Epigenetics

Lecture VII:
Small RNA and RNA interference
Ningning Wang
1/11/2012

Sequences variation and inheritance
DNA sequences( C, A, T, G)
Chromatin modifications variation
DNA methylation
histone modifications
small RNA variation
Molecular biology research

How did they work with small RNAs?
RNAi !

RNA Interference: Ancient Mechanism With A Promising Future
Gene Silencing!

RNAi Timeline
1990 co-suppression of purple color in plants.
1995 Guo S, and Kemphues KJ. First noticed that sense RNA was as effective as antisense RNA for suppressing gene expression in worm C. elegans
1998 Fire et al. First described RNAi phenomenon in C. elegans by injecting dsRNA into C. elegans which led to an efficient sequence-specific silencing and coined the term "RNA Interference".
2000 Zamone et al.Reported processing of long dsRNA by Rnase III (Dicer) into shorter fragments of 21-23-nt intervals in Drosophila extracts
2001 Bernstein et al. Cloned Dicer, the RNase III enzyme that is evolutionarily conserved and contains helicase and PAZ domains, as well as two dsRNA-binding domains.
2002 Tuschl T and colleagues First described RNAi in mammalian cells
2003 Paddison et al. Sui et al. Paul et al. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells.
2003 Song et al. First reported that siRNAs can be used therapeutically in whole animals
2004 Kawasaki and Taira Morris et al. First observed that siRNA silences gene at transcriptional level possibly through directing de novo DNA methylation.
2006 Nobel Prize in Medicine to Andrew Z. Fire (Stanford University) and to Craig C. Mello (University of Massachusetts Medical School) for their discovery of RNA interference - gene silencing by double-stranded RNA
2007 Smith, L.M., Pontes, O., Searle, I., Yelina, N.E., Yousafzai, F.K., Herr, A.J., Pikaard, C. and Baulcombe, D. A novel SNF2 protein associated with nuclear RNA silencing and spread of a silencing signal between cells in Arabidopsis.

1990 Richard Jorgensen and his group discovered gene silencing while working with petunias. Petunias developed areas of hypopigmentation when transduced with the gene encoding an enzyme required for pigment synthesis.
The phenomena was called
Co-suppression
Similar effects seen in fungi. called “Quelling”
http://www.pbs.org/wgbh/nova/sciencenow/3210/02.html
http://ag.arizona.edu/pls/faculty/jorgensen.html
Co-suppression: Exogenous gene suppress homologous endogenous genes
Gene made gene siliencing

Silencing of a green fluorescent protein (GFP) reporter in C. elegans occurs when animals feed on bacteria expressing GFP dsRNA (a) but not in animals that are defective for RNAi (b).
dsRNA made gene siliencing

Sense or anti-sense sRNA made gene siliencing

2008

Small interfering RNAs were soon found to not only induce mRNA degradation, but also guide other processes, including inhibition of translation, cytosine methylation, DNA elimination, and heterochromatin formation.
The greatest impact of RNAi is its application as an experimental tool to knock down gene expression in cells and intact organism.
Most processes known to be controlled by RNAi include synthesis of dsRNA from DNA or RNA, cleavage of dsRNAs by distinct RNases into small RNAs, small RNAs then are involve in specific biological process.
After the Hamilton and Baulcombe report in 1999, short RNAs were soon found in all other systems where dsRNA was able to induce gene silencing.

How did small RNA work?
PTGS!

Compartmentalization of processes that necessitates transport
replication
post-transcriptional gene silencing(PTGS)
transcriptional gene silencing(TGS)

Gene Silencing
Term used to describe switching off of a gene that would normally be turned on, i.e. silencing a gene that would normally be expressed.
Can be transcriptional (TGS) (DNA methylation, histone modification) or post-transcriptional (PTGS) ) (RNAi) .

Post-transcriptional gene silencing (PTGS)
It identifies long double stranded RNA molecules (dsRNA) such as those generated during virus replication, transposons mobilization or aberrant RNA synthesis.
These molecules act as trigger signals for the PTGS machinery, guiding the cell to an alert state-and the dsRNA molecules are destroyed

RNAi (PTGS)
Gene silencing mechanisms have since been found in many organisms including plants, fungi, worms, Drosophila, mice and humans
The best characterized mechanism of post transcriptional gene silencing is RNA interference or RNAi

Hannon G (2002). RNA interference. Nature 418: 244-251.
The effects of a dsRNA in a cell

How did small RNA generate?
Exogenous or endogenous!

Small RNA: non_coding, ssRNA, 18-30nt
rasiRNA
miRNA
nat-siRNA
ta-siRNA
lsiRNA (>30nt)
Now……

DICING AND SLICING
The basic pathway of RNA silencing is similar in many animals and plants. An RNA dependent RNA polymerase (RdRP) generates double stranded RNA from a single stranded precursor. The double stranded RNA is cleaved into 21-25nt fragments by nucleases of the Dicer family. These RNA fragments, known as mico (mi)RNAs and short interfering (si)RNAs, are separated into single stranded molecules and one of the two strands then becomes bound to an Argonaute nuclease. This RNA then forms base paired duplex structures with longer RNAs and thereby guides Argonaute to its target. Argonautes are sometimes referred to as Slicer. RNA silencing is therefore a combination of Dicing and Slicing.

Transcriptional silencing and epigenetics
The siRNA targeting mechanism is best understood in systems in which RNA is the target and the silencing is posttranscriptional. However there are also systems in which the targeting mechanism targets DNA and the silencing mechanism operates at the transcriptional level (Jones et al., 2001).
In these systems the transcriptional silencing is associated with methylation of the target DNA or modification of its associated histones or both.
The transcriptional silencing effect in these systems may persist for longer than the initiator silencing RNA and, in extreme examples, for many generations (Jones et al., 2001).
These are examples therefore in which RNA triggers a transgenerational effect. Because this is a heritable effect without changes to the sequence of DNA it is epigenetic rather than genetic.

See the RNA Family database—http/www.sanger.ac.uk/software/rfam/

DNA Methylation Mediated by a MicroRNA Pathway, Yijun Qi et al, Cell, 2010, 14May

rasiRNA

rasiRNA
Heterochromatin found near centromeres and telomeres are largely composed of short tandem DNA repeats and transposable elements. Heterochromatin structure is plastic and had the ability to regulate the expression of near by genes.
rasiRNA Can
Shut down expression of transposable elements.
Histone methylation.
DNA methylation modification.
rasiRNA functions via partially overlapping the components of siRNA pathway.
Instead of loading into RISC, rasiRNA load another complex called RITS (Inducible Transcriptional Silencing). Produced by the dicerlike DCL3 in A. thaliana.

thought