1. rasiRNAs: a class of small regulatory RNAs
Jasmine Lau
4 May 2009

2. The Family of small RNAs
Small noncoding regulatory RNAs
Shorter length
20-25 nucleotides
Longer length
24-27 nucleotides
small interfering RNAs
(siRNAs)
microRNAs (miRNAs)
piwi-interacting RNAs (piRNAs)
rasiRNAs – repeat-associated siRNA
Other piRNAs
rasiRNAs
rasiRNAs
= Repeat-associated small interfering RNAs
 sequence-specific gene silencing (RNAi mechanism)
 forms RNA-protein complexes with Piwi subfamily proteins
 sequences correspond to genomic repeats and transposons

3. First hints of rasiRNAs
2001
Trypanosoma brucei
parasitic protist species
causes African trypanosomiasis (sleeping sickness)
2002
Abundant nucleotide long siRNA-like RNAs
Represented 2 retrotransposons in the genome
Both sense and antisense polarity
2003
2004
Regulatory mechanism to silence retrotransposon transcription and mobilization?
2005
2006
2007
2008
Present

4. First hints of rasiRNAs
2001
Trypanosoma brucei
parasitic protist species
causes African trypanosomiasis (sleeping sickness)
2002
Abundant nucleotide long siRNA-like RNAs
Represented 2 retrotransposons in the genome
Both sense and antisense polarity
2003
2004
Regulatory mechanism to silence retrotransposon transcription and mobilization?
2005
Nicotiana and Arabidopsis thaliana plants
2006
24-26 nucleotide long endogenous siRNA species
Represented 2 retrotransposons in Nicotiana genome,
1 retrotransposon in Arabidopsis genome
Both sense and antisense polarity
2007
2008
Linked to DNA methylation and systemic silencing Mechanism to keep retrotransposon silent?
Present

5. rasiRNAs: new class of RNAs
2001
Drosophila melanogaster small RNA profiling
2 distinct classes of small RNAs:
21-23 nt RNAs
24-26 nt RNAs
2002
2003
Sequences correspond to genomic repeats or transposons
2004
Sequences correspond to genomic repeats/transposons
 Class of RNAs called repeat-associated siRNAs
(rasiRNAs)
2005
Also described in zebrafish, fission yeast
2006
Sense & antisense orientation of repetitive sequence elements:
Long Terminal Repeat (LTR) and non-LTR retrotransposons
DNA transposons
Satellite and microsatellite sequences
Complex repeats
2007
2008
Present

6. rasiRNAs: subclass of piRNAs
2001
Longer length
24-27 nucleotides
2002
2003
piwi-interacting RNAs (piRNAs)
2004
24-26 nucleotide long RNAs
BUT lack transposon or repetitive seqeuences
2005
Other piRNAs
rasiRNAs
2006
2007
2008
Present

7. rasiRNA biogenesis
Argonaute protein family
Ago subfamily
Piwi subfamily
Ago1
Ago2
Ago3
Aubergine (Aub)
Piwi
Associate with miRNAs
Associate with siRNAs
Associate with rasiRNAs, present in germline cells and early embryos
Dicer-dependent production to produce double-stranded precursor
Dicer-independent production

8. rasiRNA biogenesis ? ? AGO3 Adenine at nucleotide 10 Sense transcript:5’ 3’ A
10 complementary nucleotides
Antisense transcript: U 3’ 5’
Uridine at 5’ terminal end
PIWI/
AUB
Zucchini/ squash?
2’-O-Me
AGO3 ?
AGO3 5’ 5’ 3’ A 3’
Pimet A
AGO3
To add in? It also remains unclear how the ping-pong biogenesis mechanism is initiated. In the fly, aub and possibly piwi are accumulated at the posterior pole in the oocytes and remains in the polar granules in the early embryos. It is then deposited in the pole cells which are the progenitor germ cells. Hence, this suggests that rasiRNAs which are associated with Piwi proteins can be transmitted via the germline for the next generation, acting as ‘primary seeds’ to launch the rasiRNA biogenesis cycle in the embryos. 5’ 3’ 5’ 3’ A A 3’ U 5’ U 3’ 5’
PIWI/
AUB
2’-O-Me
Pimet U 3’ 5’ 3’ U 5’ ?
PIWI/
AUB
PIWI/
AUB
Zucchini/ squash?

9. What do rasiRNAs do? Present in fission yeast, plants, fly, zebrafish
Functions mainly in the germline and early embryo development in fly and zebrafish, found at very low levels in the adult fly.
Maintaining genomic stability
Silencing and suppressing mobilization of retrotransposons and other selfish genetic transposable elements
Guide transcriptional and post-transcriptional gene silencing of repetitive sequence elements
Guide chromatin silencing to establish and maintain heterochromatin
Also in particular for flies:
Maintaining germ cell self-renewal
Establishing axis polarity of the early embryo
Regulating pole cell formation and oocyte maturation
Maintaining telomeres

10. How rasiRNAs work Fission yeast :
Gene silencing and establishing heterochromatin
Transcriptional silencing complex in nucleus
 Ago1, Chp1 and Tas3
Binds DNA/ chromatin, induce DNA and histone methylation
Dicer cleaves long dsRNA
Plants :
Similar mechanism to yeast (above),
also repress repetitive transposable elements
Induce systemic gene silencing,
Histone H3 lysine-9 methylation, DNA methylation
 Direct chromatin modifications
Ago4-dependent mechanisms

11. How rasiRNAs work Downstream effector mechanisms and pathways
Drosophila melanogaster:
Induce histone H3 Lysine 9 methylation and association of HP1 chromatin binding protein with heterochromatin
rasiRNAs associate with piwi Argonaute proteins, and RNA helicases (Spindle-E and Armitage)
Downstream effector mechanisms and pathways
are not yet known
 Literature between piRNAs and rasiRNAs are merging

12. piwi-interacting RNAs (piRNAs)
Summary: rasiRNAs
rasiRNAs
= Repeat-associated small interfering RNAs
Longer length
24-27 nucleotides
 sequence-specific gene silencing (RNAi mechanism)
piwi-interacting RNAs (piRNAs)
 forms RNA-protein complexes with Piwi subfamily proteins
Other piRNAs
rasiRNAs
 sequences correspond to genomic repeats and transposons