This presentation mainly focus on Medical Biochemistry postgraduates.

1. POST-TRANSCRIPTIONAL PROCESSING
POST-TRANSCRIPTIONAL PROCESSING

2. TRANSCRIPTION ??

3. TERMS TO REMEMBER
• Template strand : The DNA strand that is read to make the RNA strand
• Non-Template strand : The 5’ to 3’ DNA strand complementary to the template strand, and
having the same polarity as the resulting RNA strand
• Introns : Protein-coding genes typically have non-amino acid–coding sequences & is derived
from intervening sequence—a sequence that is not translated into an amino acid sequence.
introns are removed in the processing of pre-mRNA to mature RNA.
• Exons : Between the other sequences that are present in mRNA & is derived from expressed
sequence. Exons include the 5’ and 3’ UTRs, as well as the amino acid-coding portions.

4. WHY POST-TRANSCRIPTIONALPROCESSING ??
• In prokaryotes, the RNA that is synthesized during DNA transcription is ready for translation
into a protein.
• Eukaryotic RNA from DNA transcription, however, is not immediately ready for translation.
• So, they undergo Post-transcriptional process, where the portions of the RNA that are not
supposed to be translated into proteins are cut out of the sequence.
• Helps increase the efficiency of protein synthesis by allowing only specific protein- coding RNA
to go on to be translated.
• Protein synthesis could be significantly slowed.
• The nascent RNA, also known as primary transcript, needs to be modified to become functional
tRNA’s, rRNA’s, and mRNA’s.
• The modification is very much essential to eukaryotic systems.
• RNA’s are processed during this transport.
• Processing gives them characteristics they need to be functional in the cytoplasm

5. What is PTP??
• The mRNA formed & released from the DNA template – Primary Transcript
• Also known as heteronuclear mRNA or hnRNA.
• It undergoes extensive editing → Mature mRNA
• This process includes;
1. 7 methylguanosine capping at 5’ end
2. Addition of poly A tail at 3’ end
3. Splicing
4. Methylations
5. Alternative RNA processing
• These processing occurs in nucleoplasm.

6. POST-TRANSCRIPTIONAL
MODIFICATION
Prokaryotes Eukaryotes
•mRNA not subjected to PTP
•Translation started with
transcription
•tRNA & rRNA undergo PTP
•1° transcript or heteronuclear
RNA
•hnRNA undergoes PTP

8. Eukaryotic Transfer RNA Processing
• Transfer RNA precursors are transferred into mature RNA by following alterations:
1. Cleavage of a 5’ leader sequence
2. Splicing to remove intron
3. Replacement of 3’ terminal UU by CCA
4. Modification of several bases.

9. Eukaryotic rRNA Processing
• This is very similar to that of prokaryotes.
• 3 of the eukaryotic rRNAs (28S,18S & 5.8S) synthesized from long precursor – Preribosomal
(45S) RNAs
• Fourth,5S rRNA is produced by transcription of 5s gene by RNA polymerase III
• No tRNA sequences in the precursor, unlike prokaryotes
• The 45 S precursors are cleaved & trimmed to produce mature rRNA species.
• Spacer sequences are removed by cleavage of 45 S rRNA by endonucleases.
• 5.8S rRNA base pairs with 28S rRNA during formation of ribosomal subunits & is completed
before transport from the nucleus.

11. Eukaryotic mRNA Processing
• Mature mRNA is formed from extensive processing of a large precursor –hnRNA transcript
product of RNA polymerase II.
• hnRNA (primary transcript) is modified after transcription.
• Primary transcript are very long (MW - >107 )
• Molecular weight of mature mRNA – 1-2 ˟ 106
• Half-life of mRNA ;
 In cytoplasm poly A tails are slowly shortened.
 mRNA is rapidly degraded ,when poly A tail is completely removed.
 Half life of mRNA molecule may be determined in part by rate of degradation of its poly A tail.

12. Likely order of mRNA Processing

13. METHYLATION & POLY-A
(5’ & 3’ Modification)

14. 7 METHYLGUANOSINE 5’ CAPPING
1.In Nucleus
• Guanosine triphosphate attached to 5’ end by 5’-5’ unusual linkage (capping) by enzyme
Guanyltransferase.
2. In Cytosol
• Methylation with methyl group from S-adenosyl-methionine using guanine 7 methyl tranferase.
3.Functions
• Initiation of translation
• Impo.for binding of ribosome
• Stabilize the mRNA
• Prevents attack of 5’ → 3’ exonuclease
• Eukaryotic mRNA lacking the cap are not translated efficient.

16. 1.In Nucleus
•3’ end of eukaryotic mRNAs are polynucleated (poly A) & called TAIL
•Pre-mRNAs become modified by the addition of a sequence called a poly(A) tail
•Poly A tail added at 3’ by polyadenylate
polymerase
•The length of Poly A tail may be 20 -250 nucleotides long.
•No DNA template for the poly(A) tail
•.mRNA molecules with 3’ poly(A) tails are called Poly(A) + mRNAs
2.In Cytoplasm
•Protects the 3’ end of the mRNA by buffering coding sequences against early degradation by
exonucleases.
3.Functions
•Poly A tail & its binding protein PAB -1 is
required for efficient
•Initiation of translation by ribosome's.
• In processes that regulate the stability of mRNA.
.
ADDITION OF POLY A TAILAT 3’ END

17. •Addition of the poly(A) tail is signaled when mRNA transcription proceeds past
the poly(A) site, a site in the RNA transcript that is about 10 to 30 nucleotides
downstream of the poly(A) consensus sequence 5’-AAUAAA-3’
•A number of proteins, including CPSF protein, CstF protein, and two cleavage
factor proteins, then bind to and cleave the RNA at the poly(A) site
•The enzyme Poly(A) polymerase (PAP) + CPSF, adds A nucleotides to the 3’ end
of the RNA using ATP as the substrate to produce the poly(A) tail. .
•Poly(A) binding protein II (PABII) molecules bind to the poly(A) tail as it is
synthesized.
•After cleavage by the endonuclease, a poly(A) polymerase adds about 200 to 300
adenylate residues to the 3’ end of the transcript.
•ATP is the donor of the adenylate residue.

21. • Most genes are composed of exons & introns.
• Process by which introns are excised & exons are liked to form the functional mRNA -
SPLICING.
• This must be very accurate & sensitive
• 15% of all genetic diseases due to mutations are due to splicing defects.
• Aberrant splicing causes some forms of Thalassemia.

22. SPLICE SITE
 Consensus sequences at the intron/exon boundaries of the hnRNA are AGGU.
 All introns begin with 5’ GU &end with 3’AG
 The consensus sequences at the 5’ splice in vertebrates is AGGUAAGU
 At the 3’ end of intron, it is stretch of 10 pyrimidine (U or C) ,followed by any base & then by
C & ending with invariant C.
 Introns have internal site located between 20 & 50 nucleotides upstream of the 3’ splice site –
Branch Site.

23. SMALL NUCLEAR RNAS (SNRNAS)
 Size ranges from 90-300 nucleotides
 Take part in the formation of spliceosomes
 Located in the nucleus
 Complex with specific protein – small nuclear ribonucleoprotein particles (snRNPs).
SPLICEOSOMES
 SnRNPs with hnRNAs at the exon-intron junction → Spliceosomes
 Takes place in the nucleus.
 Cuts are made at both end of introns & removed
 Exon- exon are ligated at G-G residues.

24. SPLICING MECHANISM
• Splicing of hnRNA is a complicated & a multistep process.
• Requires spliceosomes.
• Snurps are involved in the formation of spliceosome.
• They are rich in uracil & are identified by numbers preceded by a U (U1,U2,U4,U5 &U6),are
essential for splicing mRNA precursors.
• Formation of spliceosomes ;
a. SnRNAPs
b. Other proteins called splicing factors
c. The mRNA precursor being processed.
• Splicing begins with recognition of 5’ splice site by U1 snurp, then binds the branch site in the
intron containing adenine nucleotide residue.

25. • Pre assembled U4,U5 & U6 complex joins this complex of U1,U2 & mRNA precursor forming
a looped structure and a spliceosome formation takes place.
• Functions of Snurps involved in the splicing of hnRNA;
1) U1 – Binds with 5’ splice site & then 3’ splice site
2) U2 – Binds the branch site of the introns
3) U4 – Masks the catalytic activity of U6
4) U5 – Binds the 5’ splice site
5) U6 – Catalyses splicing

27. CHEMISTRY OF SPLICING PROCESS
• Splicing starts with cleavage of the phosphodiester bond between the upstream exon (exon-1)
& the 5’ end of the intron.
• Phosphate attached to G at the 5’ end of the intron forms a 2’,5’ –phosphodiester bond
between 2’ hydroxyl group of th adenine nucleotide at branch site of the intron & the 5’
terminal phosphate of intron.
• Cleavage occurs at the end of the first exon that continues to be held in place by the
spliceosome.
• This reaction is called Transesterification.
• Generates new 3’-hydroxyl group at 3’ end of exon-1
• The adenylate residue is also joined to 2 other nucleotides by normal 3’,5’ phosphodiester
bonds. Hence, a new branch is generated at this site.

28. • Second cleavage occurs at the 3’ end of intron after the AG sequence.
• Newly formed 3’ hydroxyl terminus of exon 1 attacks the phosphodiester bond between exon
2 and 3’-end of the intron (3’-splice site)
• This is a second Transesterification reaction.
• Splicing is done by two Transesterification reactions
• Exons 1 & 2 are joined.
• Intron is released in the form of lariat.
• No.of phosphodiester bond remains same during the steps.

30. ALTERNATIVE SPLICING
• hnRNA with multiple exons is spliced in different ways to yield different mRNAs & different
proteins.
• By selecting the exons in a given hnRNA it is possible to generate different mRNA from the
same section of genomic DNA.
• It provides a mechanism for expanding the versatility of genomic sequence.

32. RNA Editing
• It is a change in the base sequence of RNA after transcription by process other than RNA
splicing.
• Involves the enzyme mediated alteration of base sequence of RNA in the nucleus before
translation.
• Process may involve INSERTION, DELETION or SUBSTITUTION of nucleotides in RNA
molecule.
• The substitution of on nucleotide for another can result in tissue specific differences in
transcript. E.g. gene of apolipopreotein B, ApoB gene.

33. REVERSE TRANSCRIPTION
• Viral DNA polymerase is Reverse Transcriptase.
• Retrovirus is a subgroup of RNA viruses.
• HIV is retrovirus.
• Here, the RNA acts as a template.
• Based on this, RNA dependant DNA polymerase will make a new DNA strand.