GENE SILENCING

Gene silencing is a general term describing epigenetic (epigenetics is the study of inherited
changes in phenotype (appearance) or gene expression caused by mechanisms other than changes
in the underlying DNA sequence) processes of gene regulation. The term gene silencing is
generally used to describe the "switching off" of a gene by a mechanism other than genetic
modification. That is, a gene which would be expressed (turned on) under normal circumstances
is switched off by machinery in the cell.

Genes are regulated at either the transcriptional or post-transcriptional level.

 Transcriptional gene silencing is the result of histone modifications, creating an

environment of heterochromatin around a gene that makes it inaccessible to
transcriptional machinery (RNA polymerase, transcription factors, etc.).

 Post-transcriptional gene silencing is the result of mRNA of a particular gene being

destroyed or blocked. The destruction of the mRNA prevents translation to form an active
gene product (in most cases, a protein). A common mechanism of post-transcriptional
gene silencing is RNAi.

Both transcriptional and post-transcriptional gene silencing are used to regulate endogenous
genes. Mechanisms of gene silencing also protect the organism's genome from transposons and
viruses. Gene silencing thus may be part of an ancient immune system protecting from such
infectious DNA elements.

Genes may be silenced by DNA methylation during meiosis, as in the filamentous fungus
Neurospora crassa

Transcriptional Gene Silencing: Post-transcriptional Gene Silencing:

 Genomic Imprinting * RNA interference

 Paramutation * Nonsense mediated decay
 Transposon silencing
 Transgene silencing Meiotic gene silencing:
 Transcriptional gene silencing
 position effect * Transvection
 RNA-directed DNA methylation * Meiotic silencing of unpaired DNA

Cellular components of gene silencing:

 Histones
 Chromatin and heterochromatin
 MicroRNA
 siRNA
 dsRNA
  Dicer
 Transposons

Small interfering RNA (siRNA)

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing
RNA, is a class of double-stranded RNA molecules, 20-25 nucleotides in length, that play a
variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi)
pathway (post-transcriptional gene regulation), where it interferes with the expression of a
specific gene. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related
pathways, e.g., as an antiviral mechanism or in shaping the chromatin structure of a genome.

siRNAs have a well-defined structure: a short (usually 21-nt) double strand of RNA (dsRNA)
with 2-nt 3' overhangs on either end:

Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group. This structure is the result
of processing by dicer, an enzyme that converts either long dsRNAs or small hairpin RNAs into
siRNAs.[3]

RNAi induction using siRNAs or their biosynthetic precursors

RNA interference refers to the inhibition of gene expression by small double-stranded RNA
molecules (and also microRNA). This phenomenon was initially demonstrated in C. elegans, in
which the injection of dsRNA molecules inhibited complementary gene expression.

siRNAs can be exogenously (artificially) introduced into cells by various

Transfection methods to bring about the specific knockdown of a gene of interest. But the
Transfection of an exogenous siRNA can be problematic because the gene knockdown effect is
only transient, particularly in rapidly dividing cells. One way of overcoming this challenge is to
modify the siRNA in such a way as to allow it to be expressed by an appropriate vector, e.g., a
plasmid.

The process of producing effective endogenous siRNA is regulated by three

enzymes. Plasmid vectors rely on the use of two RNA Polymerase III promoters (U6 involved
in gene splicing and H1) to drive transcription of the siRNA molecule. The target sequence (19
to 29 nucleotides) is placed in a sense and antisense orientation with a small spacer group in
between (short hairpin RNA or shRNA). Once transcribed, a hairpin structure is formed that can
be recognized and cleaved by Dicer (ribonuclease III) to form short ds siRNA molecules.
Argonaute proteins are then required to bind siRNA molecules to form a complex known as
RISC (RNA-induced silencing complex). RISCs may then promote epigenetic silencing by target
RNA cleavage. Though protein translation may be knocked down considerably, siRNA does not
normally eliminate the expression of a gene target completely.

siRNA action Micro RNA action

Micro RNA (miRNA)

MicroRNAs (miRNAs) are post-transcriptional regulators that bind to complementary sequences

in the three prime untranslated regions (3' UTRs) of target messenger RNA transcripts (mRNAs),
usually resulting in gene silencing. miRNAs are short ribonucleic acid (RNA) molecules, on
average only 22 nucleotides long.

Most microRNA genes are found in intergenic regions and contain their own miRNA gene
promoter and regulatory units. As much as 40% of miRNA genes may lie in the introns of
protein and non-protein coding genes or even in exons. After the transcription of a miRNA gene
follows the nuclear cleavage of the pri-miRNA performed by the Drosha RNase III
endonuclease. This enzyme cuts both strands of the pri-miRNA near the stem loop and generates 
~60–70 nt miRNA precursor (pre-miRNA). This pre-miRNA is transported to the cytoplasm by
the export receptor Exportin-5 . The nuclear cut by Drosha defines one end of the mature
miRNA and cytoplasmic cut by Dicer, also RNase III endonuclease, defines the opposite one.
Dicer  recognizes the pre-miRNA and cuts both of its strands at about two helical turns away
from the base of the stem loop. Then one of this ~22 nucleotide miRNA duplex arms is chosen
and mature miRNA is associated with RNA-induced silencing complex RISC. RISC acts to
repress the translation of target mRNA by mechanisms of translational repression or mRNA
cleavage.

Limitations of RNAi

 Because RNAi intersects with a number of other pathways, it is not surprising that on
occasion nonspecific effects are triggered by the experimental introduction of an siRNA.
  When a mammalian cell encounters a double-stranded RNA such as an siRNA, it may
mistake it as a viral by-product and mount an immune response.

 Furthermore, because structurally related microRNAs modulate gene expression largely

via incomplete complementarity base pair interactions with a target mRNA, the
introduction of an siRNA may cause unintended off-targeting.

Possible therapeutic applications and challenges

Given the ability to knock down essentially any gene of interest, RNAi via siRNAs has generated
a great deal of interest in both basic[6] and applied biology.

 There are an increasing number of large-scale RNAi screens that are designed to identify
the important genes in various biological pathways. Faster identification o gene function.

 Because disease processes also depend on the activity of multiple genes, it is expected
that in some situations turning off the activity of a gene with an siRNA could produce a
therapeutic benefit.

However, applying RNAi via siRNAs to living animals, especially humans, poses many
challenges. Experimentally, siRNAs show different effectiveness in different cell types in a
manner as yet poorly understood: some cells respond well to siRNAs and show a robust
knockdown, whereas others show no such knockdown (even despite efficient transfection).