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What is the role of ARGONAUT proteins in gene silencing?

AGO proteins bind small ds-RNAs and their targets. It cleaves away one of the strands and retains the other.

They are the catalytic components of the RNA-induced silencing complex (RISC).

Once it has a hold of a ss-RNA it will search for complementary mRNA. It then binds any complementary fragments and undergoes RNA silencing.

They are named after the argonaute1 mutant of Arabidopsis; ago1 has thin radial leaves and was named for the octopus Argonauta which it resembles. The mutant version of the plant is unable to undergo normal development, indicating that ago1 is very important.


What features distinguish silencing RNAs (siRNAs) and microRNAs (miRNAs)?

- ds-RNA that can be viral in origin, can result from transposons (non-coding regions of the genome)
- recognised by Dicer that cleaves it up into small fragments
- Recognised by AGO: matches the small RNA it holds to a complementary mRNA and prevents it from being translated
- alternatively, as RNA Pol is transcribing DNA the AGO can match the RNA as it is transcribed to its own and starts transcriptional silencing e.g. recruiting enzymes that methylate histones or DNA

- origin is from non-coding regions of the DNA: MIR genes (micro RNA genes)
- this RNA is single stranded but within it has complementary regions so folds in on itself and forms a hairpin
- now ds-RNA and recognised by Dicer for cleaving
- recognised by AGO which prevents translation of complementary mRNA from elsewhere in the genome


In Arabidopsis, as miR156 levels rise, levels of its target mRNA SPL decline. What relationship would you expect to find in animals between the miRNA and its target mRNA? Why?

- after Dicer has cleaved the dsRNA there are two options, of which one is more common in animals
- Because the match-up between miRNAs and their targets in animals is looser (i.e. not as accurate) the mRNA tends to undergo translational interference. The mRNA/microRNA will go to P bodies to be degraded.
- in plants the typical pathway is mRNA slicing because there is a much higher level of complementarity. AGO can cleave the mRNA transcript and target it for destruction.
- therefore mRNA levels will not decline in animals in the same way as in plants


What are the principle types of RNAs produced in cells?

- mRNAs: messenger RNAs, code for proteins
- rRNAs: ribosomal RNAs, form the basic structure of the ribosome and catalyse protein synthesis
- tRNAs: transfer RNAs, central to protein synthesis as adaptors between mRNA and amino acids
- snRNAs: small nuclear... function in a variety of nuclear processes, including the splicing of pre-mRNA
- snoRNAs: small nucleolar... used to process and chemically modify rRNAs
- scaRNAs: small cajal... used to modify sno and snRNAs
- miRNAs: micro... regulate gene expression typically by blocking translation of selective mRNAs
- siRNAs: small interfering... turn off gene expression by directing degradation of selective mRNAs and the establishment of compact chromatin structures
- other noncoding RNAs: function in diverse cell processes, including telomere synthesis, X-chromosome inactivation, and the transport of proteins into the ER


What are small RNAs?

- a pool of 21 to 24 nt RNAs that generally function in gene silencing
- They contribute to post-transcriptional gene silencing by affecting mRNA translation or stability
- They contribute to transcriptional gene silencing through epigenetic modifications to chromatin


What are the core components of RNA silencing?

RNA silencing uses a set of core reactions in which double-stranded RNA (dsRNA) is processed by Dicer and its homologues (Dicer-likes) into short RNA duplexes.

These small RNAs subsequently associate with members of the ARGONAUTE family of proteins to confer silencing.


What is Dicer?

Inn siRNA and miRNA biogenesis, Dicer or Dicer-like (DCL) proteins cleave double-stranded RNA into ~21-24 nt fragments (depending on the exact protein).

Dicer's structure allows it to measure the RNA it is cleaving. It chops RNA into uniformly-sized pieces.

Dicer has a flat, positively charged surface that attracts the negatively charged dsRNA. The dsRNA lines up very nicely against the protein. The ends of the RNA are held in the Paz domain down the bottom. It is the distance between the Paz domain and the Ribonuclease domain (the bit that cleaves the RNA) that determines the length of the fragments.


Where are AGOs located?

miRNAs and AGO proteins are located in cytoplasmic processing or P-bodies which also contain enzymes associated with mRNA decay.


What is the role of siRNAs?

siRNAs protect cells by:
- suprressing invading viruses
- silencing aberrant RNA transcripts
- silencing transposons and repetitive elements
- maintaining some genes in a silent state


What is TMV?

Tobacco Mosaic Virus is a typical plant virus.
It is made up of coat protein (CP) subunits surrounding the single-stranded RNA genome.
The genome encodes CP and an RNA-dependent RNA polymerase (RdRP) that replicates the TMV genome and a movement protein so that it can move around plant cells.


How does TMV replicate?

The virus enters the cytoplasm.
Removal of CP allows genome to be translated.
RdRP copies the RNA genome forming a double stranded intermediate. (this is important because it is what can be recognised by gene silencers)
A single stranded genome is formed which is then packaged with CP before repeating the process.


What is viral induced gene silencing?

Double-stranded RNA is cleaved by DCL to produce siRNA which associates with AGO to silence virus replication and expression.


How can you tell if plants have recovered from a viral infection and become resistant?

Young leaves produced on a virus-infected plant can be symptom-free, indicating that the plant has recovered from the infection.
However scientists were curious as the whether the older leaves had been able to subdue the infection or if the younger leaves had developed the silencing mechanism themselves.
So they inoculated the younger leaves with the virus and showed they were also resistant to subsequent infection by the same virus indicating they already had the machinery to silence it.


What experiment was performed to show the development of viral resistance in plants?

day 1: inoculate leaf with virus or water
day 22: inoculate younger leaf with virus or water
day 32: isolate RNA from leaf inoculated on Day 22. Measure viral RNA level

Low RNA levels in the experiments where the older leaf was inoculated with the virus first show that the first viral infection has induced RNA silencing, preventing subsequent viral replication.


How are small RNAs correlated with viral-induced gene silencing?

A small RNA homologous to viral RNA is present in inoculated leaves and distal, "systemic" leaves, but not mock-infected leaves.
You see a build up of the small RNA over time indicating that gene silencing is occurring.
This build up is shown even in the leaf that is not infected.


What does virus infection cause (re: siRNA)?

Systemic siRNA accumulation


How have viruses developed mechanisms to overcome RNA silencing?

Viruses have suppressor proteins that interfere with RNA silencing.
By interfering with RNA silencing, viral suppressor proteins can interfere with the plant's viral defence mechanism.
Suppressors act on various steps of the process.
This has however helped scientists discover how proteins like AGO and Dicer actually work.


Give a summary of viral-induced gene silencing.

- RNA-mediated gene silencing is an important tool in plant defence against viruses
- siRNAs interfere with viral replication
- siRNAs act systemically to aid in host plant recovery and resistance
- Most viruses produce RNA silencing suppressor proteins that target components of the plant's siRNA defence pathway; these proteins are important tools for dissecting RNA silencing pathways.


How can siRNA be integrated into research?

By creating a 21 - 24 nt sequence scientists can take advantage of the process and silence any gene in the genome as opposed to relying on random mutations.

Say you have a gene family which all do similar things - knocking out one probably won't affect the organism. If you can knock out five or ten you have a much better chance of seeing the impact.
only has to match a relatively small region


What are micro RNAs?

- miRNAs are thought to have evolved from siRNAs, and are produced and processed somewhat similarly
- Multi-cellular organisms have large numbers of miRNAs, some are conserved across a broad range of organisms
- miRNAs are encoded by specific MIR genes but act on other genes - they are trans acting regulatory factors
- miRNAs regulate a wide range of developmental and physiological events


when were the first miRNAs discovered?

- miRNAs were discovered in studies of developmental progressions in the nematode C. elegans
- a miRNA encoded by lin-4 is required for proper larval development
- lin-14 is required to specify division of a group of cells during development
- lin-4 represses lin-14 (a repressor)


What is an example of where miRNAs function in plants?

miRNAs are involved in vegetative phase change, which is the the transition from juvenile to adult growth in plants.
In arabidopsis, juvenile plants have very rounded leaves. As they develop into the adult and reproductive stage you get a change in leaf shape. Juvenile leaves are rounder, less serrated, and have hairs (trichomes) only on the upper surface. Adult leaves also have trichomes on the lower surface.
This transition is regulated by miR156. Over expression of the miRNA causes the plants to stay in the juvenile state.
The target of miR156 is SPL, a promoter of phase change.
SPL (Squamosa promoter binding protein like) = a family of transcription factors
In wild-type plants, miR156 expression decreases with plant age, allowing SPL to accumulate and promote phase change
The binding site of miR156 is in the 3' UTR.
If you alter SPL so that the binding site is not there/destroy it then you get SPL promoting precocious phase change despite levels of miR156.


Give a summary of miRNAs.

- miRNAs are small 21-24 nt RNAs produced from longer precursors by Dicer/Dicer-like proteins
- In animals, miRNAs typically act by blocking protein translation
- In the nematode C. elegans, lin-4 suppression of lin-14 is required for developmental progression
- In plants, miRNAs typically act by slicing their target mRNAs
- miR156 promotes juvenile phase by preventing SPL mRNA accumulation
- SPL promotes juvenile-to-adult phase change and flowering


In what process are miRNAs largely involved?

Nearly half of the targets of plant conserved miRNAs are transcription factors (regulate very important roles).
- some miRNAs are highly conserved and important gene regulators.


What is the function of circRNAs?

- as these circular RNAs can contain exons, these can act as micro RNA 'sponges', preventing them from getting their targets.