Transcript LECT34 RNAproc

RNA Processing
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Capping
Polyadenylation
Introns vs exons
Splicing
Genomic vs cDNA
Ribosomal RNA processing
t-RNA processing
Prokaryotes vs Eukaryotes
PRO: All three classes of RNA are synthesized by one
polymerase.
EU: There are 3 major RNA polymerases. Pol 1
synthesizes rRNA; Pol 2 synthesizes mRNA; Pol 3
synthesizes tRNA.
PRO: mRNA undergoes hardly any posttranscriptional
processing. It is translated as it is synthesized.
EU: mRNA is capped, polyadenylated, spliced
PRO: mRNA contains no introns
EU: mRNA contains intervening sequences (introns)
that are removed during processing
Added Guanine
mRNA Capping
CH3
RULE: Capping the 5’ end
of mRNAs serves 2 purposes.
First, the cap protects the mRNA
from 5’-exonuclease activity.
Second, the cap interacts with
eIF-2, a translation initiation
factor required to position the
mRNA on the ribosome
5’-5’ triphosphate
Gppp + pppApNpNp…
CH3
CH3
GpppApNpNp… + pp + p
Polyadenylation
In eukaryotes, mRNA
is polyadenylated by
an enzyme system
that cuts the RNA 30 bs
downstream from an
AAUAAA, then
adds A to the 3’ end
at the cleavage site
Thus, poly A is not
coded in the DNA, but
is added after
transcription
Dialogue
Q: How should one picture a typical mammalian gene?
A: Mammalian genes have both introns and exons.
Only the exons encode information that will appear
in a protein.
Q: What are introns?
A: Introns appear in unprocessed mRNA. The term is
a shortened version of the words “intervening sequences”
Q: How do exons differ from introns?
A: One could say that the typical mammalian gene has
7 or 8 exons in a length of about 15 kb. The exons are
short, (100-200 bp) whereas introns are large (>1000 bp)
Q: What happens to introns?
A: During nuclear processing, the introns are spliced
out and exons are joined together in a linear continuum
Q: How is this accomplished?
A: Cells have mechanism that recognize introns. The
most common is a spliceosome that recognizes the
boundaries of intron-exon junctions and knows were to
cleave and splice
Q: What is involved in the recognition?
A: Small RNA molecules that work with spliceosomes,
The RNA hybridizes to the residues at the splice junctions
and tells the enzyme where to cut.
Stages in the Life of a Typical mRNA
DNA
Transcription via RNA Pol II
Primary Transcript
Capping and polyadenylation
Capped-polyadenylated mRNA
Splicing
Mature mRNA
Chick Ovalbumin
Introns form loops
that help with their
excision and splicing
Intron I is generally
quite large in mammalian
mRNAs
1,872 nucleotides (24.3%)
represented as exons
Dialogue on mRNA Splicing
What can you tell me about splicing?
Ans: Splicing occurs in two steps. First the junction at the
5’-end of the intron is broken. This is called the 5’-splice site
Then what happens?
Ans: The -OH on the 3’ end of the liberated exon becomes a
nucleophile and attacks the 3’-splice site of the intron. This is
the second step. The two exons are now joined.
What happens to the intron?
Ans: The intron is set free. Because a 2’-OH on an
adenosine caused the initial cleavage, there is a loop in the
intron (called a lariat)
Does this happen automatically
Ans: Sometimes. Some RNAs are capable of self-splicing.
Most of the time the splicing occurs through a large complex
called a spliceosome.
What is a spliceosome?
Ans: A spliceosome is a giant 50-60S particle composed of
splicing proteins, pre-mRNAs and small nuclear RNA proteins
or “SNURPS”.
What do the snRNPs do?
Ans: The 5’-end of some of the snRNPs complements bases
at the splice junctions. The snRNPs probably locate the
exon-intron boundaries which tend to be constant for all
eukaryote mRNAs. There are about 6 of them U1-U6. Not
all functions are known.
OH2’
Exon 1
Exon 2
Adenosine
pApA pGpU
pApG pGp
C A
U1-snRNP
Intron
O
pApA
pGpU
pApG pGp
OH
O
Exon 1
Exon 2
pApA pGp
Spliced exons
pGpU
pApG -OH3’
Intron with lariat
Ribosomal RNA
Q: Is ribosomal RNA processed the same way as mRNA?
A: No
Q: How is it different?
A: In bacteria, r-RNA is not spliced, it is only cut. All
processing is done with a special class of RNAases
Q: What about eukaryotes?
A: Eukaryotes employ basically the same mechanism,
but they also can engage in self-splicing
Q: What is self-splicing?
A: Self-splicing implies that a pre-rRNA can carry
out its own splicing without the need of a spliceosome.
Q: Does this mean the RNA is acting as its own
nuclease?
A: Yes. It is called a ribozyme in recognition of its
catalytic activity.
Q: How does self-splicing work?
A: In self-splicing 3’-OH group on the donor exon is
primed by an attack by GMP, GDP, or GTP.
Q: Then what?
A: As before, the freed -OH attacks the phosphate at the
3’ end and forms a new linkages that joins to two segments
Bacteria rRNA
5’
RNase:
Ribosomal RNA Primary transcript
1700
150 200
16S
4S
III
III
PF
2920
300
23S
III
Pre 16S rRNA
5S
Primary processing
Pre-5S
rRNA
5’
RNase:
3’
FP E
III P
Pre 23S rRNA
4S
3’
M16
M16
D M23
M23
D
M5
Secondary processing
5S rRNA
16S rRNA
Number 5’
of bases
tRNA(s)
23S rRNA
tRNA(s)
3’
1541
2904
120
Stages in the Life of a Eukaryotic
Ribosomal RNA
DNA (300 randomly repeated copies of rRNA genes in the genome
that are transcribed via RNA pol I and processed
in the nucleolus)
45S RNA
spacers
Primary Transcript 5’
3’
18S
5.8S
28S
Methylation at 110 sites
Heavily methylated
Methylated Transcript
RNase III,
RNase P
tRNA and 5S sequences
are not part of the 45S transcript
Self-splicing pre-rRNA
Any guanine nucleotide (GMP, GDP,GTP) sets it off
Processing t-RNA
Cut
Spliced
out
Amino acid
attachment site
Note: mature t-RNA has
the highest number
of odd bases
methyladenosine
2’-methylguanosine
See p345
in Strategies
dihydrouridine
isopentenyladenosine
Anticodon loop

pseudouridine