RNA Splicing
Processing of Primary RNA
Transcripts into mRNA
Possible post-transcriptional
controls on gene expression.
Only a few of these controls are
likely to be used for any one
gene.
Amount of DNA in the genomes of various organisms.
The Relationship Between Gene Size and mRNA Size
Species
Average exon number Average gene length (kb)
Hemophilus influenzae
Methanococcus jannaschii
1
1
1.0
1.0
S. cerevisiae
1
1.6
Filamentous fungi
3
1.5
C. elegans
4
4.0
D. Melanogaster
4
11.3
Chicken
9
13.9
Mammals
7
16.6
-----------------------------------------------------------------------SOURCE: Based on B. Lewin, Genes 5, Table 2-2. Oxford University Press.
------------------------------------------------------------------------
Average mRNA length (kb)
1.0
1.0
1.6
1.5
3.0
2.7
2.4
2.2
Synthesis of a primary RNA transcript (an mRNA precursor) by RNA polymerase II.
This diagram starts with a
polymerase that has just
begun synthesizing an
RNA chain.
Recognition of a poly-A
addition signal in the
growing RNA transcript
causes the chain to be
cleaved and then
polyadenylated as shown.
In yeasts the polymerase
terminates its RNA
synthesis almost
immediately thereafter, but
in higher eukaryotes it
often continues
transcription for thousands
of nucleotides.
The reactions that cap the 5' end of each RNA molecule synthesized by RNA polymerase II.
The final cap contains a novel 5'-to-5' linkage
between the positively charged 7-methyl G residue
and the 5' end of the RNA transcript. Polymerase I
and III transcripts are not capped. The indicated
reaction occurs almost immediately following
initiation of each RNA chain. The letter N is used
here to represent any one of the four ribonucleotides,
although the nucleotide that starts an RNA chain is
usually a purine (an A or a G). (After A.J. Shatkin,
Bioessays 7:275-277, 1987. © ICSU Press.)
The first two reactions are catalyzed by a capping
enzyme that associates with the phosphorylated
CTD of RNA polymerase II shortly after
transcription initiation. Two different
methyltransferases catalyze reactions 3 and 4. Sadenosylmethionine (S-Ado-Met) is the source of the
methyl (CH3) group for the two methylation steps;
the guanylate (G) is methylated first, then the 2’
hydroxyl of the first one or two nucleotides (N) in
the transcript. [See S. Venkatesan and B. Moss, 1982,
Proc. Nat’l. Acad. Sci. USA 79:304.]
Capping enzyme:
Phosphatase+
Guanyl
transferase
Methyltransferases
Structure of the 5’ methylated cap of eukaryotic mRNA.
The distinguishing chemical features are the 5’ 5’ linkage of 7-methylguanylate to the initial
nucleotide of the mRNA molecule and the
methyl group on the 2’ hydroxyl of the ribose of
the first nucleotide (base 1). Both these features
occur in all animal cells and in cells of higher
plants; yeasts lack the methyl group on base 1.
The ribose of the second nucleotide (base 2) also
is methylated in vertebrates. [See A. J. Shatkin,
1976, Cell 9:645.]
Model for cleavage and polyadenylation of
pre-mRNAs in mammalian cells.
Cleavage-and-polyadenylation specificity factor (CPSF)
binds to an upstream AAUAAA polyadenylation signal.
CStF interacts with a downstream GU- or U-rich
sequence and with bound CPSF, forming a loop in the
RNA; binding of CFI and CFII help stabilize the complex.
Binding of poly(A) polymerase (PAP) then stimulates
cleavage at a poly(A) site, which usually is 10 – 35
nucleotides 3’ of the upstream polyadenylation signal.
The cleavage factors are released, as is the downstream
RNA cleavage product, which is rapidly degraded.
Bound PAP then adds ≈12 A residues at a slow rate to
the 3’-hydroxyl group generated by the cleavage
reaction.
Binding of poly(A)-binding protein II (PABII) to the
initial short poly(A) tail accelerates the rate of addition
by PAP. After 200 – 250 A residues have been added,
PABII signals PAP to stop polymerization.
Overview of RNA processing in eukaryotes using the β-globin gene as an
example.
The β-globin gene contains three proteincoding exons (red) and two intervening
noncoding introns (blue). The introns
interrupt the protein-coding sequence
between the codons for amino acids 31
and 32 and 105 and 106. Transcription of
this and many other genes starts slightly
upstream of the 5’ exon and extends
downstream of the 3’ exon, resulting in
noncoding regions (gray) at the ends of
the primary transcript. These regions,
referred to as untranslated regions (UTRs),
are retained during processing. The 5’ 7methylguanylate cap (m7Gppp; green
dot) is added during formation of the
primary RNA transcript, which extends
beyond the poly(A) site. After cleavage at
the poly(A) site and addition of multiple
A residues to the 3’ end, splicing removes
the introns and joins the exons. The small
numbers refer to positions in the 147-aa
sequence of β -globin.
Early evidence for the existence of introns in eukaryotic genes.
The evidence was provided by the "R-loop
technique," in which a base-paired
complex between mRNA and DNA
molecules is visualized in the electron
microscope. An unusually abundant
mRNA molecule, such as β-globin mRNA
or ovalbumin mRNA, is readily purified
from the specialized cells that produce it.
When this single-stranded mRNA
preparation is annealed in a suitable
solvent to a cloned double-stranded DNA
molecule containing the gene that encodes
the mRNA, the RNA can displace a DNA
strand wherever the two sequences match
and form regions of RNA-DNA helix.
Regions of DNA where no match to the
mRNA sequence is possible are clearly
visible as large loops of double-stranded
DNA. Each of these loops (numbered 1 to
6) represents an intron in the gene
sequence.
Consensus sequences for RNA splicing in higher eukaryotes.
The sequence given is that for the RNA chain; the nearly invariant GU and AG
dinucleotides at either end of the intron sequence are highlighted in red, as is the
conserved A at the branch point. The numbers below the nucleotides represent
percent conservation.
Splicing of exons in pre-mRNA occurs via
two transesterification reactions.
In the first reaction, the ester bond
between the 5’ phosphorus of the
intron and the 3’ oxygen (red) of
exon 1 is exchanged for an ester
bond with the 2’ oxygen (dark blue)
of the branch-site A residue. In the
second reaction, the ester bond
between the 5’ phosphorus of exon 2
and the 3’ oxygen (light blue) of the
intron is exchanged for an ester
bond with the 3’ oxygen of exon 1,
releasing the intron as a lariat
structure and joining the two exons.
Arrows show where the activated
hydroxyl oxygens react with
phosphorus atoms.
Structure of the branched RNA chain that forms
during nuclear RNA splicing.
The nucleotide shown in yellow is the
A nucleotide at the branch site. The
branch is formed in step 1 of the
splicing reaction, when the 5' end of
the intron sequence couples
covalently to the 2'-OH ribose group
of the A nucleotide, which is located
about 30 nucleotides from the 3' end
of the intron sequence. The branched
chain remains in the final excised
intron sequence and is responsible
for its lariat form.
Analysis of RNA products formed in an in vitro splicing reaction
A nuclear extract from HeLa cells was incubated
with a 497-nucleotide radiolabeled RNA (bottom)
that contained portions of two exons (orange and
tan) from human β-globin mRNA separated by a
130-nucleotide intron (blue). After incubation for
various times, the RNA was purified and subjected
to electrophoresis and autoradiography, along with
RNA markers (lane M). The number of nucleotides
in the various species is indicated. Much of the
slower-migrating starting RNA (497) was correctly
spliced, yielding a 367-nucleotide product. The
excised intron (130*) migrated slower than expected
based on its molecular weight, indicating that it is
not a linear molecule. Likewise, one of the reaction
intermediates (339*) exhibited an anomalously slow
electrophoretic mobility. Additional analysis
indicated that in both cases the intron had a lariat
structure resulting in the slow mobility. The 252**
band, an aberrant product of the in vitro reaction, is
greatly reduced in reactions in which the RNA is
capped. [From B. Ruskin et al., 1984, Cell 38:317;
photograph courtesy of Michael R. Green. See also
R. A. Padgett et al., 1984, Science 225:898.]
The spliceosomal splicing cycle.
The splicing snRNPs (U1, U2, U4,
U5, and U6) associate with the premRNA and with each other in an
ordered sequence to form the
spliceosome. Although ATP
hydrolysis is not required for the
transesterification reactions, it is
thought to provide the energy
necessary for rearrangements of the
spliceosome structure that occur
during the cycle. The branch-point
A in pre-mRNA is indicated in
boldface. [See S. W. Ruby and J.
Abelson, 1991, Trends Genet. 7:79;
adapted from M. J. Moore et al.,
1993, in R. Gesteland and J. Atkins,
eds., The RNA World, Cold Spring
Harbor Press, pp. 303-357.]
Diagram of interactions between pre-mRNA, U1 snRNA, and U2 snRNA
early in the splicing process.
The 5’ region of U1 snRNA initially base-pairs with nucleotides at the 5’ end of the intron (blue)
and 3’ end of the 5’ exon (dark red) of the pre-mRNA; U2 snRNA base-pairs with a sequence
that includes the branch-point A, although this residue is not base-paired. The yeast branchpoint sequence is shown here. Secondary structures in the snRNAs that are not altered during
splicing are shown in diagrammatic line form. The purple rectangles represent sequences that
bind snRNP proteins recognized by anti-Sm antibodies. For unknown reasons, antisera from
patients with the autoimmune disease systemic lupus erythematosus (SLE) contain these
antibodies. Such antisera have been useful in characterizing components of the splicing reaction.
[See E. J. Sontheimer and J. A. Steitz, 1993, Science 262:1989; adapted from M. J. Moore et al., 1993,
in R. Gesteland and J. Atkins, eds., The RNA World, Cold Spring Harbor Press, pp. 303-357.]
The RNA
components of
snRNPs
are essential for
mRNA splicing
The two known classes of self-splicing intron sequences.
The group I intron sequences
bind a free G nucleotide to a
specific site to initiate
splicing, while the group II
intron sequences use a
specially reactive A
nucleotide in the intron
sequence itself for the same
purpose. The two
mechanisms have been
drawn in a way that
emphasizes their similarities.
Both are normally aided by
proteins that speed up the
reaction, but the catalysis is
nevertheless mediated by the
RNA in the intron sequence.
The mechanism used by
group II intron sequences
forms a lariat and resembles
the pathway catalyzed by
the spliceosome. (After T.R.
Cech, Cell 44:207-210, 1986. ©
Cell Press.)
Major Points
1. Primary eukaryote RNA transcripts are processed by 5’ Capping,
3’polyA and internal intron removal by RNA splicing
2. Many post-transcriptional steps can be regulated to form mRNA
3. 5’ cap influences protein translation and 3’polyA tail effects stability
4. Exon coding seq. are interrupted by intron seq. that must be spliced
out of primary transcripts to form mature mRNA
5. Intron removal involves self-splicing RNA seq. and splicesomes :
protein/RNA complexes (RNP’s) containing special small U RNA’s
6. Splicing occurs via 2 trans-esterification reactions and involve an
intermediate branched RNA formed by linking 5’Phos of the intron
to an A residue near the 3’ end of the intron via a 2’ OH
7. Two classes of self-splicing introns: Group I ( uses G-OH) and
Group II which uses the internal A residue to form a lariat & branch