Biogenesis and the regulation of
Small RNAs
Genomics
Evren Coşkun Becit
Guidelines
► Types of sRNAs and sRNA Biogenesis
► sRNA driven regulatory mechanisms
► Conclusion
► Future perspectives
Biogenesis of miRNA
Transcribed from MIR gene transcripts
and are cleaved from stem-loop hairpins
formed from RNA polymerase II
transcripts.
► miRNAs are processed via the action of
the RNAse III enzymes (Drosha and Dicer
in animals, DCL in plants)
► After cropping and exportation to the
cytoplasm, miRNAs are further diced and
are loaded into the Argonaute proteins.
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7methylgua
nosine
Drosha–Di George syndrome critical
region gene 8 (DGCR8) acts as a ruler to
measure the cleavage site.
Canonical intronic miRNAs are processed
co-transcriptionally before splicing.
► Non-canonical intronic small RNAs are
produced from spliced introns and
debranching.
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Biogenesis of small RNAs in animals. Kim VN, Han J,
Siomi MC. Nat Rev Mol Cell Biol. 2009 Feb;10(2):126-39.
miRNa biogenesis pathways in human and flies
Mechanisms of miRNA-mediated gene regulation
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Following Dicer cleavage, the resulting
~22-nt RNA duplex is loaded onto an Ago
protein. The guide strand remains as a
mature miRNA whereas the passenger
strand is cleaved by AGo and degraded.
The strand with relatively unstable
(thermodynamically) base pairs at the 5′
end typically survives.
AGo-miRNA then incorporates into RISC.
miRISC binds to target mRNA at 3`UTR
which leads to translation repression of
target genes. The imperfect base-pairing,
resulting in a bulge structure, avoids AGo2
cleavage.
Translation-repressed mRNA and miRISC
are concentrated in P-bodies for storage
or mRNA decay.
Stored mRNAs can be released and reenter translation pathways.
Small RNAs: regulators and guardians of
the genome. Chu CY, Rana TM. J Cell Physiol. 2007
Nov;213(2):412-9.
miRNA target recognition
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Slicing-dependent reduction in mRNA
accumulation, and/or translational
repression.
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Central bulge results in active AGomiRNA complexes.
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Seed pairing in animal differes from
cannonical plant pairing in size.
Classification and comparison of small RNAs from plants.
Axtell MJ. Annu Rev Plant Biol. 2013;64:137-59.
miRNA mediated translation repression
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Post-initiation repression mechanism.
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Initiation-repression mechanism: Binding
of AGo to m7G-cap prevents the
recruitment of eIF4E(translation initiation
factor). miRISC recruits elF6 and elF6
blocks 40s subunit association which in
turn hinders the 80s assembly. Thereby
translation initiation is blocked.
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Destabilisation of mRNA: mRNA
recognition by miRNA destabilizes the
target by inducing its deadenylation and
decay.
Small RNAs: regulators and guardians of
the genome. Chu CY, Rana TM. J Cell Physiol. 2007
Nov;213(2):412-9.
Biogenesis of piRNA
Primary precursor piRNAs are transcribed
from intergenetic repetitive elements,
active transposon genes and piRNA
clusters. It is postulated that piRNAs use
nuclease activity of the Piwi proteins for
their processing.
► piRNA biogenesis involves primary and
secondary processing mechanisms.
Primary processing is not well defined.
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Primary processing generates antisense
piRNAs which have a 5ʹ uridine (5ʹ U).
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Sense transposon mRNA is cleaved to
produce sense piRNAs, which have a
strong adenine bias at position 10 (10A).
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Antisense piRNA is produced by
AGO3-mediated cleavage of antisense
primary piRNA transcripts.
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Only loaded PIWI is imported into the
nucleus.
RNA interference in
the nucleus: roles for small RNAs in transcription, epigenetics and beyond.
Castel SE, Martienssen RA. Nat Rev Genet. 2013 Feb;14(2):100-12.
piRNA Transposon silencing
piRNAs silence the TEs through the cleavage
of TE-derived transcripts or DNA methylation
of the genomic loci to cause transcriptional
silencing.
► In follicle cells flamenco cluster generates
piRNAs.
► In oocytes and surrounding nurse cells, Active
transposons are post-transcriptionally
silenced and nuclear PIWI promotes
transcriptional silencing by methylation of
histone H3 at lysine 9 (H3K9me) and
heterochromatin protein 1A (HP1A)
localization.
► HP1A homologue Rhino binds to
heterochromatic piRNA clusters in place of
HP1A and promotes transcription.
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RNA interference in
the nucleus: roles for small RNAs in transcription, epigenetics and beyond.
Castel SE, Martienssen RA. Nat Rev Genet. 2013 Feb;14(2):100-12.
Biogenesis of siRNA
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Exogenous small interfering RNAs are
derived from double-stranded dsRNAs or
viral RNAs.
Endogenous siRNA (endo-siRNA) precursors
are derived from repetitive sequences,
sense–antisense pairs or long stem-loop
structures.
siRNA precursors are transferred to
cytoplasm and are loaded onto Dicer-TRBP
complex to generate 21- to 22-nt siRNA
duplexes.
siRNA duplex is loaded onto AGo while the
passenger strand is removed.
Active siRISC recognizes and cleaves its
target mRNA.
Biogenesis of small RNAs in animals. Kim VN, Han J,
Siomi MC. Nat Rev Mol Cell Biol. 2009 Feb;10(2):126-39.
Exo- and Endo-sirNa biogenesis pathways in human
siRNA mediated transposon silencing
In male germ line transposon transcripts are
processed into 21 nt siRNAs and mobile
siRNAs direct post-transcriptional gene
silencing (PTGS) in the sperm nuclei.
► In female gametophyte DME1 expression
and MET1 repression yields to Transposon
activation. This activates the RNA-directed
DNA methylation (RdDM) pathway and
produces 24 nt siRNAs.
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RNA interference in
the nucleus: roles for small RNAs in transcription, epigenetics and beyond.
Castel SE, Martienssen RA. Nat Rev Genet. 2013 Feb;14(2):100-12.
siRNA-directed DNA methylation
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RNA-DEPENDENT POLYMERASE 2 (RDR2)
physically associates with RNA Pol IV to
produce dsRNA.
In the cytoplasm siRNA is facilitated by
HSP90, and the loaded AGO4 is then
imported back into the nucleus.
AGO4 targets nascent RNA Pol V transcripts
and forms the RNA-directed DNA
methylation (RdDM) complex.
PolV associated protein KTF1 interacts with
AGO 4 and 5-methylcytosine.
Interaction between DRM2 and AGO4 (via
RDM1) creates a positive-feedback loop
between AGO4 localization and DNA
methylation.
After it has been localized, DRM2 catalyses
methylation of cytosine in all sequence
contexts.
RNA interference in
the nucleus: roles for small RNAs in transcription, epigenetics and beyond.
Castel SE, Martienssen RA. Nat Rev Genet. 2013 Feb;14(2):100-12.
The RNA-dependent DNA methylation pathway in Arabidopsis thaliana
Conclusion
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miRNAs with imperfect
complementarity to their targets
cause translational repression.
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piRNAs which target transposon
transcripts in animal germ lines.
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siRNAs which have perfect
complementarity to targets and
cause transcript degradation and
effect heterochromation
formation.
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The miRNA biogenesis pathway is
well studied in comparison to
piRNA and siRNA pathways, yet
many questions remain still
unanswered.
Biogenesis of small RNAs in animals. Kim VN, Han J,
Siomi MC. Nat Rev Mol Cell Biol. 2009 Feb;10(2):126-39.
Future perspectives
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Since siRNAs can be employed as a biological tool to knock down specific genes, the
most important issue in designing siRNAs is how to increase their potency and ensure
specific gene silencing.
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New mechanisms of gene regulation and genome stability as well as to enhance the
potential for new RNAi-based therapies.
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Understanding the underlying mechanisms that effect the small RNA stability will
benefit the design and utilization of small RNAs for genetic manipulations.
References
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Kawaji H1, Nakamura M, Takahashi Y, Sandelin A, Katayama S, Fukuda S, Daub CO, Kai C, Kawai J, Yasuda J, Carninci
P,Hayashizaki Y. Hidden layers of human small RNAs. BMC Genomics. 2008 Apr 10;9:157.
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Kim VN, Han J, Siomi MC. Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol. 2009 Feb;10(2):126-39.
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Daniel Klevebring, Nathaniel R Street, Noah Fahlgren, Kristin D Kasschau, James C Carrington, Joakim Lundeberg, Stefan
Jansson. Genome-wide profiling of Populus small RNAs. BMC Genomics. 2009; 10: 620.
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Vaughn MW, Martienssen R. It's a small RNA world, after all. Science. 2005 Sep 2;309(5740):1525-6.
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Ji L, Chen X. Regulation of small RNA stability: methylation and beyond. Cell Res. 2012 Apr;22(4):624-36.
►
Castel SE, Martienssen RA. RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond.
Nat Rev Genet. 2013 Feb;14(2):100-12.
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Billi AC, Fischer SE, Kim JK. Endogenous RNAi pathways in C. elegans. WormBook. 2014 May 7:1-49.
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Ba Z, Qi Y. Small RNAs: emerging key players in DNA double-strand break repair. Sci China Life Sci. 2013 Oct;56(10):933-6.
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Chu CY, Rana TM. Small RNAs: regulators and guardians of the genome. J Cell Physiol. 2007 Nov;213(2):412-9.
►
Axtell MJ. Classification and comparison of small RNAs from plants. Annu Rev Plant Biol. 2013;64:137-59.
►
Kim VN, Han J, Siomi MC. Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol. 2009 Feb;10(2):126-39.
Thank you for your patience
Additional information
Regulation of small RNA stability: methylation and beyond.
Ji L, Chen X. Cell Res. 2012 Apr;22(4):624-36. Epub 2012 Mar 13.
Factors effecting sRNA stability
Regulation of small RNA stability: methylation and beyond.
Ji L, Chen X. Cell Res. 2012 Apr;22(4):624-36. Epub 2012 Mar 13.
3′ uridylation generally marks small RNAs for degradation and reveal other 3′ tailing events that influence small RNA stability.
Regulation of small RNA stability: methylation and beyond.
Ji L, Chen X. Cell Res. 2012 Apr;22(4):624-36. Epub 2012 Mar 13.
RNA interference in
the nucleus: roles for small RNAs in transcription, epigenetics and beyond.
Castel SE, Martienssen RA. Nat Rev Genet. 2013 Feb;14(2):100-12.