Post transcriptional Gene Silencing in Fishes, Slides of Molecular biology

Download Post transcriptional Gene Silencing in Fishes and more Slides Molecular biology in PDF only on Docsity!

Post transcriptional gene silencing

By,

Subashini V

FBT-PB1-

By,

Subashini V

FBT-PB1-

FBT-

ADVANCES IN MOLECULAR BIOLOGY

FBT-

ADVANCES IN MOLECULAR BIOLOGY

INTRODUCTION

RNA silencing is a novel gene regulatory mechanism that limits

the transcript level by either suppressing transcription

(Transcriptional Gene Silencing) or by activating a

sequence-specific RNA degradation process

(Posttranscriptional Gene Silencing [PTGS] / RNA

interference [RNAi]).

The natural functions - protection of the genome against

invasion by mobile genetic elements such as viruses and

transposons as well as orchestrated functioning of the

developmental programs of eukaryotic organisms.

Other names, including co-suppression , and quelling.

PTGS is heritable, although it can be modified in subsequent

cell divisions or generations, it is an epigenetic phenomenon

PTGS – an overview

• Posttranscriptional gene silencing, also known as RNA silencing or RNA interference, is a

type of gene silencing that works through sequence-specific degradation of RNA.

• Though the transcription of the gene is not affected, the protein synthesis from mRNA

stops due to unstable or inaccessible mRNA.

• Posttranscriptional gene silencing is induced by the deliberate production of double-

stranded RNA.

• Double-stranded RNA triggers the cleavage of homologous mRNA. Small interfering RNAs

or micro RNAs that have homologous sequences to transcribed regions of genes guide the

sequence-specific degradation of mRNA.

Non coding RNAs involved in PTGS

• Two types of small ribonucleic acid (RNA) molecules – microRNA (miRNA) and small

interfering RNA (siRNA) – are central to RNA interference.

• So far, most of the research on small regulatory RNAs has focused on the siRNAs and

microRNAs (miRNAs), although other small RNAs, like small hairpin RNAs (shRNA),

small temporal RNAs (stRNA) and the germ line-specific PIWI-interacting RNAs, have

also recently been described in a range of animals including the zebrafish Danio rerio.

• These small RNAs can direct enzyme complexes to degrade messenger RNA

(mRNA) molecules and thus decrease their activity by preventing translation, via post-

transcriptional gene silencing.

• Transcription can be inhibited via the pre-transcriptional silencing mechanism of RNA

interference, through which an enzyme complex catalyzes DNA methylation at

genomic positions complementary to complexed siRNA or miRNA.

siRNAs

• Small RNA species (siRNA) of 21-25 nucleotides of both sense and antisense polarity.
• siRNAs are formed and accumulated as double-stranded RNA molecules of defined chemical

structures - the signature of any homology-dependent RNA-silencing event.

• The siRNAs resemble breakdown products of an E. coli RNAse III-like digestion. In particular,

each strand of siRNA has 5’-phosphate and 3’-hydroxyl termini and 2 to 3 nucleotide 3’ overhangs.

• siRNAs could also act as guide RNAs for cognate mRNA degradation.

Components of PTGS

Dicer

• It belongs to RNase III family that show specificity for dsRNAs and cleave them with 3’ overhangs of 2 to 3 nucleotides

and 5’ phosphate and 3’ hydroxyl termini named Dicer (DCR).

• Evolutionarily conserved in worms, flies, fungi, plants, and mammals.
• Four distinct domains: an aminoterminal helicase domain, dual RNase III motifs, a dsRNA binding domain , and a

PAZ domain (a 110-amino-acid domain present in proteins like Piwi, Argo, and Zwille/Pinhead), which it shares with the

RDE1/QDE2/Argonaute family of proteins.

• Cleavage by Dicer is thought to be catalyzed by its tandem RNase III domains. Each monomer of the Dicer enzyme

possesses two catalytic domains, with one of them deviating from the consensus catalytic sequences.

RNA-Dependent RNA Polymerase

RNA-dependent RNA polymerases (RdRPs) play a role in both

triggering and amplifying the silencing effect. Transgenic and virus-

infected animals show an accumulation of aberrant transgenic and viral

RNAs. The RdRP enzymes might recognize these aberrant RNAs as

templates and synthesize antisense RNAs to form dsRNAs. The RdRP

is also perhaps responsible for sustaining PTGS at the maintenance

level even in the absence of the dsRNA that initiates the RNAi effect.

Unwinding of double-stranded siRNA ( Helicase !?)

Ribonuclease component cleaves mRNA ( Nuclease !?) and Cleaved

mRNA is degraded by cellular exonucleases

(A) Dicer-dependent small RNA

synthesis. (B) Dicer-independent

small RNA synthesis.

(C) Heterochromatin formation-

Small RNAs from the regions

guide histone H3K9 methylation.

siRNA mediated gene silencing

• Two-step model for the mechanism of gene silencing induced by double-stranded RNA.
• Step I - dsRNA is cleaved by the Dicer enzyme to produce siRNAs. A putative kinase seems

to maintain 5’ phosphorylation at this step. The siRNAs have also been proposed to be

responsible for nuclear DNA methylation (F) and systemic spread of silencing.

Amplification might occur due to the presence of RdRP.

• Step II - the siRNAs generated in step I bind to the nuclease complex (RISC). A helicase

present in the complex might activate RISC by unwinding the siRNAs. The antisense

component of siRNA in the RISC guides the complex towards the cognate mRNA, resulting

in endonucleolytic cleavage of the mRNA, RNA-dependent DNA methylation.

miRNA mediated gene silencing

Primary miRNA (pri-miRNA) transcripts are generated by

RNA polymerase II ( 1 ).

Drosha and its double-stranded RNA (dsRNA)-binding

protein cofactor DGCR8 bind to a pri-miRNA and release

a ∼60- to 75-nt-long hairpin precursor (pre)-miRNA ( 2 ),

which is exported from the nucleus to the cytoplasm ( 3 ).

Dicer and a family of dsRNA-binding proteins (TRBPs in

mammals) cleave the pre-miRNA ( 4 ), yielding an 18- to

21-nt-long RNA duplex consisting of the passenger strand

(blue), and the guide strand (red), which is also the

mature miRNA.

The double-stranded miRNA duplex is loaded into an

Argonaute protein ( 5 ), and the passenger strand is either

cleaved by Argonaute slicing activity and released ( 6 ) or

released independently of slicing when the two strands

are mismatched.

The mature single-stranded miRNA (red), Argonaute, and

the Argonaute-interacting protein GW182 constitute the

core components of miRISC ( 7 ).

Target Recognition

(a) siRNA is usually fully

complementary to the coding

region of its target mRNA; (b)

miRNA is partially

complementary to its target

miRNA. Complementary binding

usually occurs at the seed region

(nucleotides (nt) 2–7 of the 5'

end) of miRNA and the 3' UTR of

the target mRNA.

RNAi in fishes

The highly conserved nature of the molecules of RNAi implies a strong selective pressure to uphold RNAi

pathway in fishes and higher vertebrates.

Other indirect evidence for the persistence of the RNAi pathway in fishes comes from studies of the B2 protein

from fish betanodaviruses. These studies revealed that the B2 protein retains the ability to bind dsRNA. In

alphanodavirus from insects, this ability makes B2 protein able to suppress RNAi in the insect cells. Accordingly,

B2 of the betanodaviruses can also be anticipated to work as a defence mechanism against RNAi in fish

cells.

siRNA-mediated gene silencing in fish cells lack the controls needed to give strict proof of the RNAi concept in

fishes, a problem that is shared with many studies in mammalian cells.

Furthermore, the experiments in fishes have so far been biased in their choice of models and methods.

Most studies have been performed in fish embryos in assays where developmental genes have been targeted.

As embryo development relies on the correct timing of developmental gene expression, the non-specific effects

seen in fish embryos treated with synthetic regulatory RNAs, whether smaller or longer dsRNAs, might be

caused by saturation of the endogenous RNA regulatory mechanism responsible for endogenous gene

regulation.

RNAi in aquaculture