1. Posttranscriptional Modifications of RNABy
Amr S Moustafa, MD, PhD
Medical Biochemistry and Molecular Biology Department
Faculty of Medicine, Ain Shams University

2. Intended Learning Outcomes
Understand posttranscriptional modifications of rRNA and tRNA in both prokaryotes and eukaryotes
Understand posttranscriptional modifications of mRNA in eukaryotes only

3. Posttranscriptional Modifications of Prokaryotic and eukaryotic rRNA

4. The pre-ribosomal RNAs
The rRNA of both prokaryotic and eukaryotic cells are synthesized from long precursor molecules called pre-ribosomal RNAs.
The 23S, 16S and 5S rRNA of prokaryotes are produced from a single RNA precursor molecule.
The 28S, 18S and 5.8S rRNA of eukaryotes are produced from a single precursor molecule.
The 5S rRNA of eukaryotes is synthesized by RNA polymerase III and modified separately.

5. eukaryotic Ribosomal RNA
The pre-rRNA is cleaved by
ribonucleases (RNases)
to produce the required
rRNA species
(28S, 18S and 5.8S)

6. Mature ribosomal rNAs (rRNA)

7. The Eukaryotic and Prokaryotic Ribosomes
Proteins associate with rRNA prior to and during its posttranscriptional modifications

8. Posttranscriptional Modifications of Prokaryotic and eukaryotic tRNA

9. Prokaryotic and Eukaryotic processing of tRNA1 2 3 4
&
Trimming (5’- and 3’-ends) by RNases
Addition of CCA to 3’-end by nucleotidyl transferase
Removal of an intron from the anticodon loop
Modification of bases

10. Prokaryotic and Eukaryotic processing of tRNA
1. Similar to rRNA, tRNA is posttranscriptionally modified by cleavage of the original, longer precursor by ribonucleases:
Both the 5′- and the 3′-ends of the molecule must be trimmed.
2. Addition of a CCA by nucleotidyltransferase to the 3′-terminal end of tRNA.
3. An intron must be removed from the anticodon loop.
3. Modification of bases at specific positions to produce “unusual bases”:
e.g., dihydrouracil, pseudouracil, thymine and methylated bases.

11. The Mature tRNA 5’ end is based paired for RNA stability)
3’ end is single stranded and has CCA site for amino acid attachment
has 4 single stranded loops: D-loop, TC loop, anticodon loop and extra loop.
has modified or unusual bases: dihydrouracil (DHU) ribothymine (rTMP), pseudouridine () [ribose attached to C5 of uracil instead of N1].
Extra loop

12. Posttranscriptional Modifications of
Eukaryotic mRNA

13. The pre-mRNA in Eukaryotes
5’-UTR
3’-UTR
Introns
& Exons
Newly synthesized mRNA before being modified
Parts: 5’-UTR *Introns and exons 3’-UTR
*Exception: few primary mRNAs contain no introns (only exons)
e.g., histone mRNA
Pre-mRNA or hnRNA or primary transcript

14. Posttranscription modification of Eukaryotic mRNA in the nucleu
5’- capping: addition of 7-methylguanosine triphosphate cap
3’-end addition of poly-A tail
Removal of introns and splicing of exons

15. Postranscriptional Modification of mRNA: 5′ “Capping”
This process is the first step of processing.
The cap is a 7-methylguanosine triphosphate attached “backward "to the 5′-terminal end of the mRNA, forming an unusual 5`→5` triphosphate linkage
Requires the nuclear enzyme guanylyltransferase.

16. 5′ “Capping”

17. Why? 5′ “Capping”
The addition of 7-methylguanosine “cap” permits the initiation of translation, and helps stabilize the mRNA.
Eukaryotic mRNA lacking the cap are not efficiently translated.

18. Postranscriptional Modification of mRNA: Addition of 3’-poly-A tail
Most eukaryotic mRNA have a chain of 40–200 adenine nucleotides attached to the 3′-end (downstream of a polyadenylation signal AAUAAA)
This poly-A tail is not transcribed from the DNA.
This poly-A tail is added after transcription by the nuclear enzyme, polyadenylate polymerase, using ATP as the substrate.
Exception: mRNA of histones and some interferons have no poly-A tail

19. Addition of a poly-A tail

20. Why? (poly-A tails) These tails help to stabilize the mRNA
Facilitate their exit from the nucleus.
After the mRNA enters the cytosol, the poly-A tail is gradually shortened.

21. Postranscriptional Modification of mRNA: Removal of Introns and Splicing of Exons
Maturation of eukaryotic mRNA usually involves the removal of RNA sequences, which do not code for protein (introns, or intervening sequences) from the primary transcript.
The remaining coding sequences, the exons, are joined together (spliced) to form the mature mRNA.
Exception: mRNA of histones has no introns

22. Primary transcript of some genes can be as large as 1
Primary transcript of some genes can be as large as 1.7 megabases (1,700,000 bases), while the mature (processed) mRNA are under 10 kilobases (10,000 bases)

23. Removal of introns and joining of exons

24. Mechanism of Removal of introns And Splicing of exons
Splicing process is carried out by splicesomes
(small nuclear ribonucleoprotein particles)
Which are formed of proteins and snRNA (snRNP, or “snurps”).

25. Alternative splicing of mRNA molecules

26. Alternative splicing of mRNA molecules:
It produces multiple variations of the mRNA and, therefore, of its protein product.
This appears to be a mechanism for producing a diverse set of proteins from a limited set of genes.

27. Alternative splicing of mRNA molecules:
For example, different types of muscle cells all produce the same primary transcript from the tropomyosin gene.
However, different patterns of splicing in the different cell types produce seven tissue-specific tropomyosin protein molecules (isoforms).

28. Alternative splicing of mRNA molecules

29. Clinical correlation Genetic diseases
Systemic lupus erythematosus:
A fatal inflammatory disease, results from an autoimmune response in which the patient produces antibodies against host proteins, including snRNP.
β-Thalassemia:
A hemolytic disease caused by defective production of β-globin gene due to splice site mutation of its mRNA

30. The mature mRNA of eukaryotes
Smaller than the primary transcript
Parts: 5’-end cap: 7 methylguanosine triphosphate
5’-UTR
Exons (coding region)
3’-UTR
3’-end poly-A tail
Mature mRNA, eukaryote

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