Part 5

RNA Processing

Chapter 26, Lehninger 6 ed., 7ed.

Chapter 29, Stryer 7 ed., 8 ed.

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 Contents
• Types of RNA processing
• Discovery of mRNA splicing剪接
• Group (class) I and II intron and self-splicing
mechanism
• Splicing of spliceosomal intron for mRNA
• Splicing of group IV intron for tRNA
• Splicing problem causing disease
• Differential (alternative) RNA splicing
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 RNA Processing is common
• Virtually all RNA molecules in eukaryotes are
processed to some degree after synthesis.
• Primary transcript: a newly synthesized RNA
molecule.
– The immediate product of RNA polymerase II is
referred to pre-mRNA (precursor-to-messenger RNA)
• Nearly all mRNA precursors in higher eukaryotes
are spliced.
– Introns are excised and exons are joined to form
mature mRNAs.
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 Processing of mRNA in eukaryotes
Types of mRNA processing:
• 5’ Capping
– A modified residue called a 5’ cap is added at the 5’ end.
– The 5′ cap (red) is added before synthesis of the primary transcript is
complete.
• 3’ Tailing
– The 3’ end is cleaved, and 80 to 250 A residues are added to create a
poly (A) “tail”.
• RNA editing
– Change of nucleotides in mRNA after transcription
• Splicing
– Elimination of intervening sequences (introns)
– Joining of structural sequences (exons)
→ forming a continuous sequence that specifies a functional
polypeptide.
– Splicing can occur either before or after the cleavage and
polyadenylation steps.

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 Discovery of mRNA splicing: Finding of R-loops

• Chicken ovalbumin gene is hybridized with ovalbumin mRNA

• The gene pairs with complementary regions of RNA and seven
introns (A-G) loop out of the RNA –DNA hybrid

Fig. 20-5
Watson 4th ed.
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mRNA is shorter than genomic DNA: by hybridization
experiment

• Digest chromosomal DNA from chicken oviduct or erythrocyte

with EcoRI. It is known that there is no such restriction enzyme
recognition site in the ovalbumin cDNA
• separate restriction fragments by gel electrophoresis
• transfer the restriction fragments to a piece of membrane
• hybridize DNA with labeled cDNA, which is reverse transcibed
from the ovalbumin mRNA
• several radioactive bands are found, indicating that the genomic
DNA has EcoRI sites
• The total length of the bands is larger than the cDNA,
implicating some sequences in the gene are not found in the
cDNA

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Demonstration of interrupted structure of the ovalbumin gene by
hybridization experiment

Fig. 20-4 Watson 4th 7ed.

 Organization of chicken ovalbumin gene

• Total length: 7700bp

• Intron内含子– segment that does not specify the mRNA: 7, from
about 250bp to 1600bp
• Exon外顯子– protein-coding sequences and the untranslated 5’ and
3’ residues present in the mRNA: 6, from 47bp to 1043bp

Fig. 20-5
Watson 4th ed.

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 mRNA splicing
• Signal conserved from yeast to human
– Intron begins with GU and ends with AG.
– A branch site is located 20-50 nucleotides upstream of
the 3’ splice site.
– Introns vary from 50 to 10,000 nucleotides.
– Specific sequences near the splice sites in both introns
and exons also important for splicing regulation.

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 Classes (Groups) of introns
• Four Classes in total
• Group I introns:
– Found in nuclear, mitochondrial and chloroplast genes
that code for rRNAs, mRNAs, and tRNAs.
• Group II introns:
– Generally found in mitochondrial or chloroplast mRNA
in fungi, algae, and plants.
Similarity of Group I and Group II:
– No high energy cofactors (e.g. ATP) are required for
splicing.
– Self-splicing: no protein enzymes are involved.
– Two transesterification reaction轉酯化反應steps are
involved.
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 1st step of transesterification for Group I introns

Note: 3’ OH of a guanosine molecule acts as nucleophile, attacking the

phosphodiester linkage between U and A residues at an exon-intron junction of an
mRNA. This guanosine is not used as a source of energy.
 Splicing mechanism of
Group I introns

1st step
1st transesterification

2nd transesterification

2nd step
1st transesterification
Splicing mechanism
of Group II introns
The chemistry is
similar to that of
group I intron
splicing, except for
the nucleophile in
the first step, which
is the 2’ hydroxyl
group of an A
2nd transesterification residue within the
intron and the
formation of a lariat
like intermediate.

Self-splicing implies
that RNA has
catalytic function.
 Class III - Spliceosomal introns
• Most introns are not self-splicing
• The third and largest class of introns includes
those found in nuclear mRNA primary
transcripts
– These are called spliceosomal introns, because
their removal occurs within and is catalyzed by a
large protein complex called a spliceosome剪接體
– Intron is spliced by the same lariat-forming
mechanism as the group II introns
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 Spliceosome
• The spliceosome is made up of specialized
RNA-protein complexes, small nuclear
ribonucleoproteins (snRNPs, often
pronounced “snurps”)
• Each snRNP contains one of a class of
eukaryotic RNAs, 100 to 200 nucleotides long,
known as small nuclear RNAs (snRNAs).
– U1, U2, U4, U5, and U6 are generally found in
abundance in eukaryotic nuclei
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Small nuclear ribonucleoprotein particles (snRNPs) in
the splicing of mRNA precursors

The U1 snRNP binds to the sequences near the 5’ splice site of

nuclear mRNA introns. Addition of the U2, U4, U5 and U6
snRNPs leads to formation of the spliceosome.
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Splicing mechanism in mRNA primary transcripts
• Spliceosomal introns generally have the dinucleotide
sequence GU at the 5’ end and AG at the 3’ end, and
these mark the sites where splicing occurs.

• Formation of spliceosome
– The snRNPs together contribute five RNAs and about 50
proteins to the core spliceosome.
– Perhaps 50 additional proteins are associated with the
spliceosome at different stages in the splicing process.
– ATP is required for assembly of the spliceosome, but RNA
cleavage-ligation reactions do not seem to require ATP.
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Pairing between U RNA in the spliceosome and the splice junctions

• The U1 snRNA has a sequence • U2 is paired to the intron at a

near its 5′ end that is position encompassing the A
complementary to the splice residue (shaded pink) that becomes
site at the 5′ end of the intron. the nucleophile during the splicing
• Base pairing of U1 to this reaction.
region of the primary • Base pairing of U2 snRNA causes a
transcript helps define the 5′ bulge that displaces and helps to
splice site during spliceosome activate the adenylate, whose 2′ OH
assembly. (Ψ is pseudouridine). will form the lariat structure through
a 2′,5′-phosphodiester bond.
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Splicing mechanism in
mRNA primary transcripts
(part 1)
• Assembly of spliceosomes: The
U1 and U2 snRNPs bind, then
the remaining snRNPs (the U4-
U6 complex and U5) bind to
form an inactive spliceosome.
• Internal rearrangements
convert this species to an
active spliceosome in which U1
and U4 have been expelled and
U6 is paired with both the 5′
splice site and U2.

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 Splicing mechanism in mRNA
primary transcripts (part 2)
• The formation of active
spliceosome is followed by the
catalytic steps, which parallel those
of the splicing of group II introns.

https://www.youtube.com/watch?v=CdwLKwseP9Q
Coordination of splicing and
transcription
• Some components of the splicing
apparatus seem to be associated
to the CTD of RNA polymerase II.
• The first splice junction is
synthesized, it is bound by an
associated spliceosome.
• The second splice junction is then
captured by this complex as it
passes, facilitating the
juxtaposition of the intron ends
and the subsequent splicing
process.
• After splicing, the intron remains in
the nucleus and is eventually
degraded.
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Eukaryotic mRNA splicing involves which of the following?

(a)The formation of 2’-5’ phosphodiester bonds

(b)A sequence-specific endoribonuclease that hydrolyzes the
phosphodiester bond at the junctions of the intron with the
exon
(c)The spliceosome
(d)The coupling of phosphodiester bond formation to ATP
hydrolysis
(e)The formation of lariat intermediates

A. a, b, c
B. a, c, e
C. b, c, d
D. b, d, e
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Which one of the following is not true for mRNA splicing?
A. Exons are used for polypeptide synthesis.
B. Introns are complementary to their adjacent exons and
will form hybrids with them.
C. The mRNA is originally synthesized in the nucleus, but
ends up in the cytoplasm.
D. The splicing that yields a mature mRNA occurs at very
specific sites in the RNA primary transcript.

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 Class IV intron
• Found in certain tRNAs
• Splicing reaction requires ATP and endonuclease
• Splicing endonuclease cleaves the phosphodiester binds at both ends of
the intron
• The two exons are joined by RNA ligase

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 Slicing problem causes disease

• An A-to-G mutation within the first intron of the gene for human hemoglobin
血红蛋白β chain create a new 5’ splice site (GU).
• Both 5’ splice sites are recognized by the U1 snRNP; so splicing may
sometimes create a normal mature mRNA and an abnormal mature mRNA
that contains intron sequences.
• Results in thalassemia地中海貧血. 25
 Differential RNA processing
• Some eukaryotic mRNA transcripts can be
processed in more than one way to produce
different mRNAs and thus different
polypeptides.
– The primary transcript contains molecular signals
for all the alternative processing pathways,
• Alternative modes of gene expression allow the
production of more than one protein from a
single gene.
→This allows for greater complexity in the proteins
generated from our gene complement.
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 Differential RNA processing
• The pathway favored in a given cell is
determined by processing factors, RNA-binding
proteins that promote one particular path.
• Complex transcripts can have either:
– (i) more than one site for cleavage and
polyadenylation多腺苷酸化, or
– (ii) alternative splicing patterns, or both.
→ In both mechanisms, different mature mRNAs are
produced from the same primary transcript.

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Alternative processing of
complex transcripts in
eukaryotes
Mechanism 1

• Alternative cleavage and

polyadenylation patterns.
• Two poly(A) sites, A1 and A2, are shown.
• If there are two or more sites for
cleavage and polyadenylation, use of
the one closest to the 5’ end will
remove more of the primary transcript
sequence.
• This mechanism, called poly (A) site
choice, generates diversity in the
variable domains of immunoglobulin
heavy chains.

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Alternative processing of
complex transcripts in
eukaryotes
Mechanism 2

• Two different 3′ splice sites are

shown.
• Alternative splicing patterns
produce, from a common
primary transcript, different
forms of the myosin肌球蛋白
heavy chain at different stages of
fruit fly development.

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 Differential RNA processing
– Both mechanisms come into play when a single RNA
transcript is processed differently to produce two
different hormones.
• e.g. the calcium-regulating hormone calcitonin降鈣素in rat
thyroid and calcitonin-gene-related peptide (CGRP) for
vasodilation and for the transmission of pain in rat brain.
– Alternative processing of the calcitonin gene transcript
in rats:
• The primary transcript has two poly(A) sites; one
predominates in the brain, the other in the thyroid.
• In the brain, splicing eliminates the calcitonin exon (exon 4); in
the thyroid, this exon is retained.
• The resulting peptides are processed further to yield the final
hormone products: calcitonin-gene-related peptide (CGRP) in
the brain and calcitonin in the thyroid. 30
Alternative processing of the calcitonin gene transcript in rats

6kb

31
32aa 37aa for α form