1. The Small RNA World

2. What are small RNAs?
Small RNAs are a pool of 21 to 24 nt RNAs that generally function in gene silencing
Small RNAs contribute to post-transcriptional gene silencing by affecting mRNA stability or translation
Small RNAs contribute to transcriptional gene silencing through epigenetic modifications to chromatin
AAAAA
RNA Pol
Histone modification, DNA methylation
The red symbol represents the small RNA. The green symbol with the AAAAA indicates an mRNA. The drawing on the bottom represents double stranded DNA being transcribed by RNA Polymerase to produce a transcript. Association of a small RNA with this transcript helps to target the enzymes that covalently modify chromatin (histone modifying enzymes and DNA methyltransferases) to confer silencing.

3. The core of RNA silencing: Dicers and Argonautes
RNA silencing uses a set of core reactions in which double-stranded RNA (dsRNA) is processed by Dicer or Dicer-like (DCL) proteins into short RNA duplexes.
These small RNAs subsequently associate with ARGONAUTE (AGO) proteins to confer silencing.
DICER
AGO
The Dicer or (in plants) Dicer-like enzymes cleave dsRNA. Small RNAs associate with ARGONAUTEs (AGOs) to target them to their targets.
Silencing

4. Dicer and Dicer-like proteins
In siRNA and miRNA biogenesis, Dicer or Dicer-like (DCL) proteins cleave long dsRNA or foldback (hairpin) RNA into ~ 21 – 25 nt fragments.
Note the two strands of the RNA molecule. The cleavage sites are indicated by yellow arrows.
Dicer’s structure allows it to measure the RNA it is cleaving. Like a cook who “dices” a carrot, Dicer chops RNA into uniformly-sized pieces.
From MacRae, I.J., Zhou, K., Li, F., Repic, A., Brooks, A.N., Cande, W.., Adams, P.D., and Doudna, J.A. (2006) Structural basis for double-stranded RNA processing by Dicer. Science 311: Reprinted with permission from AAAS. Photo credit: Heidi

5. The Arabidopsis ago1 mutant and the octopus Argonauta argo
Argonaute proteins
The Arabidopsis ago1 mutant and the octopus Argonauta argo
ARGONAUTE proteins are named after the argonaute1 mutant of Arabidopsis; ago1 has thin radial leaves and was named for the octopus Argonauta which it resembles.
ARGONAUTE proteins bind small RNAs and their targets.
Reprinted by permission from Macmillan Publishers Ltd: EMBO J. Bohmert, K., Camus, I., Bellini, C., Bouchez, D., Caboche, M., and Benning, C. (1998) AGO1 defines a novel locus of Arabidopsis controlling leaf development. EMBO J. 17: 170–180. Copyright 1998; Reprinted from Song, J.-J., Smith, S.K., Hannon, G.J., and Joshua-Tor, L. (2004) Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305: 1434 – with permission of AAAS.

6. RNA silencing - overview
siRNA-mediated silencing via post-transcriptional and transcriptional gene silencing
AGO
AAAn
AGO
AGO
RNA Pol
DCL
DCL
MIR gene
RNA Pol
AGO
miRNA -mediated slicing of mRNA and translational repression
mRNA
AAAn
MicroRNAs are encoded by MIR genes, fold into hairpin structures that are recognized and cleaved by DCL (Dicer-like) proteins.

7. siRNAs – Genomic Defenders
siRNAs protect the genome by
Suppressing invading viruses
Silencing sources of aberrant transcripts
Silencing transposons and repetitive elements
siRNAs also maintain some genes in an epigenetically silent state
This image shows the hypersensitive response in a virus-infected tobacco leaf. siRNAs contribute to viral gene silencing.
Reprinted by permission from Macmillan Publishers, Ltd: Nature. Lam, E., Kato, N., and Lawton, M. (2001) Programmed cell death, mitochondria and the plant hypersensitive response. Nature 411: Copyright 2001.

8. Viral induced gene silencing (VIGS) - overview
AGO
Most plant viruses are RNA viruses that replicate through a double-stranded RNA intermediate.
DCL
Viral ssRNA
Viral dsRNA
Virus-encoded RNA-dependent RNA polymerase
(RdRP)
Double-stranded RNA is cleaved by DCL to produce siRNA which associates with AGO to silence virus replication and expression.

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

10. Silencing of transgenes
Transgenes introduced into plants are frequently silenced by the siRNA pathway
Silencing can be triggered by:
Very high levels of gene expression
dsRNA derived from transgenes
Aberrant RNAs encoded by transgenes
Transgenes are silenced post-transcriptionally and transcriptionally

11. Example: Manipulation of gene expression to modify pigmentation
Anthocyanins
Chalcone synthase (CHS)
Wild-type petunia producing purple anthocyanin pigments
Chalcone synthase (CHS) is the enzyme at the start of the biosynthetic pathway for anthocyanins
Photo credit Richard Jorgensen; Aksamit-Stachurska et al. (2008) BMC Biotechnology 8: 25.  

12. Hypothesis: sense RNA production enhances pigmentation and antisense RNA production blocks pigmentation
Sense RNA
Antisense RNA
Sense construct:
PRO
ORF
Endogenous gene
mRNA
Transgene
Protein translated
Extra protein translated
Antisense construct:
Sense-antisense duplex forms and prohibits translation

13. Surprisingly, both antisense and sense gene constructs can inhibit pigment production
Plants carrying CHS transgene
CaMV 35S pro : CHS
CaMV 35S pro :
CHS
Sense
Antisense OR
Photo credit Richard Jorgensen

14. Silenced tissues do not express endogenous or introduced CHS
Transgene RNA
Endogenous gene RNA
Purple flowers
White flowers
This phenomenon, in which both the introduced gene and the endogenous gene are silenced, has been called “co-suppression”.
The gel shows the two species of RNA produced in the plants. The upper band corresponds to the transgene RNA, and the lower the endogenous gene RNA. This experiment shows that the transgene induces silencing of the endogenous gene as well as the transgene, no CHS production from either gene, and no pigment production.
Napoli, C., Lemieux, C., and Jorgensen, R. (1990) Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2: 279–289.

15. Co-suppression is a consequence of siRNA production
Wild-type
Protein translated
mRNA
mRNA
PRO
ORF
Endogenous gene
Sense RNA
Sense construct
Co-suppressed transgenic
PRO
ORF
Co-suppression
Endogenous gene
AGO
AGO
AAAn
siRNA produced
The transgene triggers siRNA production which silences both genes.
AGO
mRNA
AAAn
De Paoli, E., Dorantes-Acosta, A., Zhai, J., Accerbi, M., Jeong, D.-H., Park, S., Meyers, B.C., Jorgensen, R.A., and Green, P.J. (2009). Distinct extremely abundant siRNAs associated with cosuppression in petunia. RNA 15: 1965–1970.

16. Most siRNAs are produced from transposons and repetitive DNA
Most of the cellular siRNAs are derived from transposons and other repetitive sequences. In Arabidopsis, as shown above, there is a high density of these repeats in the pericentromeric regions of the chromosome.
Abundance of small RNAs
Abundance of transposon/ retrotransposons
Chromosome
Centromere
Deep sequencing methods allow the population of siRNAs in a cell to be mapped onto their regions of homology. The abundance of small RNAs and transposons/ retrotransposons along each of the five Arabidopsis chromosomes is shown relative to the centromere (circle on each line representing the chromosomes). In Arabidopsis these repetitive elements occur primarily in pericentromeric regions. Transposons and repetitive elements are more dispersed in organisms with larger genomes.
Kasschau, K.D., Fahlgren, N., Chapman, E.J., Sullivan, C.M., Cumbie, J.S., Givan, S.A., and Carrington, J.C. (2007) Genome-wide profiling and analysis of Arabidopsis siRNAs. PLoS Biol 5(3): e57.

17. microRNAs - miRNAS
miRNAs are thought to have evolved from siRNAs, and are produced and processed somewhat similarly
Plants have a small number of highly conserved miRNAs, and a large number of non-conserved miRNAs
miRNAs are encoded by specific MIR genes but act on other genes – they are trans-acting regulatory factors
miRNAs in plants regulate developmental and physiological events

18. microRNAs - miRNAS
microRNAs slice mRNAs or interfere with their translation
MIR gene
RNA Pol
DCL
AGO
AAAn
RNA Pol
AGO
AAAn
AAAn
AGO
mRNA
AAAn

19. MIR genes are transcribed into long RNAs that are processed to miRNAs
miRNAs are encoded by MIR genes
The primary miRNA (pri-miRNA) transcript folds back into a double-stranded structure, which is processed by DCL1
The miRNA* strand is degraded
DCL 3' 5'
miRNA
miRNA*
pri-miRNA
MIR gene
mRNA target

20. miRNAs and vegetative phase change
Vegetative phase change is the transition from juvenile to adult growth in plants.
Vegetative phase change
Germination
zygote
EMBRYONIC PHASE
JUVENILE PHASE
ADULT PHASE
REPRODUCTIVE PHASE

21. In Arabidopsis, SPL9 directly activates transcription of MIR172b
Phase change may involve a temporal cascade of miRNAs and transcription factors
In Arabidopsis, SPL9 directly activates transcription of MIR172b
miR156
SPL
miR172
AP2-like

22. miRNAs contribute to developmental patterning
miRNA concentration gradient
miRNA distribution patterns can spatially restrict the activity of their targets
miRNAs can move between cells to spatially restrict activity of their targets
Low Med High
HIGH MED LOW
Activity of target
HIGH MED LOW
Low Med High
In some cases the movement of miRNAs from cell to cell establishes a gradient

23. In roots, miR165/6 moves from endodermis into vascular cylinder
PHB
Movement of miR165/6 inwards from the endodermis in which it is produced helps to establish the radial pattern of the root
Reprinted by permission from Macmillan Publshers Ltd. Scheres, B. (2010). Developmental biology: Roots respond to an inner calling. Nature 465: ; Carlsbecker, A. et al., (2010) Cell signalling by microRNA165/6 directs gene dose-dependent root cell fate. Nature 465:

24. Conclusions
Small RNAs contribute to the regulation and defense of the genome, and confer silencing specificity through base-pairing
siRNA targets include repetitive-rich heterochromatin, transposons, viruses or other pathogens
miRNAs and tasiRNAs targets include regulatory genes affecting developmental timing or patterning, nutrient homeostasis and stress responses