Transcript RNA interference - genemol de Jean

RNA interference
http://en.wikipedia.org/wiki/RNA_interference
Definition:
RNA interference (RNAi) is a mechanism where the presence of certain fragments of ds RNA
interferes with the expression of a particular gene which shares a homologous sequence
with this dsRNA.
Before RNAi was well characterized, it was called by other names, including post transcriptional
gene silencing (TGS) and transgene silencing. Only after these phenomena were characterized at
the molecular level was it obvious that they were the same phenomenon.
The use of RNA to reduce expression in plants has been a common procedure for many years.
Single-stranded antisense RNA was introduced into plant cells that hybridized to the cognate,
single-stranded, sense messenger RNA. While scientists first believed that the resulting dsRNA
helix could not be translated into a protein, it is now clear that the dsRNA triggered the
RNAi response. The use of dsRNA became more widespread after the discovery of the
RNAi machinery, first in petunias and later in roundworms (C. elegans).
RNAi is a specific process, known as the RNA interference machinery. It appears that the machinery,
once it finds a double-stranded RNA molecule, cuts it up with an endonuclease (Dicer), separates
the two strands, and then proceeds to destroy other single-stranded RNA molecules that are
complementary to one of those sequences. dsRNAs direct the creation of small interfering RNAs
(siRNAs) which target RNA-degrading enzymes (RNAses) to destroy transcripts complementary
to the siRNAs.
The life cycle and replication of many RNA viruses involves a double-stranded RNA stage,
so it is likely that part of the RNA interference machinery evolved as a defense against these viruses.
The machinery is however also used by the cell itself to regulate gene activity:
certain parts of the genome are transcribed into microRNA, short RNA molecules that fold back
on themselves in a hairpin shape to create a double strand. When the RNA interference machinery
detects these double strands, it will also destroy all mRNAs that match the microRNA,
thus preventing their translation and lowering the activity of many other genes
This mechanism was first shown in the "JAW microRNA" of Arabidopsis; it is involved in the
regulation of several genes that control the plant's shape. The mechanism has also been show
in many other eukaryotes; by now, more than 200 microRNAs have been detected in humans.
RNAi has been linked to various cellular processes, including the formation of centromeric structure
and gene regulation, through microRNAs and heterochromatin formatio
siRNA
siRNA (small interfering RNA)
http://en.wikipedia.org/wiki/Small_interfering_RNA
Small interfering RNA (siRNA), sometimes known as short interfering RNA, are a class
of 20-25 nucleotide-long RNA molecules that interfere with the expression of genes.
They are naturally produced as part of the RNA interference (RNAi) pathway by the enzyme Dicer.
They can also be exogenously (artificially) introduced by investigators to bring about th
knockdown of a particular gene.
siRNA's have a well defined structure.
Briefly, this is a short (usually 21-nt) double-strand of RNA (dsRNA) with 2-nt overhangs
on either end, including a 5' phosphate group and a 3' hydroxy (-OH) group.
Transfection of an exogenous siRNA is problematic, since it is only transient, and the
dsRNA structure cannot easily be permanently maintained.
One way of overcoming these problems is to modify the siRNA in such a way as to allow it
to be expressed by an appropriate vector, e.g. a plasmid. This is done by the introduction
of a loop between the two strands, thus producing a single transcript, which can be processed
into a functional siRNA.
This transcription cassette usually uses an RNA polymerase III promoter, which direct the
transcription of small nuclear RNA's, such as U6 or H1. It is assumed (although not known
for certain) that the resulting short hairpin RNA (shRNA) transcript is processed by Dicer.
Introduction of too much siRNA can result in non-specific events due to activation of the interferon
pathway. Most papers suggest that this is probably due to activation of the dsRNA sensor PKR,
although retinoic acid inducible Gene I (RIG-I may also be involved
One method of reducing the non-specific effects is by turning the shRNA into a micro RNA.
Micro RNA's are naturally occurring, and, as such, tolerated better by the cell.
By engineering an siRNA sequence into an miRNA structure, non-specific effects can
potentially be eliminated.
The mediators of RNA interference are 21- and 23-nucleotide small interfering RNAs (siRNA).
siRNAs bind to a ribonuclease complex called RNA-induced silencing complex (RISC) that
guides the small dsRNAs to its homologous mRNA target.
Consequently, RISC cuts the mRNA approximately in the middle of the region paired
with the antisense siRNA, after which the mRNA is further degraded.
http://www.qbiogene.com/products/transfection/app-sirna.shtml
miRNA
miRNA (micro-RNA)
http://en.wikipedia.org/wiki/MiRNA
A miRNA (micro-RNA) is a form of single-stranded RNA which is typically 20-25 nucleotide long.
It is thought to regulate the expression of other genes.
miRNAs are RNA genes which are transcribed from DNA, but are not translated into protein.
The DNA sequence that codes for an miRNA gene is longer than the miRNA itself. This DNA sequence
includes the miRNA sequence and an approximate reverse complement. When this DNA sequence is
transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse-complement base
pair to form a double stranded RNA hairpin loop; this forms a primary miRNA structure (pri-miRNA).
In animals, the nuclear enzyme Drosha cleaves the base of the hairpin to form pre-miRNA.
The pre-miRNA molecule is then actively transported out of the nucleus into the cytoplasm by Exportin 5,
a carrier protein. The Dicer enzyme then cuts 20-25 nucleotides from the base of the hairpin to release
the mature miRNA.
In plants, which lack Drosha homologues, pri- and pre-miRNA processing by Dicer probably takes
place in the nucleus, and mature miRNA duplexes are exported to the cytosol by Exportin 5.
miRNAs are RNA genes which are transcribed from DNA, but are not translated into protein.
The DNA sequence that codes for an miRNA gene is longer than the miRNA itself.
This DNA sequence includes the miRNA sequence and an approximate reverse complement.
The pre-miRNA molecule is then actively transported out of the nucleus into the cytoplasm by Exportin 5,
a carrier protein.
The Dicer enzyme then cuts 20-25 nucleotides from the base of the hairpin to release
the mature miRNA.
In plants, which lack Drosha homologues, pri- and pre-miRNA processing by Dicer probably takes
place in the nucleus, and mature miRNA duplexes are exported to the cytosol by Exportin 5.
The function of miRNAs appears to be in gene regulation
For that purpose, a miRNA is complementary to a part of one or more messenger RNAs (mRNAs).
Animal miRNAs are usually complementary to a site in the 3' UTR whereas plant miRNAs are
usually complementary to coding regions of mRNAs. The annealing of the miRNA to the mRNA the
inhibits protein translation, but sometimes facilitates cleavage of the mRNA.
This is thought to be the primary mode of action of plant mIRNAs.
In such cases, the formation of the double-stranded RNA through the binding of the miRNA
triggers the degradation of the mRNA transcript through a process similar to RNA interference (RNAi),
though in other cases it is believed that the miRNA complex blocks the protein translation
machinery or otherwise prevents protein translation without causing the mRNA to be degraded.
miRNAs may also target methylation of genomic sites which correspond to targeted mRNAs.
Gene silencing in plants occurs at both transcriptional (TGS) and posttranscriptional (PTGS) levels.
Double stranded RNA (dsRNA), which is processed to short interfering RNAs (siRNA) of
21-24 nucleotides, plays a key role as an inducer in both silencing processes.
The siRNAs corresponding to promoter sequences direct the silencing machinery to bloc
transcription of homologous promoters (TGS), whereas
those corresponding to transcribed sequences direct the silencing machinery to degrade
homologous RNAs (PTGS). TGS accompanied by chromatin methylation and remodeling;
and PTGS associated with RNA degradation can neutralize endogenous and exogenou
invaders such as transgene, viruses and transposon.