The Small RNA World www.plantcell.org/cgi/doi/10.1105/tpc.110.tt0210

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.

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

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: 195 -198. Reprinted with permission from AAAS. Photo credit: Heidi

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. http://youdpreferanargonaute.com/2009/06/ 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 – 1437. with permission of AAAS.

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.

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: 848-853. Copyright 2001.

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.

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

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

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.  

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

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

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.

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.

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.

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

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

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

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

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

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

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: 299-300; Carlsbecker, A. et al., (2010) Cell signalling by microRNA165/6 directs gene dose-dependent root cell fate. Nature 465: 316-321.

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