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RNA INTERFERENCE
SLIDES 1, 2 The natural functions of RNA interference are as follows: (1) antiviral
mechanism, (2) regulation of gene expression (?). There is not too much data on the
regulatory role of RNA interference in vivo. RNA interference (RNAi) is a mechanism in
molecular biology where the presence of certain fragments of double-stranded RNA (dsRNA)
interferes with the expression of a particular gene. RNAi appears to be a highly potent and
specific process which is actively carried out by special mechanisms in the cell, known as the
RNA interference machinery. While the complete details of how it works are still unknown, it
appears that the machinery, once it finds a double-stranded RNA molecule, cuts it up,
separates the two strands, and then proceeds to destroy other single-stranded RNA molecules
that are complementary to one of those segments. dsRNAs direct the creation of small
interfering RNAs (siRNAs*) which target RNA-degrading enzymes (RNases) to destroy
transcripts complementary to the siRNAs. The genetic information of many viruses is held in
the form of double-stranded RNA, so it is likely that 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 microRNAs, 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 micro RNA, thus preventing their translation and lowering the activity
of many other genes. RNAi has been linked to various cellular processes, including the
formation of centromeric structure and gene regulation, through microRNAs and
heterochromatin formation. The effectiveness of RNAi lies in two processes: (1) Cleavage of
mRNA by the RISC complex; (2) amplification of the original signal by RNA-dependent
RNA polymerase by utilizing mRNA as a template and the complementary strand of siRNAs
as a primer for the amplification step in the first part shows that transcription and translation,
and the second part presents the mechanism of RNA interference. Together: DICER forms
siRNAs from double-stranded RNA molecules. The fate of siRNAs can be as follows. 1. The
siRNA bind to RISC, and after being single stranded, it lads the RISC to the target mRNA,
where the RISC cut the mRNA at a single point, which is followed by the degradation of
mRNA by Razes. 2. The single stranded siRNS detaches (or not attaches to) from the RISC,
and binds directly to the mRNA. This binding is recognized by an RNA-dependent RNA
polymerase, which utilizes the siRNA as a primer and the mRNA as a template for the
synthesis of second RNA strand. The newly formed double-stranded RNA will serve as a
substrate for DICER, which will create new and new siRNAs. This amplification step makes
RNA interference so effective. RNA interference participates in gene expression regulation as
an antiviral defense mechanism.
SLIDE 3 RNA interference (RNAi) is an evolutionary ancient mechanism that is used in animals, plants, and
even single-celled fungi to protect cells against viruses and transposable elements. Both viruses and active
transposable elements can produce long double-stranded RNA, at least transiently during their life cycles. Long
double-stranded RNA is not normally found in cells and, for many organisms, it triggers an RNAi pathway. A
cytoplasmic endoribonuclease called Dicer cuts the long RNA into a series of short double-stranded RNA pieces
known as short interfering RNA (siRNA). The siRNA produced is on average 21 bp long, but asymmetric
cutting produces two-nucleotide overhangs at their 3’ ends. The siRNA duplexes bound to by different
complexes that contain an argonaute-type endoribonuclease (Ago) and some other proteins (scanning 284. old).
Thereafter, the two RNA strands are unwound and one of the strands is degraded by argonaute, leaving a singlestranded RNA bound to the argonaute complex. One type of the argonaute complex is known as RNA-induced
silencing complex (RISC). In this case, after the single-stranded guide RNA binds to complementary long
single-stranded RNA, the argonaute enzyme will cleave the RNA, causing it to be degraded. Viral and
transposon RNA can be inactivated in this way. Another class of argonaute complex is the RNA-induced
transcriptional silencing (RITS) complex. Here, the single-stranded RNA guide binds to complementary RNA
transcripts as they emerge during transcription by RNA polymerase II. This allows the RITS complex to position
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itself on a specific part of the genome and then attracts protein such as histone methyltransferases (HMTs) and
sometimes DNA methyltransferases (DNMTs), which covalently modify histones in the immediate region. This
process eventually causes heterochromatin formation and spreading; in some cases the RITS complex can induce
DNA methylation. As a result, gene expression can be silenced over long periods to limit, for example, the
activities of transposons. Although mammalian cells have RNAi pathways, the presence of double-stranded
RNA triggers an interferon response that causes non-specific gene silencing and cell death. Therefore, when
RNAi is used as an experimental tool to produce gene silencing of pre-selected target genes, short doublestranded RNAs are used to trigger RNAi-based gene silencing.
SLIDE 4 Fire and Mello wanted to silence the mex3 gene of C. elegans (a worm species). Beside antisense
RNAs they inoculated double stranded RNAs, which were homologous to a certain region of mex3 mRNA. To
their surprise, the double-stranded RNA proved to be much more efficient for down-regulation of mex3
expression than the antisense RNA. Remark: the activity of mex3 protein can be visualized by a chromogenic
substrate (the cell turns blue). Furthermore, if transgenic C. elegans (expressing green fluorescent proteins; GFP)
were fed with E. coli expressing double stranded RNAs homologous to GFP mRNA, down-regulation of GFP
was observed in most cells of C. elegans. Thus, RNA interference (RNAi) can be transmitted from cell-to-cell, at
least in the case of worm. If a gene participating an RNAi was knock out (deleted) in these worms, the GFP
expression was not silenced.
SLIDE 5 Technologies for selective down-regulation of genes. Earlier: (1) antisense technology (RNA,
DNA oligonucleotides) and (2) knock-out technology. Today: knock-down technology via RNS interference.
MINIMAL REQUIREMENTS: SLIDES 1 - 2
EXTRA REQIREMENTS: SLIDES 3 - 5
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