Download M1 - Biochemistry Transcription III / mRNA Processing

Transcription III / mRNA Processing
Learning Objectives:
1. To know that prokaryotic mRNAs are virtually unprocessed
and that eukaryotic pre-mRNAs are drastically modified.
M1 - Biochemistry
2. To know the cap structure, its mechanism of addition, and
functions.
Transcription III / mRNA Processing
3. To know the polyA recognition sequence, how the polyA
tail is added and its functions.
PH Ratz, PhD
(Resources: Lehninger et al., 5th ed., Chapters 26 & 27)
4. To have a sense how introns and exons were discovered,
revealing that mature RNAs lacked sequences present in
the gene.
5. To describe the constituents and mechanism of action of
the spliceosome.
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Overview of post-transcriptional processing of mRNAs:
A. Eukaryotic mRNA primary transcripts are processed before they
become “mature” transcripts.
6. To understand the concepts of alternative splicing and
RNA editing, revealing that one gene can produce more
than one type of functional protein.
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Overview of post-transcriptional processing of mRNAs:
B. Prokaryotic mRNA primary transcripts are not processed, but
rather, are used directly as codons in protein synthesis (translation) as
they emerge from the transcription apparatus.
Usually > 1 gene
In prokaryotes, a set of adjacent genes
is often transcribed as a unit, and
therefore, the mRNA transcript may
carry information for several proteins.
1
“mature” transcript
2
In eukaryotes, a mRNA transcript
usually carries the information of a
single gene.
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Figure 6.21. Alberts,
Molec Biol Cell, 4th
1 transcript
…thus, usually, > 1 protein
Figure 6.21. Alberts,
Molec Biol Cell, 4th
RNA splicing can
occur after 3’
polyadenylation
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Comparison of mature mRNA transcript structures
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Eukaryotic mRNAs - Processing events include:
(1) cap addition to the 5’ end
RNA Pol II
}
Figure 6.22(a) Alberts,
Molec Biol Cell, 4th
Synthesis of the cap occurs after about
25 nucleotides have been synthesized
by Pol II, and is carried out by enzymes
(cap synthesizing complex) tethered
to the multiply phosphorylated carboxy
terminal domain (CTD) of Pol II. The
cap-binding complex (CBC) replaces
the cap-synthesizing complex, and the
cap remains tethered to the CTD
through association with the CBC.
CTD
Prokaryotic:
•5’ and 3’ mRNA transcript ends are unmodified
•mRNAs contain instructions for numerous proteins
Eukaryotic:
• 5’ cap, 3’ polyA tail
• Nearly always contain information for only a single protein
(but note protein isoform formation via intron excision)
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Figure 26.13(c) Lehninger, 5th ed.
Eukaryotic mRNAs - Processing events include:
(1) cap addition to the 5’ end
Eukaryotic mRNAs - Processing events include:
(1) cap addition to the 5’ end
What # is this?
5’
(“reverse”)
5’
The cap derives from a GTP that is
added to the 5’-most nucleotide of
the primary transcript. In the
process 3 Pi’s are lost and an
unusual 5’ to 5’ triphosphate bond is
formed. The position #7 N of the G
is then methylated and occasionally the 2’ hydroxyls of nucleotides
1 and 2 are also methylated.
Cap addition involves the
attachment of a GTP in an
unusual 5’-to-5’ bond to the 5’
end of the primary transcript,
methylation of the 7 position of
the guanine, and sometimes
methylation of several 2’ OH’s.
The overall decoration of the 5’
end is shown at left.
3’
5’
Figure 26.13(a) Lehninger, 5th ed.
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Eukaryotic mRNAs - Processing events include:
(1) cap addition to the 5’end
(2) removal of the intervening sequences “introns” with a splicing
mechanism.
(3) site-specific cleavage and polyA addition to the 3’ end
Eukaryotic mRNAs - Processing events include:
(1) cap addition to the 5’end
(2) removal of the intervening sequences “introns” with a splicing
mechanism.
(3) site-specific cleavage and polyA addition to the 3’ end, and
Only ~1.5% of the human
genome is translated into
protein (“exons”, or coding
segments of a gene). “Introns”
are intervening nontranslated
DNA sequences.
interrupt, the “exons,” which contain the
linear array of codons that direct the
sequence of amino acids in proteins.
Hence the introns must be removed
and the exons spliced together in the
correct order to maintain the fidelity of
the genetic code.
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10
Figure 26.18 Lehninger, 5th ed.
histone pre-mRNAs, whose genes lack
introns). These “introns” separate, or
(note that, although not shown here, the 5’ cap is actually bound to the CTD of Pol II)
Binds to
Pol II CTD
- Synthesis of the mRNA transcript by Pol II goes
beyond
the
cleavage
signal
sequence
((5’)AAUAAA upstream, & a poorly defined
sequence rich in G & U downstream, from the
cleavage point).
- An enzyme complex including a cleavage
sequence recognition protein, endo-nuclease,
polyadenylate polymerase, and poly(A) lengthregulating protein cleaves at the cleavage point
(between AAUAAA & G&U regions), then adds
nucleotide (“A’s”).
-Poly(A) tail serves as a binding site for a
- 1) protein that protects the 3’ terminal transcript
from accidental exonuclease activity, and
- 2) a protein that interacts with the cap
complex to facilitate initiation of translation
Most eukaryotic heteronuclear primary
transcripts (precursor mRNAs) contain
intervening sequences derived from
corresponding sequences in the gene
(an exception is the absence of splicing of
Figure 26.12 Lehninger, 5th ed.
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Figure 26.13(b) Lehninger, 4th ed.
Eukaryotic mRNAs
- Processing events include:
(1) cap addition to the 5’ end
(2) (3) site-specific cleavage and poly(A)
(80-250 A’s) addition to the 3’ end
The RNA Pol II highly phosphorylated CTD not only
has the “capping factors” bound to it, but also bound
are polyadenylation factors and splicing factors
Figure 6-23 Alberts et al., “Molecular Biology of the Cell, 4th
The unusual bonding of the cap:
1) Protects 5’ ends of the mature
product against adventitious exonucleolytic cleavage.
2) Facilitates splicing.
3) Permits binding of eukaryotic
translational initiation factors to
the cap that promotes binding of the
small ribosomal subunit to the start
codon on the mRNA.
Snapshot of the human genome – types of
sequences. Figure 24.8 Lehninger 5th ed.
There are 4 classes of introns:
The 1st 2 classes only are
designated by Group #s. These
are self-splicing introns found
mostly in mitochondrial and
chloroplast & some nuclear
mRNA (& in archebacteria >
eubacteria). The 4th class is
mostly within tRNAs.
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The following diagram shows an overview of the eukaryotic mRNA
processing including the removal of lettered introns and splicing
together of exons L (encodes a peptide sequence that targets the protein
for export from the cell) and 1-7. Note in this example that ~3/4 of the
total primary transcript length is removed during processing.
The existence of intronic sequences was determined in part by
electron microscopy of mature RNA complexed to its template
DNA strand, revealing unhybridized DNA loops that were not
represented in the mature transcript. The EM below shows such
an analysis of the ovalbumin gene.
Mature
Taken from Lehninger et al. “Principles of
Biochemistry” 3rd ed.
Figure 26.19 Lehninger, 5th ed.
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Self-Splicing: It was during the characterization of intron removal that the
existence of self-splicing RNA was discovered (in 1982) in non-mammalian
systems. In the absence of any protein subunits, RNA displayed the ability to
accurately remove its own intron, giving rise to the term “ribozyme” (RNA
with catalytic activity like that of protein enzymes).
“The discovery that RNAs could have catalytic functions was a milestone in our
understanding of biological systems” - from Lehninger, 4th ed. Until the discovery of
ribozymes, all biological catalysts were known to be proteins. Relatively few catalytic
RNAs exist in modern-day cells.
S ÅÆ P
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As an example of a self-splicing
(ribozyme-catalyzed) reaction, see
the Group II intron splicing
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mechanism in this Figure.
Note that the process is initiated via the
attack by a 2’ OH of an “A” nucleotide
within the intron on the 5’ splice site to
form a lariat structure.
catalysts
Figure 6.5(a&c) Lehninger, 5th ed.
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Catalysts enhance reaction rates by lowering activation energies…reaction reaches
equilibrium more quickly when a catalyst is present.
3’ OH is a nucleophile -causes
phosphodiesterase hydrolysis
Spliceosome assembly: The remainder of the process showing the
involvement of the other snRNPs, the cleavage of the 5’ splice site, the attack
on the 3’ splice site, and the release of the lariat intron is diagrammed below.
After splice site recognition by
binding of U1 & U2 (energyrequiring)…..
Base pairing here
forms a bulge that
displaces and
activates adenylate
for it’s 2’ OH attack
Figure 26-17(a) (& 26.23, pseudouridine) Lehninger 5th ed.
5’
2’ 3’
The new 3’ OH of the 5’ exon attacks
the new 5’ end of the 3’ exon
expelling the now lariat-structured
intron. The mature product contains
the contiguous set of codons
specifying the proper amino acid
sequence of the encoded protein.
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S ÅÆ P
Adenylate nucleotide that is an attacking nucleophile
5’
Figure 26-16
Lehninger 5th ed.
2
MOST INTRONS ARE NOT SELF-SPLICING. The 3rd and largest class of
introns includes those found in eukaryotic nuclear mRNA transcripts.
This class is NOT given a “group” designation, but called…
Spliceosome introns: The splicing chemistry is the same as in the lariatforming Group II introns, except the process is not self-splicing. Instead there
is a number of small nuclear ribonucleoproteins (“snRNPs” sometimes called
“snurps”) that catalyze the process. The large aggregate of catalytic snurps
is referred as the “spliceosome.” The process is initiated by the U1 and U2
small nuclear RNAs (snRNAs) that have some complementarity to the 5’
splice site and the region around what will become the attacking A nucleotide.
There is sequence specificity around the intron/exon boundaries. For
example virtually all introns of this type start with 5’-GU and end with AG-3’.
Trans-esterification
leaves a free 3’ OH
the U4/U6 complex and the U5
bind (also energy-requiring) to
form an inactive spliceosome.
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Figure 26-17(b) Lehninger 5th ed.
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Certain spliceosome components are bound
to CTD of Pol II. Thus, Pol II serves as a
scaffold for spliceosome assembly.
Now you can see why the 5’ Cap of the
mRNA transcript is attached to the CTD of
Pol II.
A complex & energy-requiring
snurp rearrangement involving
U1 & U4 displacement leads to
an active spliceosome. Note
that U6 is now paired with the
5’ exon (splice-site) and U2
Figure 26-17(b) Lehninger 5th ed.
Lariat formation and removal of
lariat-shaped intron occurs by the
same chemistry and catalytic steps
as in the splicing of group II introns.
Note that the GU and AG are part of
the intron. Note also that ATP does
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not seem to be required at this point.
An aside: Thalassemia, a form of anemia common in the Mediterrarean
countries, is caused by errors in the splicing process
Normal red blood cells
contain correctly spliced
beta-globin, an
important component in
hemoglobin that takes
up oxygen in the lungs.
The red blood cells in
thalassemia patients are
distorted and sometimes
immature, containing a
nucleus. This is due to a
point mutation in the betaglobin gene, which causes
an error in splice site
selection. A faulty betaglobin protein is made,
leading to severe anemia.
Arrows mark two
examples of sites where
point mutations causing
thalassemia occur in the
beta-globin gene.
This keeps the mRNA transcript close-by so
that splicing occurs efficiently. Once the
spliceosome is assembled after the 1st splice
and near Pol II’s catalytic domain for mRNA
synthesis, the next intron will be close to the
spliceosome to be efficiently captured as it
passes by….etc.
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Figure 26-17(c) Lehninger 5th ed.
Why are there introns???? Don’t know, but an evolutionary advantage may be
that splicing errors potentially can lead to functionally different but useful
proteins that come about without having to make a new gene.
Alternative (differential) splicing of a precursor-RNA from the same genes
can, for some genes, be matured to mRNAs with alternative structures in a
tissue-specific manner! This then leads to differential protein expression in
different tissues (NOTE THAT THIS IS NOT 1 GENE = 1 PROTEIN!).
An example where alternative splicing has a dramatic consequence is
somatic sex determination in the fruit fly Drosophila melanogaster. The
female-specific sxl-protein is a key regulator that controls a cascade of
alternative RNA splicing decisions that finally result in female flies.
Mature mRNA for femalespecific sxl-protein
Mature mRNA for malespecific sxl-protein
From Roberts & Sharp 1993 lectures “Nobelprize.org”
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From Roberts & Sharp 1993 lectures “Nobelprize.org”
An excellent example of alternative splicing is the calcitonin gene:
- produces calcitonin (lowers blood Ca2+ levels) in the thyroid
- produces CGRP (calcitonin gene-related peptide, a vasodilator), in neurons.
- low CGRP levels, perhaps due to faulty splicing, have been noted in
individuals with Raynaud Syndrome.
-The multiple poly(A)
recognition sites direct
the poly(A) addition at
different locations.
“4” is part of the
intron in brain, &
thus, is eliminated
RNA Editing Changes the Sequence of mRNAs. Editing commonly
occurs during mRNA maturation, & is carried out by enzymes that
recognize a particular C (usually) that is hydrolytically deaminated to a
U. An example is the apo B gene transcript that produces apoB100
normally in the liver, but is edited to apoB48 in the intestine. The apoB100
is a component of lipoprotein complexes (LDL, VLDL, IDL) that transport
lipids in the serum. However, in the intestine, the primary transcript is
edited, converting a CAA Gln codon to a UAA Stop. Hence the intestinal
protein product (apoB48) is truncated in the translation process to form a
version lacking the C-terminal end. This shortened version carries
primarily chylomicrons in the intestine. This gene and its different modes
of expression are diagrammed below.
-Splicing occurs
AFTER poly(A)
addition.
Note that there also
are post-translational
modifications.
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Figure 26-21. Alternative splicing of the calcitonin transcript Lehninger 5th ed.
Binds lipids
Binds LDL Rs
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From Lodish et al. “Molecular Cell Biology” copyright © W.H. Freeman 2004; Fig 12-17