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Post-transcriptional modifications:
Eukaryotic cells modify their RNA after transcription
RNA transcripts in eukaryotes are modified, or processed,
before leaving the nucleus to yield functional mRNA.
Eukaryotic RNA transcripts can be processed in two
1. Covalent alteration of both the 3’ and 5’ ends.
2. Removal of intervening sequences.
Primary transcript – general term for initial RNA transcribed
from DNA
Pre-mRNA – primary transcript that will be processed to
functional mRNA
Eukaryotic cells modify their RNA after transcription
RNA processing: RNA splicing
Split genes and RNA splicing
RNA splicing – RNA processing that removes introns and joins exons
from eukaryotic pre-mRNA;
produces mature mRNA that will move into the cytoplasm from the
•Enzymes excise introns and splice exons to form mRNA with
continuous coding sequence.
•RNA splicing also occurs during post-transcriptional processing
of tRNA and rRNA.
RNA splicing
Spliceosome – a large molecular complex that catalyzes RNA
splicing reactions.
•As the spliceosome is assembled, one type of snRNP base pairs
with a complementary sequence at the 5’ end of the intron.
•The spliceosome precisely cuts the RNA transcript at the specific
splice sites at either end of the intron, which is excised as a loop.
•The intron is released and the adjacent exons are immediately
spliced together by a spliceosome.
RNA splicing
Each end of the intron has short boundary sequences that accurately
signal the RNA splicing sites.
Small nuclear ribonucleoproteins (snRNPs) – complexes of proteins
and small nuclear RNAs that are found only in the nucleus.
Some participate in RNA splicing.
They are composed of:
Small nuclear RNA – this molecule has less than 300 nucleotides.
Protein – each snRNP possesses several different proteins. There
are different types of snRNPs with different functions.
The roles of snRNPs (small
nuclear ribonuclearproteins)
and spliceosomes in mRNA
The R-looping experiments
reveal the introns of
Loops-introns of the gene that
can not hybridize to DNA
Most of higher eukaryotic genes coding for mRNA, tRNA and
some coding for rRNA are interrupted by unrelated regions called
The other parts of the genes are called exons.
Exons contain information that appears in the functional mRNA
Genes for mRNA have 0 to 60 introns
Genes for tRNA have 0 to 1 intron.
mRNA synthesis
1 – primary transcript, hnRNA; contains introns
2 – mRNA maturation – part of maturation –
removal of introns, splicing
How does the cell deal with introns during transcription?
The process of cutting out the introns from immature RNA and
stitching together the exons is called splicing
The outline of splicing
The introns are transcribed.
Experiment with mouse beta-globin mRNA and its precursor.
Splicing signals are important
•Splicing has to be precise.
•Splicing signals in nuclear mRNA are remarkably uniform
•The first two bases of intron are always GU.
•The last two are always AG.
•The 5’ and 3’ splice sites have consensus sequences that extend
beyond the GU and AG motifs.
•The consensus sequences are important for splicing
•Mutations leads to abnormal splicing
Intron-exon boundaries
Consensus sequence:
Y is pyrimidine C or U
Yn – string of 9 pyrimidines
R – purine, A or G
A – special A that participates in forming branched splicing
N – any base
Yeast mRNA – a bit different from mammals
Simplified mechanism of nuclear mRNA precursor
1. The 2’-hydroxyl group of
adenine within the intron attacks
the PDF bond linking the first
exon to the intron.
This attack breaks the bond
between the exon 1 and intron,
yielding the free exon 1 and the
lariat-shaped intron-exon 2
intermediate with the GU at the
5’ end of the intron linked
through the PDB to the
breakpoint A.
The lariat is a consequence of
the internal attack of one part of
the RNA precursor on another
part of the same molecule.
Simplified mechanism of nuclear mRNA precursor
2. The free 3’-hydroxyl
group on exon 1 attacks
the PDF bond between the
intron and exon2. This
yields the spliced product
and the lariat-shaped
NB – several lines of evidence demonstrate that nuclear mRNA
precursors are spliced via lariat-shaped, or branched intermediate.
A signal at the branch
Special regions within introns are also important for splicing
Experimental evidence: removal of a region between 35 and 70
bp upstream of the introns 3’ splice site blocked splicing
Special intron regions contain the branch-point adenine nucleotide
UACUAAC – in yeast
Higher eukaryotes – sequence more variable.
It appears to tell splicing machinery which AG to select as a 3’ splice site.
Splicing takes place on a particle called spliceosome
Yeast and mammalian once have sedimentation coefficients 40S
and 60S.
Snurps – agents to recognize the critical splicing signals.
snRNPs – U1,U2, U4, U5 and U6.
Splicing substrate+U1= CC
U2, ATP  A
U4/U6 and U5 B1
U4 diss. From U6
U6 displ. U1 at 5’ splice site
U1 and U4 exit
U6 bps withU2 =B2
1st splicing stepC1
2nd splicing stepC2
Splicing RNA exits,
Lariat held by I.
Alternative splicing is important
Different RNA and thus protein can be produced:
difference between membrane-bound and secreted protein
Drosophila – of products 3 genes involved in sex
determination are alternatively spliced
Other kinds of RNA primary transcripts, such as some of those giving
rise to tRNA and rRNA, are spliced by mechanisms that do not involve
spliceosomes, however, as with mRNA splicing, RNA is often
involved in catalyzing the reactions.
Ribozymes – RNA molecules that can catalyze reactions by breaking
and forming covalent bonds, they are called ribozymes to emphasize
their catalytic activity.
RNA has catalytic capacities
In early 1970 it was discovered that folded domains of RNA
molecules have catalytic capacities just as the folds (helices and
beta-strands) of proteins do.
Less is understood about the 3D structure of the catalytic sites,
but catalytic activities were identified.
RNA has catalytic capacities
Example - the ability of RNA to catalyze the cutting of an RNA chain. The
example of the mode of action of such ribozyme (RNA enzyme):
the substrate chain is base-paired with a site on the ribozyme, and the
cleavage is induced at the precise nucleotide in the substrate.
Other reactions –eg. phosphotransferase reaction, which unites two distant
segments of RNA chain after cleavage has occurred is called splicing.
5’ G C G C C
3’ C G C G G
Nicking site
U C G A G C 3’
A G C U C G 5’
Functional and evolutionary importance of introns
Introns may play important role in the cells.
Intron sequences direct the synthesis of different proteins and may
control gene activity.
The splicing process itself may help to regulate the export of mRNA
to the cytoplasm.
Introns may allow single gene to direct the synthesis of different
proteins. This can occur if the same transcript is processed differently
among various cell types in the same organism.
Functional and evolutionary importance of introns
All introns may be removed from particular transcript in one case, but
in the other one or more introns may be left in place. The proteins will
be different.
Introns play important roles in the evolution of protein diversity they increase the probability that recombination of exons will occur
between alleles.
Functional and evolutionary importance of introns
In split genes, coding sequences can be separated by long distances, so
they have higher recombination frequencies than continuously coded
genes without introns.
Exons of the split gene may code for different domains of a protein
that have specific functions, such as enzyme’s active site or a protein’s
binding site.
Protein domains – continuous polypeptide sequences that are
structural and functional units in proteins with a modular architecture.
Alteration of pre-mRNA ends
During pre-mRNA processing, both the 5’ and 3’ ends are covalently
5’ cap – Modified guanine nucleotide (guanosine triphosphate) that
is added to the 5’ end of mRNA shortly after transcription begins. It
has two important functions:
•Protects the growing mRNA from degradation by hydrolytic
•Help small ribosomal subunits recognize the attachment site on
mRNA’s 5’ ends. A leader segment of mRNA may also be part of
the ribosome recognition signal.
Leader signal – Non-coding (untranslated) sequence of mRNA from
the 5’ end to the start codon.
Alteration of pre-mRNA ends
The 3’ end, which is transcribed the last, is modified by enzymatic
addition of a poly-A-tail, before the mRNA exits the nucleus.
Poly-A tail – sequence of about 0 to 200 adenine nucleotides added
to the 3’ end of mRNA before it exits the nucleus.
•May inhibit degradation of mRNA in the cytoplasm
•May facilitate attachment to the small ribosomal subunit
•May regulate protein synthesis by facilitating mRNA’s export from
the nucleus to the cytoplasm
•Is not attached directly to the stop codon, but to untranslated trailer
segment of mRNA.
Trailer sequence – noncoding (untranslated) sequence
Pages 425-465