AQC-321
Post Transcriptional
Modification
Dr. Mamta Singh
Assistant Professor
COF (BASU), Kishanganj
Post Transcriptional Modification
Prokaryotes: RNA transcribed from DNA template and used
immediately in protein synthesis
Eukaryotes: Primary transcript (hn RNA) must undergo certain
modifications to produce mature mRNA (active
form) for protein synthesis.
“Post-transcriptional modification is a set of biological
processes common to most eukaryotic cells by which
an primary RNA transcript is chemically altered
following transcription from a gene to produce a
mature, functional RNA molecule that can then leave
the nucleus and perform any of a variety of different
functions in the cell.”
Post Transcriptional Modifications
• Post transcriptional modifications are also
responsible for changes in rRNA, tRNA and
other special RNA like srpRNA, snRNA,
snoRNA, miRNA etc.
Important Post Transcriptional Modifications
for Production of Mature mRNA
1. 5’ Capping
2. 3' maturation (Cleavage & Polyadenylation)
3. Splicing
4. Transport of RNA to Cytoplasm
5. Stabilization/Destabilization of mRNA
Likely order of events in producing a
mature mRNA from a pre-mRNA.
5’ RNA Capping
1. Occurs before the pre-mRNA is 30 nt long.
2. The modification that occurs at the 5' end of the
primary transcript is called the 5' cap.
3. In this modification, a 7-methylguanylate residue
is attached to the first nucleotide of the pre-mRNA
by a 5'-5' linkage.
4. The 2'-hydroxyl groups of the ribose residues of
the first 2 nucleotides may also be methylated.
Order of events
or “RNA triphosphatase” and enzymes in
5’ Capping
AdoMet = S-adenosylmethionine,
Product is Cap 0 the methyl donor
Product is Cap 1
5’ Cap Functions
Cap provides:
1. Protection from some ribonucleases*
2. Enhanced translation*
3. Enhanced transport from nucleus
4. Enhanced splicing of first intron for some
pre-mRNAs
*Also functions of the polyA-tail
Polyadenylation
1. String of adenine nucleotide (-AAAAAAA-3’) added at
3’ end of primary transcript is known as
polyadenylation
2. Most cytoplasmic mRNAs have a polyA tail (3’ end) of
50-250 Adenylates
– a notable exception is histone mRNAs
3. Discovered in 1971 (J. Darnell et al.)
4. Added post-transcriptionally by an enzyme, PolyA
polymerase(s)
5. It involves 2 steps a): Cleavage of RNA at 3’ end
b): Addition of adenine residue
Specific Sequences for 3’ Polyadenylation
Polyadenylation: The Proteins
Proteins required for cleavage and polyadenylation of a
new transcript.
James Manley
Proteins required for efficient cleavage of pre-mRNA:
1. CPSF (cleavage & polyadenylation specificity
factor), binds the AAUAAA
2. CstF (cleavage stimulation factor) binds to the G/U
rich region cooperatively with CPSF
3. CFI and CFII (cleavage factors I and II), RNA-binding
proteins
4. PAP (PolyA Polymerase)
5. nRNAP II (the CTD of the very large RPB1 subunit)
stimulates cleavage
Functions of the PolyA-Tail
1. Promotes mRNA stability ( protection from
ribonucelase activity)
- De-adenylation (tail shortening) can trigger
rapid degradation of the RNA
2. Enhances translation
- promotes recruitment by ribosomes
- bound by a polyA-binding protein in the
cytoplasm, PAB1
- synergistic stimulation with Cap!
Splicing of mRNA
Eukaryotic gene, the coding sequence (exon) are
separated/interrupted by non coding sequences (intron)
Split Genes (Intron & Exon)
Transcription start polyA Stop
5’ GT AG GT AG GT AG GT AG
Gene
3’
promoter
Open reading frame
Initial exon
Internal exon
Internal coding exon
Terminal exon
Translation Translation
Start Stop
mRNA 5’ 3’
5’ untranslated Protein 3’ untranslated
region coding region
region
Splicing (Removal of Introns)
Spliceosome
Alternative Splicing
Alternative splicing can generate multiple
polypeptides from a single gene (Protein A)
T he mRNA for Protein A is made by splicing together exons 1, 2 and 3:
exon1 intron1 exon2 intron2 exon3
Primary transcript:
single stranded RNA 5' and 3' end processing
cap AAAA
Precursor to
mRNA
splicing
mRNA cap AAAA
1 2 3
translation
2 Protein A
1
3
Alternative splicing can generate multiple
polypeptides from a single gene ( Protein B)
Or, by an alternative pathway of splicing that skips over exon2, Protein B can be
made:
exon1 intron1 exon2 intron2 exon3
Precursor cap AAAA
to
mRNA
splicing
mRNA cap AAAA
1 3
translation
1 3 Protein B
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• Content used for educational purpose only
