RNA: Types, Structure and
Function
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Topic outline
• Introduction
– Nucleic acids
– DNA as master plan and RNA as working copies
• RNA classification
– Coding RNA
– Non-coding RNAs
• RNA structure & function
• RNA Interference
– Mechanism
– Biological roles and Applications
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Introduction
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Introduction: Nucleic acids
• Nucleic acids
– Molecules that store information for cellular growth and
reproduction
– These are polymers consisting of long chains of monomers called
nucleotides
• There are two types of nucleic acids:
– Deoxyribonucleic acid (DNA)
– Ribonucleic acid (RNA)
• A nucleotide consists of a nitrogenous base, a pentose sugar
and a phosphate group
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Introduction: Nucleic acids
Nucleotide
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Introduction: DNA and RNA
• The genetic master plan of an organism is contained in
DNA
• However, the master plan is expressed through the
“working copies” of DNA called ribonucleic acid (RNA)
• The copying process, during which a DNA strand serves as
a template for the synthesis of RNA, is called transcription
• Transcription produces messenger RNA (mRNA), ribosomal
RNA (rRNA), transfer RNA (tRNA), and additional RNA
molecules
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Expression of genetic information by transcription 7
Classification of RNA
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Classification of RNA
• RNAs have been classified into two groups, based on whether
the transcript is translated into protein or not:
1. Coding RNA
– Translated into protein e.g mRNA
2. Non-coding RNA (ncRNA)
– Not translated into proteins and further classified in two
groups:
• House keeping ncRNA e.g tRNA, rRNA
• Regulatory ncRNA e.g miRNA, siRNA
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Classification of RNA
• House keeping ncRNAs
– Constitutively expressed and required for normal function and
viability of cell
• Regulatory ncRNAs
– Expressed only in certain stages of organism development or as
a response to external stimuli
• Note
– Regulatory ncRNAs can affect the expression of other genes at
the level of transcription or translation
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Classification of RNA
.
RNA
Coding RNA Non-Coding RNA
•mRNA
House keeping RNA
Regulatory RNA •rRNA
•miRNA •tRNA
•siRNA •snRNA
•snoRNA
•Rnase P RNA
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Coding RNA: mRNA
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Messenger RNA
• Referred to as coding RNA because it carries genetic
information from DNA for use in protein synthesis
Polycistronic mRNA
– Carry information from more than one gene
– Characteristic of prokaryotes
Monocistronic mRNA
– Carry information from only one gene
– Characteristic of eukaryotes
• Cistron = Gene
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mRNA: Structure
It consists of:
1. Coding region
• Translated into protein
2. Untranslated regions (UTR)
– 5’ UTR:
7methyl-G cap: Bound by cap binding proteins
Translation regulation
– 3’ UTR:
Stability elements/polyadenylation signal
Subcellular localization (zip codes)
poly(A) tail
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mRNA: Structure
Structure of eukaryotic messenger RNA
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mRNA: Function
• Function: Carries genetic information for protein synthesis
from the DNA in the nucleus to the ribosomes in the
cytoplasm
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Non-coding RNAs
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1. House keeping ncRNAs
• Constitutively expressed and required for normal
function and viability of a cell
• They include:
– Transfer RNA (tRNA)
– Ribosomal RNA (rRNA)
– Small nuclear RNA (snRNA)
– Ribonuclease P RNA (RNase P RNA)
– Small nucleolar RNA (snoRNA)
– Telomerase RNA and others……
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tRNA: Structure
• Contain a high percentage of unusual (modified) bases, for
example, dihydrouracil
• Have extensive intrachain base-pairing that leads to
characteristic secondary and tertiary structure
• There is at least one specific type of tRNA molecule for each of
the twenty amino acids commonly found in proteins
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tRNA: Structure
Folded (tertiary) tRNA structure found in
tRNA secondary structure
cells 20
tRNA: Structure
D = dihydrouracil, Ψ = pseudouracil 21
tRNA: Function
• tRNA transfers a specific amino acid to the ribosome during
protein synthesis
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RNase P
• Ribonuclease P (RNase P) is a holoenzyme that cleaves the 5’
leader element of pre-tRNA to produce mature tRNA
• RNase P is made of a catalytically active ncRNA component
and protein component
• It was this enzyme that led scientists to the discovery of
ribozymes; the RNA species capable of catalytic activity
• Note: The letter P in RNase P stands for phosphodiesterase
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Action of RNase P on Pre-tRNA
RNAse P
cleavage
site
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D = dihydrouracil; Ψ = pseudouracil
RNase P
Action of RNase P on Pre-tRNA 25
rRNA: Structure
• rRNA are found in association with several proteins as
components of the ribosomes
• rRNA in eukaryotes is actually 4 separate RNA species: 28S
RNA, 18S RNA, 5.8S RNA and 5S RNA
• The 28S, 18S and 5.8S rRNA are transcribed as a long
precursor pre-rRNA of 45S
• The bacterial rRNAs (23S, 16S and 5S) are also transcribed as
one long molecule
S = Svediberg unit
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rRNA: Structure
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rRNA: Structure
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rRNA: Function
• rRNA act as ribozyme in formation of peptide bonds during
protein synthesis
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snoRNA
• snoRNA
– Small nucleolar RNAs between 60 and 300 nucleotides in length,
derived from introns
• Function:
– They recognise their target sequence by base pairing and then
recruit specialised proteins to perform nucleotide modifications to
RNAs:
– The modifications include:
1. 2’ O-ribose methylation,
2. base deaminations such as adenine to inosine conversions
3. addition of pseudouridines
• Note:
The exact purpose of these modifications are still unknown except
to say that they somehow guide the rRNA subunits to form a
functional ribosome
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snoRNA
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snRNA: Structure
• snRNA: Are small RNA molecules ranging from about 100-200
nucleotides in length
• Each snRNA forms a complex with 6-10 proteins which are
called small nuclear ribonucleoprotein particles (snRNPs,
pronounced "snurps")
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snRNA: Structure
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snRNA: Function
• snRNAs bind to pre-mRNA and each other within a larger
splicing complex known as the spliceosome to remove the
introns from the pre-mRNA
• The splicing reaction requires five snRNAs (U1, U2, U4, U5, &
U6)
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snRNA: Function
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Telomere and Telomerase RNA
• Telomeres are repetitive sequences at the ends of linear DNA
molecules in eukaryotic chromosomes
• With each round of replication in most normal cells, the
telomeres are shortened because DNA polymerase cannot
complete synthesis of the 5' end of each strand
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Telomere and Telomerase RNA
• Telomere is found on the ends of eukaryotic chromosomes
• Characterized by a 3’-overhang of single-stranded DNA
• Forms a T-loop with the help of specialized proteins to
protect itself from exonuclease activity
• Shortening of telomeres results in the loss of genetic
information and ultimately cell death
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Telomere and Telomerase RNA
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Telomerase
• A specialized form of reverse transcriptase that carries its
own internal RNA template (Telomerase RNA) to direct DNA
synthesis
• Elongates the lagging strand template from its 3’-OH end
• Adds short, repeated, guanosine-rich sequences to the ends
of chromosomes
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Telomerase
• Telomerase is thus able to replace telomere sequences that
would otherwise be lost during replication
• Normally telomerase activity is present only in embryonic
cells, germ (reproductive) cells, and stem cells, but not in
somatic cells
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Telomerase
• Most human somatic cells lack telomerase
activity
-Telomere shortening
Senescence
End-to-end chromosome fusion and cell death
• Most tumors regain the ability to produce
telomerase
-Cells become immortal
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Telomerase
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Regulatory ncRNAs
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2. Regulatory ncRNAs
• Regulatory ncRNAs are expressed only in certain stages of
organism development or as a response to external stimuli
• Regulatory ncRNAs can affect the expression of other genes
at the level of transcription or translation
• They include:
-microRNA (miRNA)
-small/short interfering RNA (siRNA)
-
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miRNA and siRNA
• microRNA (miRNA) and short interfering RNA (siRNA) are
very small RNA molecules, ranging between 21 to 25
nucleotides long
• The 2 species are quite similar, the variations come from their
source or origin
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miRNA: Structure
• MicroRNA comes from short endogenous hairpin loop
structures, synthesised by RNA pol II, often from within
introns
• The hairpin structures are cleaved in the nucleus, exported to
the cytoplasm and further processed to ~22 nt duplexes
nt = Nucleotide
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miRNA: Structure
RISC = RNA Induced Silencing Complex 47
miRNA: Function
• Function:
– Vast majority of microRNAs regulate other genes by
binding to complementary sequences in the target gene
• Mechanism of regulation:
– Perfect complementarity of binding leads to mRNA
degradation of the target gene
– Imperfect pairing inhibits translation of a target mRNA to
protein
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siRNA
• Small interfering RNA, sometimes known as short interfering
RNA, are a class of 20-25 nucleotide-long RNA molecules that
interfere with the expression of genes
• siRNAs are similar to miRNA but are produced from long
double stranded RNA molecules or giant hairpin molecules,
often of exogenous origin
• They can also be exogenously (artificially) introduced by
investigators to bring about the knock-down of a particular
gene
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siRNA
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How do miRNA and siRNA regulate
gene expression?
• Small RNAs mediate gene silencing through at least three
different mechanisms:
1. Endonucleolytic cleavage of the cognate mRNAs
2. Translational repression
3. Repression through the modification of DNA
and/or histone
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RNA Interference
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RNA Interference
• RNA interference (RNAi)
– A mechanism where the presence of certain fragments of
dsRNA interferes with the expression of a particular gene
which share homologous sequence with this dsRNA
– Is a mechanism that inhibits gene expression at the level
of translation or by hindering the transcription of specific
genes
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RNAi
• Before RNAi was well characterized, it was called by other
names, including post transcriptional gene silencing (TGS) and
transgene silencing
• Only after these phenomena were characterized at the
molecular level was it obvious that they were the same
phenomenon
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How does RNAi work?
• RNAi is a specific process, known as the RNA interference
machinery
• It appears that the machinery, once it finds a double-stranded
RNA molecule, cuts it up with an endonuclease (Dicer),
separates the two strands, and then proceeds to destroy
other single-stranded RNA molecules that are complementary
to one of those strands
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How does RNAi work?
• dsRNAs direct the creation of small interfering RNAs (siRNAs)
which target RNA degrading enzymes (RNAses) to destroy
transcripts complementary to the siRNAs
• RNA interference machinery detects these double strands, and
destroy all mRNAs that match the microRNA or siRNA
• Thus preventing their translation and lowering the activity of
many other genes
• The machinery is however also used by the cell itself to regulate
gene activity/expression
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How does RNAi work?
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How does RNAi work?
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Key players in RNAi
• RNAs:
– siRNA
– miRNA
• Enzymes:
– Dicer: Initiate RNAi
– Drosha: Cleaves base hairpin to form pre-miRNA
• RNA-Enzyme Complex (RISC):
– RNA Induced Silencing Complex that cleaves mRNA
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Biological roles of RNAi
• RNAi has the following biological roles:
1. Protection against invasive RNAs especially RNA viruses
2. Silencing of transposable elements
3. Down-regulation of expression of endogenous genes
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RNAi Applications
1. Treatment of diseases
– Cancer: Define roles of signaling molecules in tumor creation
– HIV/AIDS: RNA transcriptase is RNAi target
2. Testing hypotheses of gene function
– RNAi can be used in testing the accuracy of these predictions
using siRNA
3. Studying cell division
– Genes involved in cell division can be identified by RNAi
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RNAi Applications
4. Pathway analysis
– Treating cells with an siRNA targeting a given gene and then
monitoring the expression of other gene using microarray will
make it possible to identify genes that are associated with the
target gene
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Thank you for coming
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