- Small noncoding RNAs include miRNAs, siRNAs, and piRNAs that are 20-33 nucleotides in length and regulate gene expression through RNA degradation or translation inhibition. - miRNAs are derived from hairpin structures in RNA transcripts and regulate genes through partial base pairing, while siRNAs require perfect base pairing and are derived from double-stranded RNA. piRNAs are derived from single-stranded RNA and function in germ cells. - These small RNAs are processed by Dicer and loaded into RISC complexes containing Argonaute proteins to target RNAs for cleavage or translational repression. Their biogenesis pathways differ but generally function to reduce expression of protein-coding or transposon genes.

1. Small Noncoding RNAs:
miRNAs, siRNAs, and piRNA

2. Non-coding RNAs: Breaking the central dogma
Front Plant Sci. 2015; 6: 1001.

3. Small noncoding RNAs in the genome
• RNA transcripts range 20-30 nucleotides
• 3 flavors
• miRNAs:
• Micro RNAs (21-24 nt)
• Generated from RNA that forms a hairpin
• ~2600 miRNAs in vertebrates
• siRNAs:
• Small interfering RNA (20-25 nt)
• formed from double stranded RNA or perfect hairpins
• piRNAs: Piwi RNAs
• (26-33 nt)-
• formed from long single stranded RNA
• > 30,000 identified
Used to reduce gene
expression through
RNA degradation and
inhibition of
translation

4. Discovery of small noncoding RNAs
• Early 1990s in c elegans
• Identified 2 small RNA molecules 22 and 61 nt in the lin-4 gene that
were involved with silencing lin-14
• The 2 small RNAs were complimentary to a region within the lin-14
transcript
• Later determined that the 61 nt RNA was the precursor of a 22 nt
miRNA
• siRNAs were discovered a few years later in plants. Identified 25 nt
RNA from virus involved in gene silencing.

5. Generation of small noncoding RNAs
Nature 451, 414–416
siRNA: precursor is double stranded RNA from sense and
antisense transcription or perfect hairpins. 100% base
pairing of the 2 strands
miRNA: transcribed from one strand in which the RNA
forms a hairpin. There is not 100% base pairing in the
hairpin.
piRNA: long single stranded RNA with no base pairing
All of these are processed and cleaved to form the small
mature forms of the RNAs
siRNA and miRNA use similar machinery (RNAi pathway)
while piRNA has a unique pathway.

6. Production of siRNA
• Originally believed to be an immune defense against foreign RNA
• Endogenous production from transposons, centromeric RNA, mRNA,
etc…
• Generated from all types of RNA Pol

7. siRNA pathway
Arthritis Res Ther. 2004; 6(2): 78–85.
The initial step is recognition of double stranded RNA usually between 50-
70 nt in length by the ribonuclease Dicer
Dicer cleaves this precursor RNA into a small RNA 21-25 nt in length leaving
a dinucleotide overhang on each end of the double stranded RNA.
This process requires the hydrolysis of ATP in some but not all species.
Humans do not need ATP function.
The short double stranded RNA is then loaded on the RISC complex in
which the RNA is unwound and one strand is removed.
The single stranded RNA/RISC complex interact with target RNA through
base pairing of the siRNA and target RNA. The RISC complex then cleaves
the target RNA.

8. Dicer Ribonuclease
• Humans only have 1 isoform while other organisms have more than
one (Drosophilia have 2, Arabidopsis have 4)
• Drosophilia have unique functions for their 2 isoforms. Dicer1
processes miRNA while Dicer2 processes siRNA
• Usually purifies with a partner (TRBP in humans, R2D2 in Drosophila)
which has RNA binding activity. It is unclear what these partners do
as RNA processing occurs in the absence of these binding partners

9. RNA induced silencing complex (RISC)
• Not a well defined complex- may have several
forms
• Contains Argonaute protein
• RISC loading complex (RSC) contains Dicer,
TRBP, and Ago2
• Ago2 cleaves one strand of duplex RNA and
releases it.
• Preference for strand retention is determined
by the thermodynamic stability of the 5’ end.
• Mature RISC complex has single stranded RNA
which is able to target specific RNA.
• Mature RISC complex localizes to P bodies
however it does not appear this is necessary for
RNA cleavage
• RISC binding to target RNA causes cleavage
leaving an exposed 3’ and 5’ end for
exonucleases
Pharm Res. 2008 Jan; 25(1): 72–86.

10. RISC
RISC
RdRP
RISC
Amplification of the siRNA signal
Front. Plant Sci., 14 February 2017
Not known to occur in vertebrates due to a lack of
RdRP. Mostly characterized in c elegans
RdRP is an RNA polymerase that extends RNA from an
RNA template.
In essence, the siRNA/mRNA complex allows for RdRP
to extend the siRNA to generate a new precursor that
can then be cleaved by dicer to generate new siRNAs.
A small amount of siRNA can generate a complete
silencing of a gene.

11. miRNA processing
Front. Neurosci., 09 April 2012
miRNAs are transcribed by RNA Pol II
The RNA forms a hairpin through complementary
sequences
The initial RNA transcript is called the primary miRNA (pri-
miRNA)
A complex of Drosha and DGCR8 cleave the pri-miRNA to
release a hairpin of 70-100 nt. This hairpin is called the
precursor miRNA (pre-miRNA)
The pre-miRNA is exported to the cytoplasm where it
serves as a substrate for Dicer. The subsequent steps of
dicer cleavage and RISC loading are the same as siRNA.
miRNAs can associate with other Argonaute proteins that
lack endonuclease activity

12. Drosha/DGCR8 complex
Cell. Volume 125, Issue 5, p887–901, 2 June 2006
Drosha is a ribonuclease that complexes
with DGCR8 to form the microprocessor
complex.
DGCR8 is an RNA binding protein
It is thought that structures within the
stem loop that have double stranded single
stranded junctions direct cleavage of the
pri-miRNA.
Remember miRNA create hairpins without
100% base pairing.
RNA. 2014 Jul;20(7):1068-77.

13. miRNA/RISC complex
Unlike siRNA, miRNA do not necessarily have 100% base
pairing.
If there is no base pairing in the middle of the miRNA, then
Ago2 can not cleave the one strand of the miRNA.
Also if the miRNA associates with other Ago proteins that lack
endonuclease activity, cleavage to remove the second strand
can not take place.
In these instances the RISC complex incorporates a helicase to
unwind the two strands leaving and active miRNA/RISC
complex

14. Target regulation by miRNAs
Nature 435, 745-746 (9 June 2005)
Most miRNAs do not have 100% homology to their target RNAs and thus a
single miRNA has the ability to target multiple different RNAs.
Target identification is strongest at 5’ end of the miRNA called the seed region.
This represents nt 2-8 of the miRNA. The seed region has perfect or near
perfect base pairing. It can be strengthened by additional base pairing.
If base pairing occurs in nt 9-11, Ago2 can cleave and cause degradation of the
target RNA similar to the siRNA pathway

15. Translation inhibition by miRNAs
EMBO J. 2016 Jun 1;35(11):1158-9.
The RISC complex can recruit the CCR4-NOT
complex, GW182, and DDX6.
This complex is then localized to P bodies.
CCF4-NOT has deadenylase activity
which may lead to eventual degradation of the
RNA
However, deadenylase activity is not required for
translational repression and other mechanisms
to inhibit the translational complex exist.

16. Production of miRNAs
• miRNAs are produced by Pol II
• Many are encoded in noncoding genes
• Some are encoded within introns of other genes
• Many miRNAs are produced in clusters from the same transcript
• miRNAs gene naming : mir-#
• Many miRNAs are conserved
Nucleic Acids Res. 2008 May;36(9):2811-24.

17. Piwi RNAs
• piRNAs do not have a conserved mechanism of biogenesis
• Strand specific suggesting they arise from single stranded RNA
• Usually start with a uracil.
• ~80% have intergenic sequences
• Preferentially expressed in germ cells
• May be critical in silencing transposable elements.
• Bind Piwi proteins which are members of the Argonaute family

18. piRNA biogenesis is not well understood
Several mechanisms of piRNA formation have been characterized
in different species.
5’ monophosphate nucleotides are essential for piRNA biogenesis.
It represents the regulatory licensing event for Piwi proteins
3’ methylation at the 2 position concludes biogenesis
Nat Rev Genet. 2018 Nov 16

19. Two biogenesis pathways in most animals
Nat Rev Genet. 2018 Nov 16

20. piRNA in transposon silencing.
Nat Rev Genet. 2011 Aug 18;12(9):615-27.
piRNA can be generated that target transposons
Potentially random insertion of the transposon in piRNA
clusters creates the targeting RNA to direct the piRNA
complex to transposon RNAs. These RNAs are then
cleaved allowing for further generation of piRNAs.
Nat Rev Genet. 2018 Nov 16

21. piRNA: Silencing of genes
PLoS One. 2015 May 7;10(5)
Antisense transcription creates
piRNAs complementary to mRNA
transcripts.
Targeting of the mRNA generates
new piRNAs to retarget the
antisense strand.

22. Overview of the small noncoding RNAs.
Viruses. 2014 Nov 18;6(11):4447-64