Riboswitches are the control buttons of mRNAs. They control the expression of gene by regulating transcription and translation. Gene silencing by RNA interference is a mechanism of post transcriptional regulation of gene expression that involves mainly siRNA and miRNA.

1. Riboswitches and RNA
interference
Janmoni Borah
Assistant professor
Department of Zoology
Barnagar College, Sorbhog, Assam
Email-janmoniborah16@gmail.com

2. Riboswitch
 A riboswitch is a regulatory segment of a messenger RNA molecule
that binds to a small molecule (ligand), resulting in a change in
production of the proteins encoded by the mRNA.
Riboswitches provide a mechanism to control the expression of a gene
in transcriptional and translational level.
Riboswitches generally turn off the gene(mRNA) and turns on in
exceptional cases.
Riboswitches are usually found in the 5’-UTR region of mRNA
molecules.

3. Structure

4. Riboswitches are often divided into two domains (parts)- an aptamer
and an expression platform.
The aptamer directly binds to a specific small molecule (ligand) with
very high affinity and forms a thermodynamically stable three
dimensional structure.
The expression platform acts directly on gene
expression through its ability to toggle between two different
secondary structures in response to ligand binding.

6. Types of riboswitches
Ligand type Ligand name Riboswitch family/class Discovery (year) Structure (year)
Enzyme cofactor
S-adenosylmethionine SAM/SAM-I 2003 (Winkler et al. 2003)
2006 (Montange & Batey
2006)
S-adenosylmethionine SAM/SAM-II 2005 (Corbino et al. 2005) 2008 (Gilbert et al. 2008)
S-adenosylmethionine SAM/SAM-III 2006 (Fuchs et al. 2006) 2008 (Lu et al. 2008)
Thiamine pyrophosphate TPP
2001 (Miranda-Rios et
al. 2001)
2006 (Serganov et al. 2006;
Thore et al. 2006)
Nucleotide precursor
Guanine Purine/G 2003 (Mandal et al. 2003)
2004 (Batey et al. 2004;
Serganov et al. 2004)
Adenine Purine/A
2003 (Mandal & Breaker
2004)
2004 (Serganov et al. 2004)
2'-Deoxyguanosine Purine/dG 2007 (Kim et al. 2007)
2009 (Edwards & Batey
2009)
Amino acid Lysine Lysine
2003 (Rodionov et al. 2003;
Sudarsan et al. 2003)
2008 (Garst et al. 2008;
Serganov et al. 2008)
Metal ion Magnesium Mg2+
/ykoK
Identified 2004 (Barrick et
al. 2004); validated 2007
(Dann et al. 2007)
2007 (Dann et al. 2007)

7. Mechanism of Action
The aptamer domain of the riboswitch specifically binds to a ligand
with very high affinity that leads to conformational change in the
expression platform domain.
The expression platform typically turn off gene expression in
response to the ligand. The following riboswitch mechanisms have
been experimentally demonstrated.
Riboswitch controlled formation of rho independent termination
hairpin leads to premature transcription termination.
Riboswitch mediated folding sequesters the ribosome binding site of
mRNA, thereby inhibiting translation.
The riboswitch sometimes acts as ribozyme that cleaves itself in the
presence of sufficient concentrations of its metabolite.

8. Riboswitch sequesters ribosome binding site

9. RNA interference
RNA interference (RNAi) is a biological process in which RNA
molecules inhibit gene expression, typically by causing the
destruction of specific mRNA molecule.
It is a mechanism of gene regulation and known as post
transcriptional gene silencing. Earlier, it was also known as co-
suppression, quelling etc.
It is widely found in many eukaryotes.
Andrew Fire and Craig C. Mello were awarded the Nobel prize in
Physiology and Medicine for work on RNA interference.

10. Two types of small ribonucleic acid molecules-microRNA(miRNA) and
small interfering RNA (siRNA) are central to the process of RNA
interference.
 The active form of miRNA and siRNA are double stranded RNA
molecules. A number of different proteins are required in the
interference pathway.
RNA interference has an important role in defending cells against
parasitic nucleotide sequences, viruses and transposons. It also
influences development.

11. Mechanism of RNA interference
The RNA interference pathway is initiated by the enzyme Dicer. It is a
ribonuclease enzyme. It cleaves long double stranded RNA (dsRNA)
molecules into short double stranded fragments of apprx. 21 nucleotide
siRNAs with a 2-nucleotide overhand at the 3’-end.
The dsRNA may be endogenous or exogenous in origin. When the dsRNA is
exogenous (coming from infection by a virus with an RNA genome or
laboratory manipulation), the RNA is imported directly into the cytoplasm
and cleaved to short fragments by dicer into siRNA.
The endogenous dsRNA are encoded by RNA genes in the genome. These
genes basically encode the miRNAs. They are transcribed as single strand
transcript, but later processed to form the characteristics stem-loop
structure in the nucleus and then exported to the cytoplasm.

12. In the next step, each siRNA is unwound into two single stranded
RNAs(ssRNAs), the passenger strand and the guide strand.
The passenger strand is degraded and the guide strand is
incorporated into the RNA-induced silencing Complex (RISC) by RISC
Loading Complex (RLC).
The RLC contains Dicer-2 and R2D2 and is crucial to unite Ago@ and
RISC.
After integration into the RISC, siRNAs base pair with their target
mRNA and cleave it, thereby preventing it from being used as a
translation template. The cleavage is done by Ago2 component of
RISC.
miRNA binds to the 3’-UTR of mRNA with imperfect complementarity
and instead of cleaving, it blocks the access of ribosomes for
translation.

14. siRNA
Small interfering RNA (siRNA), sometimes also known as Short
interfering RNA or silencing RNA is a class of double stranded RNA
molecules, 20-25 base pairs in length. siRNA are similar to miRNA and
operates within the RNAi pathway.
siRNA has a well defined structure. It has phosphorylated 5’- ends
and 3’-ends with two overhanging nucleotides.
siRNA production is catalysed by an enzyme called Dicer from long
dsRNAs and small hairpin RNAs.

16. miRNA
A micro RNA is a small non-coding RNA molecule containing about 22
nucleotides. It is found in plants, animals and in some viruses.
It’s main function is gene silencing.
It silences a targeted gene by any of the three mechanisms—1)
cleavage of the mRNA strand into two pieces, 2) destabilization of the
mRNA through shortening of its poly(A) tail and 3) blocking ribosome.

17. miRNA genes are usually transcribed by RNA polymerase II (Pol II). The
polymerase often binds to a promoter found near the DNA sequence,
encoding what will become the hairpin loop of the pre-miRNA. The
resulting transcript is capped with a specially modified nucleotide at the 5'
end, polyadenylated with multiple adenosine (a poly(A) tail), and spliced.
Animal miRNAs are initially transcribed as part of one arm of an ∼80
nucleotide RNA stem loop that in turn forms part of a several hundred
nucleotide-long miRNA precursor termed a pri-miRNA.
miRNA transcript is edited and processed both in nucleus and cytoplasm.
In the cytoplasm the pre-miRNA hairpin is cleaved by the RNase III
enzyme Dicer.