The document discusses various post-transcriptional processes that eukaryotic RNA undergo. It describes: 1) Introns are removed and exons are joined together in mRNA through splicing. Small nuclear RNAs and spliceosomes are involved in splicing. 2) Other processing includes adding a 5' cap and 3' poly-A tail. The tail protects the mRNA and is important for translation. 3) Transfer RNA and ribosomal RNA also undergo processing after transcription to become functional in the cell. Alternative splicing allows different mRNAs and proteins to be produced from the same initial RNA.

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.