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Processing of eukaryotic pre-mRNA
For primary transcripts
containing multiple
exons and introns,
splicing occurs before
transcription of the gene
is complete--cotranscriptional splicing.
Human dystrophin gene has 79
exons, spans over 2,300-Kb and
requires over 16 hours to be
transcribed!
Capping of the 5’ end of nascent RNA transcripts with m7G
• The
Existing in
a single
complex
Sometimes
methylated
Sometimes
methylated
cap is added
after the nascent RNA
molecules produced
by RNA polymerase II
reach a length of 2530 nucleotides.
Guanylyltransferase is
recruited and activated
through binding to the
Ser5-phosphorylated
Pol II CTD.
• The methyl groups
are derived from Sadenosylmethionine.
• Capping helps
stabilize mRNA and
enhances translation,
splicing and export
into the cytoplasm.
Polyadenylation of mRNA at the 3’ end
CPSF: cleavage and polyadenylation specificity
factor.
CStF: cleavage stimulatory factor.
CFI & CFII: cleavage factor I & II.
PAP: poly(A) polymerase.
PABPII: poly(A)-binding protein II.
RNA is cleaved 10~35-nt 3’ to A2UA3.
The binding of PAP prior to cleavage ensures
that the free 3’ end generated is rapidly
polyadenylated.
PAP adds the first 12A residues to 3’-OH
slowly.
Binding of PABPII to the initial short
poly(A) tail accelerates polyadenylation by
PAP.
Poly(A) tail stabilizes mRNA and enhances
translation and export into the cytoplasm.
The polyadenylation complex is associated with
the CTD of Pol II following initiation.
Consensus sequences around 5’ and 3’ splice sites in vertebrate pre-mRNAs
The central region of the intron, which may range from 40 bases to 500 kilobases in
length, generally is unnecessary for splicing to occur.
Thalassemia: a group of inherited anemias characterized by defective
synthesis of hemoglobin (O2-transporter with 22 subunits) is caused by
mutations in -globin gene splice sites
Normal 3’
end of intron
Normal:
5’-CCTATTGGTCTATTTTCCACCCTTAGGCTGC-3’
-thalassemia:
5’-CCTATTAGTCTATTTTCCACCCTTAGGCTGC-3’
A new 3’ splice site due to
a G to A mutation,
leading to aberrant
splicing of -globin gene.
Stop
codon
The splicing reaction proceeds in two steps
Step 1. Cleavage at the 5’ splice site and joining
of the 5’ end of the intron to the branch point A
within the intron, producing a lariat-like
intermediate.
Step 2. Cleavage at the 3’ splice site and
simultaneous ligation of the exons, resulting in
excision of the intron as a lariat-like structure.
Two transesterification reactions: (1) The 5’ P
of the intron is attacked by the 2’-OH of the
branch site Adenosine, causing cleavage of a 3’,
5’-phosphodiester bond and formation of a 2, 5’phosphodiester bond (not hydrolysis followed by
ligation). (2) The newly formed 3’-OH of exon 1
attacks the 5’ P of exon 2, causing cleavage of a
phosphodiester bond and formation of a new
bond.
Two transesterification reactions: the number
of phosphodiester bonds remains unchanged
in either reaction.
Model of spliceosome-mediated splicing of pre-mRNA
•Five snRNPs (U1, U2, U4, U5 and U6 small nuclear
ribonucleoprotein particles) containing 5 snRNAs (U1, U2, U4,
U5 and U6 small nuclear RNAs, ranging from 107 to 210
nucleotides) and their associated proteins (6-10 per snRNP)
assemble on the pre-mRNA to form the spliceosome.
•There are a total of ~100 proteins in the spliceosome, some of
which are not associated with snRNPs. These non-snRNP
proteins may contribute to the specificity of recognition of the
splice sites by snRNPs and some of them contain RNA helicase
activity to help the rearrangements of base pairing in snRNAs
during the splicing cycle.
•U4 masks the catalytic activity of U6 in the U4/U6/U5 trisnRNPs prior to the actual transesterification reactions.
•Massive rearrangements of base-pairing interactions among
various snRNAs converts the spliceosome into a catalytically
active form, which releases the U1 and then the U4 snRNPs and
brings U2 and U6 together.
•RNA molecules play key roles in directing the alignment of
splice sites (e.g. U1 and U2 base pairing with the pre-mRNA)
and in carrying out the catalysis (a U2/U6 catalytic center).
Base pairing between pre-mRNA, U1 snRNA, and U2 snRNA early in the
splicing cycle and experimental demonstration that the base pairing between
U1 and the 5’ splice site in pre-mRNA is important
Cell type-specific splicing of fibronectin pre-mRNA in fibroblasts and
hepatocytes: concept of alternative splicing
•The presence of multiple introns in many eukaryotic genes permits expression of multiple, related
proteins from a single gene by means of alternative slicing, an important mechanism for the
production of different forms of proteins, called isoforms, by different types of cells.
•Nearly 60% of all human genes are expressed as alternatively spliced mRNAs, leading to an
expansion of the coding capacity of our genome.
•Fibroblasts produce fibronection with exons EIIIA and EIIIB, which allow the protein to adhere to
proteins in the fibroblast plasma membranes and enable fibroblasts to stick to the extracellular
matrix.
•Hepatocytes produce fibronection without EIIIA and EIIIB, which circulates in the serum and is
important during the formation of blood clots.
Detection of alternative splicing by Northern blotting
•Northern blotting can be used to detect specific RNAs in complex mixtures.
•Southern blotting detects specific DNA fragments.
•Western blotting (immunoblotting) detects specific proteins with antibodies.
RNA
mixture
Transfer solution
RNA
Question:
You are using Northern blotting to analyze two mRNA samples derived from fibroblasts and
hepatocytes. What will you see if you use a probe made from exon EIIIB of the fibronectin gene?
What about using a probe made from the exon next to EIIIB?
Exonic splicing enhancers (ESEs) and SR proteins contribute to exon
definition and regulate alternative splicing
The correct 5’ GU and 3’ AG splice sites are recognized by splicing factors on the basis of their
proximity to exons. The exons contain exonic splicing enhancers (ESEs) that are binding sites for
SR proteins. When bound to ESEs, the SR proteins interact with one another and promote the
cooperative binding of the U1 snRNP to the 5’ splice site of the downstream intron, the 65- and
35-kD subunits of U2AF to the pyrimidine-rich region and 3’ AG splice site of the upstream
intron. The bound U2AF also helps recruit U2 snRNP to the branch point. The resulting RNAprotein cross-exon recognition complex spans an exon and activates the correct splice sites for
RNA splicing and this exon is retained in the final spliced mRNA.
If U1 snRNP and/or U2AF are not recruited to the splice sites on each side of an exon (no
formation of a stable cross-exon recognition complex), this exon will not be recognized and,
instead, will be excised as part of the intron.
The RNA Pol II CTD is required for the coupling of transcription with
mRNA capping, polyadenylation and splicing
1.
The coupling allows the
processing factors to
present at high local
concentrations when
splice sites and poly(A)
signals are transcribed
by Pol II, enhancing the
rate and specificity of
RNA processing.
2.
The association of
splicing factors with
phosphorylated CTD
also stimulates Pol II
elongation. Thus, a premRNA is not
synthesized unless the
machinery for
processing it is properly
positioned.
Splicing mechanisms in group I and group II self-splicing introns and spliceosome-catalyzed
splicing of pre-mRNA; discovery of self-splicing of ribosomal RNA in Tetrahymena
Occurs in pre-rRNAs from Tetrahymena
and mitochondria & chloroplast origins.
The 1st cleavage is carried out by an
external cofactor guanosine (G, 3’-OH).
The intron is released in a linear form.
Group II self-splicing introns are found in mitochondria and chloroplast
pre-mRNAs. The 1st cleavage is carried out by the 2’-OH of A within the
intron. The intron is released in the form of a lariat.
Common to all: two transesterification reactions are involved.
Catalytic introns ----- RNA as enzymes ------ Ribozymes
During evolution, there has been a transfer of
catalytic power from the intron itself to other
molecules such as snRNPs, which are specialized in
carrying out splicing reactions. Introns nowadays
are variable in size because they are no longer selfsplicing and this increases the capacity of
regulation.
The similarity between these two suggests that
the U snRNAs probably evolved from group II
introns of endosymbiotic organelles.