RNA Processing

Dr. Odette Laneuville

Different Types of Eukaryotic RNA

Types

Description

mRNA

messenger RNA, variable amounts, code for proteins

rRNA

ribosomal RNA, abundant, contribute to the structure of the

ribosomes

tRNA

transfer RNA, moderate levels, carry the amino acids and act as
adaptors during the translation process.

snRNA

small nuclear RNAs, low to moderate levels, involved in splicing

mechanisms.

snoRNA

small nucleolar RNAs, low to moderate levels involved in maturation

covalent modification of rRNAs.

miRNA

micro RNA, non-coding, low levels, control of gene expression

Others
ncRNA

non-coding RNA. Low levels, involved in telomere synthesis, X

chromosome inactivation, transport of proteins to ER.
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Eukaryotic Gene and mRNA Structure

DNA

The pre-mRNA is
an exact copy of
the coding strand
genomic DNA
(contains introns)

Precursor
RNA

Mature
mRNA

Pearson 2010

Pre-mRNA

mRNA Maturation: Overview

1. 5 Capping
2. 3 Polyadenylation
3. Splicing

mRNA maturation occurs in nucleus

after quality control check, the mRNA
is exported to cytoplasm for translation

4. RNA editing
(but relatively rare)

m7G

AAAAAn
Keegan et al. Nature Rev Genet. 2:870, 2001

Structure of a Typical Eukaryotic mRNA

Initiation codon
(translation)
(AUG)
5 Cap 5 untranslated region:
5UTR
(inverted G)

Termination codons
(translation)
(UGA, UAA, UAG)

3 untranslated
region:
3UTR

Poly(A) tail

Features and Roles of UTRs

5 UTR
- starts at the first transcribed nucleotide and ends at the AUG initiation codon
- contains regulatory elements for translation
3UTR
- starts at the first nt after the termination codon and ends at the poly(A) sequence
- can contain regulatory elements for mRNA stability and/or mRNA localization
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Pre-mRNA

mRNA Maturation: 5 Capping

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Roles of 5 cap

Protects mRNA against exoribonucleases

Increases efficiency of mRNA translation
Increases efficiency of splicing of the first exon
Increases efficiency of mRNA export from nucleus

5 Cap is Methylated (5-7-methylguanosine)

Features of 5 methyl cap
1

7
3

7-methyl guanosine (m7G) is added

co-transcriptionally by a capping
enzyme when the pre-mRNA reaches
about 30 nucleotides in length
Unusual 5 -to- 5 linkage between
ribose groups

The 2OH groups of the first two riboses

in the chain can also get methylated

Russell text Fig. 5.10 (2010)

Assembly of the 5 7-Methyl Cap (m7G)

1st nucleotide at 5 end of RNA
which is being synthesized
GTP

RNA triphosphatase activity

removes g phosphate
GMP

Guanylyltransferase
(1) removes g and b phosphates from GTP
(2) GMP transferred to b phosphate of RNA

1st and 2nd nts can

also be methylated
at 2 position of m7G
ribose
2-O-methyl

Methyltransferase adds
methyl group (CH3) at
position 7 of guanine

Watson text Fig. 3-24 (2014)

Formation of 2-O-Methyl Cap1 and Cap2

methylation of 2'-hydroxy group

of ribose of 1st nucleotide of RNA
by RNA methyltransferase to form
cap 1

and in some cases

methylation of 2'-hydroxy
group of ribose of 2d
nucleotide of RNA to form
cap 2

Kruse et al. Sci Rep 1:126 (2011)

Role of 2O-methylation
assists binding of eIF4E (eukaryotic translation initiation factor 4E)

5 m7G Cap: Capping Enzymes Summary

Nature Reviews Molecular Cell Biology 9, 810-815 (October 2008)

5 capping is coupled with transcription

CE: capping enzyme

RNMT: RNA methyltransferase

5 maturation enzymes bind to CTD of RNA pol II so they can efficiently

act on their substrate (which is the 5 end of nascent RNA)
RNA triphosphatase & guanylyltransferase activities are in one protein (CE)
in some eukaryotes (human), but separate proteins in others (yeast)

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Pre-mRNA

mRNA Maturation: Polyadenylation

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Post-transcriptional enzymatic process (but coupled with

transcription and 3 processing proteins are docked on CTD)
Addition of 20 250 adenosines at the 3 end of the pre-mRNA
Roles of polyadenylation:
Protects against degradation by exonucleases
Increases the efficiency of mRNA nuclear export
Involved in pre-mRNA transcription termination
Increases translation efficiency
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Pre-mRNA

mRNA Maturation: Polyadenylation

CPSF: binds poly (A) signal

sequence located 10-30 nts
upstream of cleavage site
CstF: binds a GU-rich
sequence downstream of
cleavage site (black arrow)
CFI/CFII: Cleavage
Factor Protein I/II

1. Polyadenylation is triggered
when the pre-mRNA is cleaved

Stop
codon
polyA signal
sequence

2. Adenosine monophosphates
are sequentially added by PAP
(polyadenylate polymerase)
3. PABII proteins bind the
nascent poly (A) tail
4. PAP stops adding As
when it can no longer
5. RNA pol II stops RNA synthesis
bind CPSF due to the
shortly after CPSF/CstF/PAP
large numbers of As
Russell text Fig. 5.11 (2010)
complex dissociates

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Co-ordination of Polyadenylation with Transcription

AATAAA
in DNA

C-terminal domain
(CTD) of RNA Pol II
[phosphorylated
state]
RNA Pol II

AAUAAA
in RNA
http://slideplayer.com/slide/4343217/

Cleavage/polyadenylation machinery moves from CTD docking site

to specific signal site (AAUAAA) in pre-mRNA

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Pre-mRNA

mRNA Maturation: mRNA Splicing

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Splicing: Process to remove introns and join exons

Intron: Non-coding sequence in a gene that is removed from
precursor RNA by splicing
Exon: Segment of a gene that codes for a protein (textbook definition)
but introns can also occur in UTR regions of protein-coding genes
and non-coding RNA genes can also have introns
Exon is segment of precursor RNA that remains in the mature RNA
whereas an intron does not
Multicellular eukaryotes typically have many introns (human)
but some eukaryotes have very few introns (yeast)

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Pre-mRNA

mRNA Maturation: mRNA Splicing

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Roles of introns:
Increase genetic diversity through alternative splicing (later slides)
Can contain regulatory elements that influence transcription
Splicing can assist expression (eg. proteins bound at spliced
junctions help mRNA export from nucleus)
Long introns can contain other genes (eg. non-coding regulatory
RNA genes)
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mRNA Splicing: Discovery of introns

Electron micrograph of human
ovalbumin mRNA:DNA hybrid (1978)

Affinity: RNA:DNA > DNA:DNA

R loop: created when an RNA molecule
dislodges the complementary DNA
strand in a DNA:DNA duplex
When researchers tried to anneal the
mRNA of adenovirus gene with its DNA
genome, they found that the DNA was
longer and there were DNA segments
that did not anneal to the mRNA (1976)
Watson text Box 14-1 (2014)

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RNA Splicing: Splice Site Recognition

3 splice site

5 splice site
Branchpoint

--(A/C)G GU(A/G)AGU---YNYURAY----(Y)nNCAG G-Upstream

exon

Polypyrimidine
tract
~ 20 50 nt

Intron
YNYURAY:
Y = pyrimidine
N = any base
R = purine
A = adenine
(branch-point site)

Downstream
exon

Splicing
reaction
--(A/C)G G-Spliced product

Intron

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Pre-mRNA

mRNA: Splicing Reaction

1st transesterification step
2OH of branchpoint A acts
as nucleophile to attack
phosphoryl group of G at 5
splice site

2d transesterification step
3 OH of upstream exon
attacks the phosphoryl
group at 3 splice site

Intron lariat
Watson text Fig. 14-4 (2014)

Spliced exons
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RNA Splicing: The Spliceosome

The spliceosome: Large catalytic complex containing numerous proteins
(~150) and 5 non-coding RNAs that participate in pre-mRNA splicing
Small non-coding RNAs (snRNAs) of the spliceosome: U1, U2, U4, U5 and U6
These snRNAs associate with RNA-binding proteins to form small
nuclear ribonucleic proteins (snRNPs or snurps)
snRNPs of the spliceosome:
U1 snRNP
U2 snRNP
U4 snRNP
U5 snRNP
U6 snRNP
U6/U2 snRNAs act
as ribozyme
(RNA with catalytic
activity)

snRNA

Length
(nt)

U1

165

Binds the 5splice site

U2

185

Binds the branch-point site and forms part of the

catalytic core

U4

116

U4 base-pairs with U6 to form U4/U6 snRNP

Modulates U6 snRNP catalytic activity

U5

145

Contributes to the transesterification process

U6

106

Catalyzes the splicing reaction with U2

snRNP function

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RNA Splicing: snRNA/pre-mRNA Interactions

U1 snRNP

.
intron 1 (5 end)

helps to expose
the 2-OH group

intron 1 (3 end)

U2 snRNP

At early stage in
spliceosome assembly:
U1 snRNA base-pairs
with 5 end of intron

U2 snRNA base-pairs with

branchpoint region
.dislodging the BBP (branch-point
binding protein)

At later stage: U6 snRNA

binds there
Interactions are dynamic
Watson text Fig. 14-6 (2014)

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RNA Splicing: snRNA/snRNA Interactions

snRNAs have complex RNA folding and interactions are dynamic

U6 snRNA base-pairs with U4 snRNA base-pair in pre-catalytic stage

and it base-pairs with U2 snRNA during catalysis

Wahl et al. Cell 136:701, 2009

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RNA Splicing: Spliceosome Assembly

Pre-catalytic steps
1. U1 snRNP binds to 5 splice site,
BBP to branchpoint, splicing protein
U2AF65 to polypyrimidine tract and
U2AF35 to 3 splice site
2. U2 snRNP binds branchpoint
and displaces BBP (branchpoint
binding protein)
3. U4/5U5/U6 tri-snRNP associates with
complex bringing 3 and 5 ends of intron
close together, U2AF65/35 are released

4. U1 snRNP is released from complex

Watson text Fig. 14-7a (2014)

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RNA Splicing: Spliceosome and Catalytic Reaction

4. Reorganization of interactions
among snRNPs and release of U4
snRNP from complex
5. First transesterification reaction
which generates branched intron
still attached to downstream exon
and the freed upstream exon (with
its 3OH nucleophile)
6. Second transesterification
reaction to join exons and
release intron as lariat
exon1

exon 2

Watson text Fig. 14-7b (2014)

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Alternative mRNA Splicing and its Outcome

Pathway 1 (upper lines)
Pre-mRNA

exon skipping

Pathway 2 (lower lines)

Blencowe Cell 126:37, 2006

Alternative splicing leads to exclusion or inclusion of particular regions of the

pre-mRNA and this typically results in different proteins from the same gene
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Different Protein Isoforms by Alternative Splicing

Human g-tropomyosin gene & transcripts:
Alternative splicing results in
different tropomyosin proteins

aTm
gTm
genes

. that are tissue-specific,

development-specific, have
different subcellular location
Neuron

Epithelial cells

Embryo
A symbols on schematic show polyadenylation sites.
Unfilled blocks show 3UTRs of various tropomyosin
mRNAs generated by alternative splicing

Many different splicing

pathways in exons 6-9 region
Blue proteins: g-tropomyosin isoforms
Pink & grey proteins: a-tropomyosin
alternatively-spliced isoforms
Gunning et al. Trends Cell Biol 15:333, 2005

Adult
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Other Consequences of Alternative Splicing

Introns within UTR regions can also undergo alternative splicing

Changes in regulatory sequences within the 5UTR may influence

translation
Changes in the 3UTR may affect sequences important for
mRNA stability and/or subcellular localization

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mRNA Editing in Humans: Deamination

U is recognized as a T
e.g., a CCC codon (proline) can
become CUC (leucine)

I is recognized as a G
e.g., a CAA codon (glutamine) can
become CGA (arginine)

Watson text (Fig.14-31, 2014)

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C-to-U RNA Editing of Apolipoprotein B pre-mRNA

In this case, editing generates two proteins that differ in their carboxy
terminal regions and have different biological functions
eg. apolipoprotein B in mammals

In liver: lipid transport in circulation,

LDL receptor binding domain

In intestine: truncated protein, role in

dietary lipid absorption
Lodish Fig. 8-19 (7th ed, 2013)

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Different Types of Eukaryote RNA

Types

Description

mRNA

messenger RNA, variable amounts, code for proteins

rRNA

ribosomal RNA, abundant, contribute to the structure of the

ribosomes

tRNA

transfer RNA, moderate levels, carry the amino acids and act as
adaptors during the translation process.

snRNA

small nuclear RNAs, low to moderate levels, involved in splicing

mechanisms.

snoRNA

small nucleolar RNAs, low to moderate levels involved in maturation

covalent modification of rRNAs.

miRNA

micro RNA, non-coding, low levels, control of gene expression

Others
ncRNA

non-coding RNA. Low levels, involved in telomere synthesis, X

chromosome inactivation, transport of proteins to ER.
29

Transcription and Processing of

Eukaryote pre-rRNA Transcripts
Transcription:
RNA pol I transcribes rRNA from
the multiple transcription units.
Each precursor contains
18S, 5.8S and 28S rRNAs.

Processing:
Cleavage of spacers and those
will later be degraded.

U3 snoRNA in involved in rRNA

maturation.

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Transcription and Processing of

Prokaryote pre-rRNA Transcripts
Transcription:
RNA pol transcribes rRNA genes.
Each precursor RNA (polycistronic)
contains
16S, 23S and 5S rRNAs + 1 to 2 tRNAs.

http://slideplayer.com/slide/6401193/

Processing:
1. site-specific methylation of pre-rRNA
using guide RNAs.
2. Nuclease cleavage with: RNase III,
RNase P, RNase E.
3. Processing with various specific
Nucleases.
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Processing of pre-tRNA Transcripts

RNA pol III (eukaryotes) transcribes tRNA genes .
Processing of 5end: cleavage by RNase P. (RNase P is a ribozyme: has catalytic activity)
Processing of 3end of tRNAs: cleavage by nuclease, the addition of
CCA terminus occurs post-transcriptionally in some cases, modification of some bases.

http://www.slideshare.net/narasimhareddy3954546/

Similar in prokaryotes: RNA polymerase rather than RNA pol III.

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Learning Objectives:
1.
2.
3.
4.
5.
6.
7.
8.

Illustrate the architecture of a pre-mRNA and of a mature mRNA.

Explain the reaction of polyadenylation of mRNA.
Explain the reaction of mRNA capping.
Why are pre-mRNAs spliced?
Identify important sequences in the pre-mRNA that are involved in splicing.
Explain the reaction of mRNA splicing.
Explain the roles and functions of snRNPs in splicing.
Explain a type of mRNA editing mechanism in humans.

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