Download RNA processing #1

RNA processing #1
Making ends of RNA
Types of RNA processing
• A) Cutting and trimming to generate ends:
– rRNA, tRNA and mRNA
• B) Covalent modification:
– Add a cap and a polyA tail to mRNA
– Add a methyl group to 2’-OH of ribose in mRNA
and rRNA
– Extensive changes of bases in tRNA
• C) Splicing
– pre-rRNA, pre-mRNA, pre-tRNA by different
mechanisms.
Cutting and Trimming RNA
• Can use endonucleases to cut at specific
sites within a longer precursor RNA
• Can use exonucleases to trim back from
the new ends to make the mature product
• This general process is seen in
prokaryotes and eukaryotes for all types of
RNA
Excision of mature rRNA and tRNA from prerRNA in E. coli
Genes:
16S rRNA
tRNA
23S rRNA
5S rRNA tRNA
Promoters
Terminators
30S pre-rRNA:
Transcription
Cleavage at
Further trimming
16S rRNA tRNA
23S rRNA 5S rRNA tRNA
RNase III cuts in stems of stem-loops
16S rRNA
23S rRNA
RNase III
No apparent primary sequence specificity - perhaps RNase III
recognizes a particular stem structure.
Endo- and exonucleases to generate
ends of tRNA
• Endonuclease RNase P cleaves to generate
the 5’ end.
• Endonuclease RNase F cleaves 3
nucleotides past the mature 3’ end.
• Exonuclease RNase D trims 3’ to 5’, leaving
the mature 3’ end.
Cleavage of pre-tRNA in E. coli
CCA at 3’ end of tRNAs
• Virtually all tRNAs end in the sequence
CCA.
• Amino acids are added to the CCA end
during “charging” of tRNAs for translation.
• In most prokaryotic tRNA genes, the CCA is
encoded in the DNA.
• For most eukaryotic tRNAs, the CCA is
added after transcription, in a reaction
catalyzed by tRNA nucleotidyl transferase.
Where is the catalytic activity in RNase P?
RNase P is composed of a 375 nucleotide RNA and a
20 kDa protein.
The protein component will NOT catalyze cleavage
on its own.
The RNA WILL catalyze cleavage by itself !!!!
The protein component aids in the reaction but is not
required for catalysis.
Thus RNA can be an enzyme.
Enzymes composed of RNA are called ribozymes.
Covalent modification of RNA
5’ and 3’ ends of eukaryotic mRNA
Add a GMP.
Methylate it and
1st few nucleotides
Cut the pre-mRNA
and add A’s
5’ cap structure
Initiating nucleotide
O
H
NH
2
+
-O
N
N
N
5' to 5' link
CH 3
N
O-
O-
N
N
O P O CH2 O N
O CH2 O P O P
O
O
O
OH OH
2'
N
H
Methylated in cap 1 and 2
N
O O CH 3
O-
Can be methylated in cap 1
NH 2
H
N
P O CH2 O
N
Methylated in cap 2
O
O
NH 2
From GTP
O O CH 3
O-
N
P O CH2 O N
N
N
O
O OH
rest of RNA
Synthesis of 5’ cap
g
5'
P
b
P
a
a
P
P
P
P
G
5'
b
A
C
g P
P-P-P
P
P
U
G
P
P
P
OH
GTP
A
G
a
P
b
P
a
5' to 5' link
P
A
C
P
U
P
G
PPi
RNA being
synthesized
RNA being
synthesized
OH
3'
OH
3'
RNA
triphosphatase
(P=phosphoryl)
3'
OH
mRNA guanylyl
transferase
P
C
U
G
RNA being
synthesized
OH
3'
methyl
transferases
Add methyl groups to N7 of capping G,
2' OH of 1st and sometimes 2nd nucleotides, etc.
Cleavage and polyadenylation at the 3’ end
Cut site
CPSF =
Cleavage and
polyadenylation
specificity factor
CstF = cleavage
stimulation
factor
CFI, CFII =
cleavage factors
PAP = polyA
polymerase
RNA is processed while being synthesized
• Tight linkage between transcription and
processing
• Processing proteins associated with CTD of
large subunit of RNA polymerase II:
– Capping enzymes: mRNA guanylyl transferase,
methyl transferases
– Cleavage and polyadenylation factors: CPSF,
CstF
– Splicing factors: SR proteins to recruit
spliceosomes (next class)
• Can visualize splicing on nascent transcripts
in EM
Functions of 5’ cap and 3’ polyA
• Both cap and polyA contribute to stability
of mRNA:
– Most mRNAs without a cap or polyA are
degraded rapidly.
– Shortening of the polyA tail and decapping are
part of one pathway for RNA degradation in
yeast.
• Need 5’ cap for efficient translation:
– Eukaryotic translation initiation factor 4 (eIF4)
recognizes and binds to the cap as part of
initiation.
Splicing of RNA
Overview of types of splicing
4 major types of introns
• 4 classes of introns can be distinguished on
the basis of their mechanism of splicing
and/or characterisitic sequences:
– Introns in pre-tRNA
– Group I introns in fungal mitochondria, plastids,
and in pre-rRNA in Tetrahymena
– Group II introns in fungal mitochondria and
plastids
– Introns in pre-mRNA
Splicing of pre-tRNA
• Introns in pre-tRNA are very short (about 10-20
nucleotides)
• Have no consensus sequences
• Are removed by a series of enzymatic steps:
– Cleavage by an endonuclease
– Phosphodiesterase to open a cyclic
intermediate and provide a 3’OH
– Activation of one end by a kinase (with ATP
hydrolysis)
– Ligation of the ends (with ATP hydrolysis)
– Phosphatase to remove the extra phosphate on
the 2’OH (remaining after phosphodiesterase )
Steps in splicing of pre-tRNA
+
OH 5’
1. Endonuclease
Intron of
10-20
nucleotides
P
2’,3’ cyclic
phosphate
+
Excised intron
2. Phosphodiesterase
3. Kinase (ATP)
4. Ligase (ATP)
5. Phosphatase
Spliced
tRNA
Splicing of Group I and II introns
• Introns in fungal mitochondria, plastids,
Tetrahymena pre-rRNA
• Group I
– Self-splicing
– Initiate splicing with a G nucleotide
– Uses a phosphoester transfer mechanism
– Does not require ATP hydrolysis.
• Group II
– self-splicing
– Initiate splicing with an internal A
– Uses a phosphoester transfer mechanism
– Does not require ATP hydrolysis
Splicing of pre-mRNA
• The introns begin and end with almost
invariant sequences: 5’ GU…AG 3’
• Use ATP to assemble a large spliceosome
• Mechanism is similar to that of the Group II
fungal introns:
– Initiate splicing with an internal A
– Uses a phosphoester transfer mechanism
for splicing