, with one strand, the guide strand, being retained for target recognition.

Argonaut proteins with guide miRNAs form the RISC complex, which binds target RNAs and promotes gene silencing.

The seed sequence of the miRNA, nucleotides 2 to 8, is crucial for target recognition in the 3' UTRs of mRNAs.

Gene silencing by miRNAs involves recruitment of factors like TRBP, which represses translation and destabilizes mRNAs.

siRNAs are derived from double-stranded RNA of various sources, including endogenous and exogenous duplexes.

siRNAs require only Dicer for processing, cleaving duplex RNA into short duplexes approximately every 20-25 base pairs.

The siRNA duplex is loaded into the Argonaut protein, with the passenger strand being cleaved and released.

siRNA-guided Argonaut complexes scan for fully complementary target RNAs, leading to cleavage and mRNA decay.

The cleavage of target RNAs by siRNAs is referred to as slicing, activating the RNAse H domain of the Argonaut protein.

Slicing products are targeted for degradation by the exosome and other RNA decay pathways in the cell.

The sorting process for miRNA duplexes is not fully understood, but helicase activity may play a role in strand selection.

The absence of extended duplex formation in miRNA-target interactions allows the miRNA to remain bound to the P domain of Argonaut.

Gene silencing mechanisms by miRNAs and siRNAs involve different binding complementarities and subsequent actions on target mRNAs.

Transcripts

two families of small regulatory rnas

called micr rnas and small interfering

rnas or Si rnas regulate the stability

and translation of mrnas in ukar both

classes of small RNA associate with a

family of proteins known as Argonaut but

differ in their origin processing

pathway targets a mechanism of action we

will first focus on micro rnas and then

point out the similarity with and

differences from SI

rnas micro rnas derive from actual genes

found in genomes across the lineages of

multicellular animals and plants these

rnas begin as primary transcripts or

Prim micr rnas and they're usually

transcribed by RNA polyas 2 the prim

micro RNA transcript folds into a stem

Loop structure that generally has some

unpaired nucleotides and single stranded

extensions are found at both the five

Prime and three prime ends the structure

serves as the initial substrate for

processing by drosa a nuclear RNA 3

enzyme drosa interacts with a

specialized RNA binding protein called

dgcr8 in humans to form what is termed

the microprocessor complex this complex

performs a cleavage reaction that

removes the five Prime and three prime

extension and liberates a 60 to 70

nucleotide transcript known as a pre

microrna the pre micr RNA generated by

the microprocessor complex is recognized

by by a nuclear export Factor called

expor in five which transports the pre

microrna to the cytoplasm for subsequent

processing in the cytoplasm a second

endonuclear reaction generally referred

to as dicing is catalyzed by dica

another rna's three enzyme daiser is

found in complex with a double stranded

RNA binding protein called the tar RNA

binding protein 2 or

trbp2 the product of this second

cleavage reaction is is called the M

mirar duplex it carries five Prime

monophosphates and three prime overhangs

of two nucleotides which is not fully

complimentary the two strands of this

duplex RNA are called the guide and

passenger

strands once the Mir mirar duplex has

been generated it is loaded into the

argonut protein that is dedicated to

microrna mediated silencing reactions if

the duplex is loaded in an appropriate

orientation one of the RNA strands

called the mere guide strand is retained

while the other strand the mere Star

passenger strand is selectively removed

the guide strand will ultimately be

involved in determining which Target

rnas will be silenced this process of

retaining one strand and removing the

other is called

sorting for imperfect M mirar duplexes

it is not certain how the passenger

strand is identified and selectively

removed though the action of a Healer

case may well be

helpful argonut proteins charge with

their guide micro rnas are referred to

as the miir risk complex the complex is

now ready to bind its Target RNA and

promote gene silencing Argonauts bound

to micr rnas typically identify

sequences with imperfect complementarity

in the three prime utrs of mrnas the

most important pairing region for the

micr RNA is referred to as the seed

sequence which generally encompasses

nucleotide positions 2 to8 of the

microrna species the absence of extended

duplex formation allows the three prime

end of the micro r& remain securely

bound to the P domain of the argonot

protein as a consequence the pee domain

of the argonot protein is generally not

positioned appropriately to cleave mrnas

that are targeted by micro

rnas once bound to targets Mi risks

appear to recruit additional factors

including in particular A protein that

contains multiple glycine tryptophan

repeats which is known as TR nc6 in

humans this protein is thought to be

involved in repressing translation and

in destabilizing the MRNA through

mechanisms that remain poorly understood

what is clear is that the repression

mechanism does not depend on the sial

activity of the argonut

protein in contrast to micrornas snas

are not generally encoded in the genome

as specific genes instead snas are

derived from double stranded RNA that

comes from several different sources

endogenous duplex RNA can arise from the

normal transcription of genomic Loi have

extensive hairpin structures or from the

analing of sense and antisense rnas that

have both been transcribed from a given

Locus exogenous sources of double

stranded RNA include viral rnas and

duplex structures that have been

synthetically introduced into cells by

scientists for experimental

purposes irrespective of their Origins

duplex rnas become substrates for an RNA

processing reaction akin to the one

described for the micr rnas but without

the first nuclear droser dependent

cleavage only dicing by the dacer enzyme

is

needed the cytoplasmic enzyme

sequentially cleav the long duplex

structures approximately every 20 to 25

base pairs The Cutting frame is set by

interactions between the double stranded

RNA and the Pas region of the daiser

protein the products of the daiser

reaction are short duplex rnas similar

to the mere mere star duplexes but fully

base paired along their length once the

SI s star duplex has been generated it

also is loaded into the appropriate

argonut protein and the sir Star

passenger strand is selectively removed

for these fully complimentary sir s star

duplexes the endogenous endonuclease

activity of the peee domain of the

algonot protein catalyzes cleavage of

the passenger strand the cleaved strands

are then easily Unwound and released

this mechanism makes use of the rna's H

fold of the peee domain and acidic

residues that are poised in the active

site for such

catalysis the resulting singl stranded

SI risk complexes can now scan for their

fully complimentary Target rnas in

contrast to the situation with the Mi

risk complexes fly complimentary binding

to the Target rnas lead to the three

prime end of the sna being displaced

from the P domain of the argonut as

extended duplex nucleic acid forms this

results in confirmational changes that

activate the cleavage activity of the

rna's h peee domain the cleavage

reaction is referred to as slicing

products of the slicing reaction are

thought to immediately become targets

for the exosome and other standard RNA

Decay Pathways in the

cell