RNA editing (cap, polyA, splicing) 1

MLAB 230 - Molecular Biology

Topic 5: in eukaryotes only!!!

RNA editing (cap, polyA, splicing)

RNA protection
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Post transcription initiation events :

➢ Once RNA polymerase is in position at the promoter, the next step of
transcription—elongation—can begin

➢ Unlike bacterial mRNAs, eukaryotic mRNAs are modified at the 5’ and 3’

ends.

➢ Some of these modifications start while the mRNA is in the elongation phase
and are necessary for the maturation, stability and translation of this mRNA.

➢ In the processing of pre-mRNA to mature mRNA, eukaryotic mRNAs undergo

three main modification :

• Capping at the 5’ end

• Polyadenylation at the 3’ end
• Splicing of exons
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5’ modification or Capping
➢ Once RNA polymerase II has made about 20 to 30 nucleotides of pre-mRNA,
a capping enzyme (guanylyltransferase) adds a guanine nucleotide, most
commonly, a methylated guanosine (m7G)—to the 5’ end.

➢ This addition is based on an unusual 5’ to 5’ linkage between both

nucleotides rather than the classical 5’ to 3’.
➢ This process is called 5’ Capping
m7G is not sensitive to exonucleases since:
1) it is methylated
2) exonuclease cleave a classical bond
5'-3', which is not the case for m7G (5'-5')

➢ The sugars of the next two nucleotides are also modified by methylation
(addition of a –CH3 methyl group) to increase protection
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5’ modification or Capping

5’ to 5’ linkage

7-methyl-guanosine
Methyl
groups
5’ end of RNA chain
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5’ modification or Capping
➢ The 5’ cap remains throughout processing and is present in the mature
mRNA.

➢ It plays two important roles:

1. The 5’ cap protects the mRNA against degradation by exonucleases

because of the unusual 5’ to 5’ linkage (exonucleases usually cleave
conventional 5’-3’ phosphodiester bonds).

2. The 5’ cap is also crucial for the binding of the ribosome as an initial step of
translation.
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3’ modification or Polyadenylation
➢ Most eukaryotic pre-mRNAs become modified at their 3’ ends by the addition
of a sequence of about 50 to 250 adenine nucleotides called a poly(A) tail.

➢ mRNA molecules with 3’ poly(A) tails are called poly(A)+ mRNAs

➢ The poly(A) tail remains when the pre-mRNA is processed to mature mRNA
and is required for efficient export of the mRNA to the cytoplasm.

➢ In the cytoplasm, the poly(A) tail protects the 3’ end of the mRNA by buffering
coding sequences against early degradation by exonucleases.
does not prevent the action of exonuclease (like m7G) but rather delays
it/buffers it by adding several A nucleotides
➢ The poly(A) tail also plays important roles in the initiation of translation by
ribosomes and in processes that regulate the stability of mRNA.

polyA tail, being at the end, plays a role in the initiation of translation where factors bind to polyA
and 5' cap and RNA bends
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How is the poly(A) tail synthesized ?

➢ Addition of the poly(A) tail is associated with the termination of transcription
of protein-coding genes.

➢ Addition of the poly(A) tail is signaled when mRNA transcription proceeds

past the poly(A) site, a site in the RNA transcript that is about 10 to 30
nucleotides downstream of the poly(A) consensus sequence 5’-AAUAAA-3’.

➢ The Poly(A) site is followed downstream by a GU/U rich region

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How is the poly(A) tail synthesized ?

Consensus
sequence

Poly(A) site
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How is the poly(A) tail synthesized ?

➢ The consensus sequence leads to the recruitement of the polyadenylation
machinery to the Poly(A) site

➢ This machinery is composed of various proteins :

• CPSF (cleavage and polyadenylation specificity factor)
• CstF (cleavage stimulation factor)
• CFI and CFII (two cleavage factor proteins)
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How is the poly(A) tail synthesized ?

Consensus
sequence

Poly(A) site
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How is the poly(A) tail synthesized ?

Consensus
sequence

Poly(A) site
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How is the poly(A) tail synthesized ?

polymerase that dsnt need a template
➢ The enzyme poly(A) polymerase (PAP), which is bound to CPSF, adds A
nucleotides to the 3’ end of the RNA using ATP as the substrate to produce
the poly(A) tail.

➢ Poly(A) binding protein II (PABII) molecules bind to the poly(A) tail as it is

synthesized to stabilize the polyadenylation complex, regulate the addition of
A nucleotides and protect the mRNA.
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How is the poly(A) tail synthesized ?

Consensus
sequence

Poly(A) site
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Termination of transcription in Eukaryotes

➢ RNA polymerase II is still synthesizing RNA although that RNA is not part of
the mRNA.

➢ RNA pol II can keep synthesizing thousands of nucleotides before being

stopped because protein-coding genes do not have specific terminator
sequences.

➢ Eukaryotic genes transcribed by RNA polymerases I and III do have specific

terminators.

➢ The process differs for each of the three RNA polymerases. The mechanism
of termination is the least understood of the three transcription stages.

like rho-dependent
➢ For RNA pol II transcribed genes, an exonuclease (Xrn2 in humans) binds to
the post-poly(A) site RNA and starts to degrade it. When it catches up to the
RNA polymerase II it destabilizes the enzyme–transcription factor–DNA
complex. RNase degrades it faster than pol --> catches up and
dissociates the complex
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Termination of transcription in Eukaryotes

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Processing pre-mRNA to mature mRNA

➢ Pre-mRNAs contain a number of introns that must be excised from each pre-
mRNA to produce a mature mRNA that can be translated into the encoded
polypeptide.
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Processing pre-mRNA to mature mRNA

➢ Consensus sequences around the splice sites provide signaling for the
splicing machinery.
consensus signals

• The only nearly invariant bases are the 5′GU and the 3′AG of the intron
• The branch-point adenosine, also invariant, is usually 20–50 bases from the 3′
splice site

➢ The central region of the intron, which may range from 40 bases to 50
kilobases in length, is generally unnecessary for splicing to occur.
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mRNA splicing
➢ The splicing events occur in a spliceosome, a complex of :
• Pre-mRNA
• Small nuclear RNA (snRNA)
• Proteins

➢ The five principal snRNAs are U1, U2, U4, U5, and U6; each is associated
with a number of proteins to form the small nuclear RiboNucleoProteins
snRNPs).

➢ Each snRNP type is abundant in the nucleus, with at least 105 copies per
cell.
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mRNA splicing
➢ U1 snRNP binds to the 5’ splice junction of the intron. This binding is
primarily the result of base pairing of U1 snRNA in the snRNP to the 5’ splice
junction.

1
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mRNA splicing
➢ U2 snRNP binds to the branch point sequence, which is located upstream of
the 3’ splice junction. This binding occurs as a result of the base pairing of U2
snRNA in the snRNP to the branch-point sequence

2
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mRNA splicing

3 ➢ A U4/U6 snRNP and a U5 snRNP

interact, and the combination binds to
the U1 and U2 snRNPs, causing the
intron to loop and thereby bringing its
two junctions close together.

Splicing sites
come close
together
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mRNA splicing
➢ U4 snRNP dissociates, resulting in
the formation of the active
spliceosome

4
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mRNA splicing

3' G is taken --> 5' G will be involved

3' and 5' A are taken --> 2'A will be involved

SO, 5'G and 2'A will link

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mRNA splicing
➢ The snRNPs in the spliceosome cleave the intron from exon 1 at the 5’ splice
junction, and the now free 5’ end of the intron bonds to a particular A
nucleotide in the branch-point sequence.

➢ Because of its resemblance to the rope cowboys use, the loopedback

structure is called an RNA lariat structure.

➢ The branch point in the RNA that produces the lariat structure involves an
unusual 2’–5’ phosphodiester bond formed between the 2’ OH of the adenine
nucleotide in the branch-point sequence and the 5’ phosphate of the guanine
nucleotide at the 5’ end of the intron. The A itself remains in normal 3’–5’
linkage with its adjacent nucleotides of the intron.

➢ Next, the spliceosome excises the intron (still in lariat shape) by cleaving it at
the 3’ splice junction and then ligates exons 1 and 2 together. The snRNPs
are released at this time.
If there is a mutation in any of the consensus
sequences of the intron, then intron is retained
➢ The process is repeated for each intron.
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number of proteins in the body are greater than the

mRNA alternative splicing number of genes coding for them --> this is due to
alternative splicing

➢ But the process might not happen on each intron and is tightly regulated to
improve gene expression and mRNA production.

➢ There are many cases in which alternative splicing (also called differential
splicing) may be used to produce different functional mRNAs.

➢ Which product is generated depends on regulatory signals mostly tissue

specific or in response to different conditions. the same mRNA may give protein A in a
certain tissue but protein B in another

➢ The products of alternative splicing are proteins (isoforms) that are encoded
by the same gene, but that differ structurally and functionally.

➢ 90% of human genes undergo alternative splicing.

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Five ways to splice RNA

UTR
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The tissue specific Calcitonin gene splicing

Alternative
polyadenylation
sites
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The tissue specific Calcitonin gene splicing

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RNA editing
➢ RNA editing involves the post-transcriptional insertion or deletion of
nucleotides or the conversion of one base to another. mostly found in
tRNA
small nucleolar RNA
➢ The functional RNA molecule has a base sequence that does not match the
base-pair sequence of its DNA coding sequence.

➢ There are three main nucleotide conversions :

• C→ U
• A→ I (inosine)
• U→ ψ(pseudouridine)
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The deamination of cytosine

➢ That process typically occurs only in certain tissues or cell types and in a
regulated manner.
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The deamination of adenosine

➢ This reaction is performed by the enzyme ADAR (adenosine deaminase
acting on RNA).
➢ Inosine can base-pair with cytosine, and thus this change can readily alter
the sequence of the protein encoded by the mRNA.
➢ This type of editing—enzymatic deamination—seems to be quite rare, but
important ex: the absence of this editing in an ion channel expressed in
mammalian brains seriously impairs brain function.
diff aa
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The conversion of uridine to pseudouridine

➢ The functions of these base and ribose
modifications are not well understood,
but since they are highly conserved,
they probably have a positive influence
on protein synthesis.

➢ Are mediated by snoRNAs in the

nucleoli.

➢ Most present in tRNA and rRNA.