The Small RNA World

Small RNAs, discovered in the 1990s, silence

genes through transcriptional gene silencing (TGS)
and post-transcriptional gene silencing (PTGS)

DNA

mRNA

Transcription
tRNA = transfer RNA
tRNA = transfer RNA
rRNA = ribosomal RNA
rRNA = ribosomal RNA
mRNA = messenger RNA
mRNA = messenger RNA
sRNA = small RNA
sRNA = small RNA
siRNA = small interfering RNA
siRNA = small interfering RNA
miRNA = microRNA
miRNA = microRNA

tRNA
rRNA

Protein

Translation

small RNAs (sRNAs)

(siRNAs and miRNAs)

2016 American Society of Plant Biologists

Small RNAs are siRNAs, miRNAs,

and phasiRNAs or tasiRNAs
siRNAs
Derived from
viral RNA, or
RNA produced
from
heterochromatin
or transposons

miRNAs

phasiRNAs

tasiRNAs

Derived from
transcription of
MIR genes

Derived from
transcription of
PHAS genes

Derived from
transcription of
TAS genes

21 24 nt

Usually 21-22 nt

Usually 21 nt

Usually 21 nt

Generally involved
in transcriptional
gene silencing

Generally involved
in posttranscriptional
gene silencing

Generally involved
in posttranscriptional
gene silencing

Generally involved
in posttranscriptional
gene silencing
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History: The discovery of small

RNAs in plants
Insights from the study of transgene silencing
Experiments to modify flower color and
to introduce two related transgenes

Insights from the study of viral resistance

Experiments to study resistance to viruses

Lindbo, J.A., Silva-Rosales, L., Proebsting, W.M., and Dougherty, W.G. (1993). Induction of a highly specic antiviral state
in transgenic plants: Implications for regulation of gene expression and virus resistance. Plant Cell 5: 17491759.
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Manipulation of chalcone synthase

expression to modify pigmentation

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

Anthocyanins

Photo credit Richard Jorgensen; Aksamit-Stachurska et al. (2008) BMC Biotechnology 8: 25.
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Hypothesis: Sense transcript

increases color, antisense lowers
Endogenous gene
PRO

ORF

Transgene
Sense construct:
PRO

mRNA

Protein translated
mRNA
Extra protein translated

Sense RNA

ORF

mRNA
mRNA

Transgene
Antisense construct:
PRO

Antisense
RNA

Sense-antisense duplex forms

and prohibits translation

ORF

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Surprisingly, both antisense and

sense gene constructs inhibited
pigment production

Plants carrying CHS transgene

Sense

OR CaMV 35S pro :

CHS

CaMV 35S pro : CHS

Antisense
Photo credit Richard Jorgensen
2016 American Society of Plant Biologists

Silenced tissues do not express

endogenous or introduced RNA
Purple
flowers

White
flowers

This phenomenon, in which both the

introduced gene and the
endogenous gene are silenced, has
been called co-suppression.

Transgene RNA

Endogenous
gene RNA
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: 279289.
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Co-suppression is a consequence of
siRNA production
Wild-type
PRO

ORF

Protein translated

mRNA

mRNA

Endogenous gene

Co-suppressed transgenic

Co-suppression
Sense RNA

Sense construct
PRO

ORF

Endogenous gene

siRNA
produced

mRNA

Gene
silencing

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: 19651970.
2016 American Society of Plant Biologists

Other transgene studies revealed

RNA-mediated DNA methylation
Small RNAs can initiate gene silencing through
covalent modifications of the DNA or its associated
histone proteins, interfering with transcription.
Transcription
Histone
proteins

DNA

Silencing

This form of silencing is

frequently associated with
stably silenced DNA
including centromeres and
transposons, but also
occurs at genes.

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Studies of viral resistance in plants

provided evidence of small RNAs
This viral-gene
expressing plant
became resistant to
the virus
Wild-type plant
showing symptoms
of viral infection

Model proposed to explain viral resistance:

Presence of transgene induces cellular factor that
targets same sequences in viral genome
Lindbo, J.A., Silva-Rosales, L., Proebsting, W.M., and Dougherty, W.G. (1993). Induction of a highly specic antiviral state
in transgenic plants: Implications for regulation of gene expression and virus resistance. Plant Cell 5: 17491759.
2016 American Society of Plant Biologists

Viral resistance involves siRNAmediated silencing

DAY 22 Inoculate
younger leaf with
virus or water

DAY 1
Inoculate
leaf with
virus or
water

DAY1
DAY22

DAY 32 Isolate RNA from

leaf inoculated on Day 22.
Measure viral RNA level.
Inoculum
Virus
Water
Water
Virus

Virus
Virus

DAY 32
(RNA)
Low RNA levels show that the first viral
infection has induced RNA silencing,
preventing subsequent viral replication.
From Ratcliff, F., Henderson, B.D., and Baulcombe, D.C. (1997) A similarity between viral
and gene silencing in plants. Science 276: 15581560. Reprinted with permission from AAAS.
2016 American Society of Plant Biologists

The biogenesis of small RNAs in

plants
Small RNAs start
as dsRNAs
MIR gene

Hairpin formed in
single stranded
RNA, like found in
the single-stranded
genome of many
RNA viruses

Double-stranded
RNA used by
viruses during
replication

Hairpin structure
formed by the
transcripts of
microRNAencoding genes

The product of an
RNA-dependent
RNA Polymerase

RNA-dependent
RNA Polymerase
(RdRP/RDR)

2016 American Society of Plant Biologists

Enzymes and
proteins
involved in
small RNA
biogenesis
and function

or

DCL cleaves dsRNA

into short duplexes

HEN1 methylates
small RNA duplexes

Small RNAs bind

AGO to interact
with targets
Reprinted from Vazquez et al. (2010). The biosynthetic pathways and biological scopes of plant small RNAs. Trends Plant Sci. 15: 337-345 with permission from
Elsevier.
2016 American Society of Plant Biologists

Viral siRNAs are produced in two

ways: Primary and secondary
Viral dsRNA
(replication intermediate)

Viral ssRNA

LCL
D
C
D

Virus-encoded
RNA-dependent
RNA polymerase
(RdRP)

RdRP
L
DC

Primary
siRNA
Silencing can spread beyond the site of
the virus by production of secondary
siRNA, which requires the action of RNAdependent RNA polymerase (RdRP).

L
DC

Secondary siRNA
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Viruses have suppressor proteins

that interfere with RNA silencing
RdRP
L
DC

By interfering with RNA silencing, the

viral suppressor proteins can interfere
with the plants viral defense
mechanism. Suppressors can act at
any step of the process.

L
DC

2016 American Society of Plant Biologists

Heterochromatin-associated siRNAs
are derived from Pol IV RNA
RNA Pol IV transcripts
are duplicated and
diced to produce
siRNAs that target
homology-mediated
silencing

Transcripts
produced by RNA
Pol V contribute to
the targeting and
silencing of
transposons

Reprinted from Fultz D., Choudury S.G., and Slotkin, R.K. (2015). Silencing of active transposable elements in plants. Curr. Opin. Plant Biol. 27: 67-76 with permission from Elsevier.
2016 American Society of Plant Biologists

MIR genes are transcribed into long

RNAs that are processed to miRNAs
MIR gene
miRNAs are encoded by MIR genes
The primary miRNA (pri-miRNA)
transcript folds back into a doublestranded structure, which is processed
by DCL1
The miRNA* strand is degraded

pri-miRNA

5'
3'

DCL
5'
3'

miRNA
miRNA*

miRNA
mRNA target

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Biological functions of small RNAs

Transposon silencing
Viral defense
Transgenerational transposon
silencing
Developmental patterning
Developmental progression
Pathogen defense and biotic
interactions

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Small RNAs can be mobile and

function non-cell autonomously

Small RNAs (or their precursors) can be

transported short-distance cell-to-cell through
plasmodesmata to induce non-cell
autonomously gene silencing
Small RNAs (or their precursors) can also be
transported long-distance via the plants vascular
system to induce systemic gene silencing
Reprinted from Dunoyer, P., Melnyk, C., Molnar, A., and Slotkin, R.K. (2013). Plant mobile small RNAs. Cold Spring Harb Perspect Biol. 5. pii: a017897.
2016 American Society of Plant Biologists

Small RNAs move from the

vegetative cells to the sperm cells

The activated transposons

in the vegetative nucleus
contribute to a burst of small
RNAs that move into the
sperm cells to ensure
transposons are silent

Reprinted by permission from Macmillan Publishers Ltd: Nodine, M. (2016). Mobile small RNAs: Spermcompanion communication. Nature Plants. 2: 16041, see also
Martnez, G., Panda, K., Khler, C. and Slotkin, R.K. (2016). Silencing in sperm cells is directed by RNA movement from the surrounding nurse cell. Nature Plants. 2: 16030.
2016 American Society of Plant Biologists

miRNAs contribute to patterning

throughout leaf development
By
Byregulating
regulatingthe
theexpression
expressionpattern
patternand
andtiming
timingofoftheir
theirtargets
targets(mostly
(mostly
transcription
factors),
miRNAs
are
key
regulators
of
leaf
development
transcription factors), miRNAs are key regulators of leaf developmentfrom
from
initiation
initiationtotosenescence
senescence

Pulido, A., and Laufs, P. (2010). Co-ordination of developmental processes by small RNAs during leaf
development. J.Exp.Bot. 61: 1277-1291, by permission from Oxford University Press.
2016 American Society of Plant Biologists

Some phasiRNAs target large gene

families: MYB, PPR and NB-LRR

Reprinted from Fei, Q., Xia, R., and Meyers, B.C. (2013). Phased, secondary, small interfering RNAs in posttranscriptional regulatory networks. Plant Cell. 25: 2400-2415.
2016 American Society of Plant Biologists

Small RNAs move

between plants and
pathogens,
symbionts, and
herbivores
Recent discoveries about crosskingdom trafficking of small RNAs have
opened up possibilities to better control
diseases and pests of crops.
HIGS (host-induced gene silencing)
involves transfer of small RNAs from
host to pest or pathogen. Small RNAs
also can move from pest to plant.
Reprinted from Weiberg, A., Bellinger, M. and Jin, H. (2015). Conversations between
kingdoms: small RNAs. Curr. Opin. Biotechnol. 32: 207-215 with permission from Elsevier.
2016 American Society of Plant Biologists

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

2016 American Society of Plant Biologists