RNA Processing: Post-Transcriptional Modification
- 1. a RNA Processing (or Post Transcriptional Modification)
- 8. RNA Processing In Eukaryotes, newly transcribed RNA undergo iteration of molecular events to convert the primary transcript into a mature mRNA. Transcription of Protein RNA Processing IN Eukaryotes Primary RNA mRNA Coding Genes
- 9. Heterogeneous Nuclear RNA(hnRNA) It refers to the large pre‐mRNAs of various nucleotide sequences that are made by RNA Polymerase II Mean Length 2000 bp
- 10. Processing of pre-RNA Post Transcriptional Modifications 1) 5’ capping 2) 3’ Cleavage/ Polyadenylation 3) Splicing 4) RNA editing Before being transported to cytoplasm where they are translated by ribosomes. After transcription, eukaryotic mRNA is altered extensively, changes are made to RNA molecules 5’ end, the 3’ end and protein coding portion.
- 11. The 5’ Cap Addition - While the pre mRNA is being synthesized, a 7-methylguanosine cap is added to the 5’ end of the growing transcript by a 5’ to 5’ phosphate linkage. - It occurs rapidly after start of transcription and much before completion. The capping is catalyzed by an enzyme guanylyl transferase, which associates with the phosphorylated carboxy terminal domain(CTD) of RNA polymerase I.
- 12. • A capping enzyme (CE) is an enzyme that catalyzes the attachment of the 5' cap to messenger RNA molecules that are in the process of being synthesized in the cell nucleus during the first stages of gene expression. • Capping enzymes are found in the nucleus of eukaryotic cells. Depending on the organism, the capping enzyme is either a monofunctional or bifunctional
- 14. 1 One subunit of capping enzyme removes a phosphate from 5’ end of hnRNA (Phosphohydrolase) 2. Other subunit) transfers Guanosine 5’- 5’ Triphosphate structure. (Guanylyl Transferase) 3 Transfer methyl groups from S-adenosylmethionine to N7 position of guanine at 5’ end of nascent RNA.
- 15. Role of 5’ Cap - Protection of mRNA from degradation -Transportation of mRNA from nucleus to cytoplasm -Binding of ribosome with mRNA.
- 16. The final cap contains a novel 5’ to 5’ linkage between the positively charged 7-methyl G residue and the 5’ end of the RNA transcript. Cap 0 ---> If mRNA has methyl group on N7 position of “G” at the 5’ end Cap 1, Cap2-->In some species, a methyl group is added to the 2nd as well as 3rd nucleoside of the capped m RNA
- 17. Steps in capping • Removal of leading phosphate group.from mRNAs 5’ terminal triphosphate group (Phosphohydrolase) • Capping enzyme (Guanylyl transferase) • Methylation of Guanine (Guanosine 7-methyl transferase) • Uses S adenomethionine
- 18. Tailing/ Polyadenylation Poly(A)polymerase was first identified in 1960 as an enzymatic activity in extracts made from cell nuclei that could polymerise ATP The poly-A tail is a long chain of adenine nucleotides that is added to a messenger RNA (mRNA) molecule during RNA processing to increase the stability of the molecule. A poly (A) tail is added to the 3 end of the pre-mRNA ′ once elongation is complete.
- 19. •The poly-A tail makes the RNA molecule more stable and prevents its degradation. •Poly(A)polymerase was first identified in 1960 as an enzymatic activity in extracts made from cell nuclei that could polymerise ATP, but not ADP, into polyadenine.
- 20. Tailing/ Polyadenylation ~ Polyadenylation – Cutting of Endonuclease enzyme and Adding of Poly A tail by poly(a) polymerase enzyme, utilizing ATP. ~ The poly A tail is added by template independent RNA polymerase called poly(A) polymerase. ~ Cuts at internal site, that creates a new 3’ end where poly A tail is added. ~ Series of 250 or more Adenine nucleotides
- 21. Requirements 1) Signal Sequence in mRNA a) 5’ AAUAAA 3’ -binding site for cleavage and polyadenylation factor) -Directs polyadenylation in mammals b) 5’ CA 3’ - binding site for Poly A polymerase -Poly A signal located immediately after 5’ CA 3’ sequence c) GU rich region cleavage stimulation factor, Binding site for Cleavage factor 1 2. maybe absent in yeast 2) Endonuclease 3) Poly (A) Polymerase 4) ATP, as a substrate
- 22. STEPS IN POLYADENYLATION 1. Cleavage - Generation of proper 3’ end structure occurs by an endonuclease (consisting of CF 1, CF 2, Cst F) that cleaves RNA Cleavage is followed by polyadenylation in a tightly coupled manner.
- 23. 2) Polyadenylation passes through 2 stages: ~ First, a rather short oligo(A) sequence(~ 10 residues) is added to 3’ end ~ 2nd phase poly (A) pol extend the oligo(A) tail to the fall ~ 200 residue length. 3) PABP (Poly-adenylation Binding Protein) helps the polymerase to the Adenosines.
- 24. PABP- influences the length of poly(A) tail, it has its role in maintaining the tail after synthesis. 4) Binding of poly(A) polymerase stimulates cleavage and then residues at slow rate to 3’-OH group generated by cleavage.
- 25. Function of Polyadenylation 1) Export of mature mRNA from nucleus. 2) Affects the stability of some mRNAs 3) Serves as a recognition sequence of ribosomes.
- 26. RNA Splicing Berget, Moore, and Sharp had discovered RNA splicing. In 1977 they independently worked with one another, Phillip Sharp and Richard Roberts both successfully demonstrated how RNA can be divided up into introns and exons. RNA is modified in the nucleus by additions to the 5' and 3' ends and by splicing to remove the
- 27. The process of removing the sequence in split genes(RNA) that correspond to introns and joining of sequences corresponding to exons, to form a continuous exons, to form a continuous
- 28. One end of the intron is referred to as the 5' splice site, and the other end is the 3’ splice site. Most introns begin with GU in pre- mRNA
- 30. • Five RNA molecules(U1, U2, U4, U5 and U6) and almost 300 proteins from the spliceosome termed as snurp U3 present in nucleolus termed as snoRNA • Small nuclear RNA (snRNAs) ranging in length from 107 to 210 nucleotides are the RNA components. These snRNAs are associated with proteins to form small particles of ribonucleoprotein.
- 31. •The intron types differ in their transesterification and structure, the source of the initial hydroxyl donor, the nature of the intermediate.
- 33. Splicing of Nuclear pre mRNA – These are the highly represented intron types which are unable to splice auto-catalytically and are identified as nuclear pre-mRNA introns. --The 5’ left splice site - donor site 3’ right splice site - acceptor site, and a branch site is involved in splicing reaction • The cutting and pasting mechanism is coordinated in splicing machinery termed as spliceosome that contains proteins often termed as U-12 mRNA Processing and
- 34. STEPS in RNA SPLICING The U1 snRNP forms base pairs with the 5’ splice junction and the BBP (Branch Point Binding Protein) and U2AF (U2 Auxillary factor) recognize the Branch-Point site The U2 snRNP displaces BBP and U2AF and forms base pairs with the branch point site consensus sequence.
- 35. STEPS in RNA SPLICING The U4/ U6 + U5 “triple” snRNP enters the reaction. In this triple snRNP, the U4 and U6 snRNAs are held firmly together by base- pair interactions.
- 36. STEPS in RNA SPLICING • Subsequent Rearrangements break apart the U4/U6 base pairs, allowing U6 to dispace U1 at the 5’ splice junction. • This creates the active site that catalyzes the first phosphoryl- transferase reaction. Additional RNA-RNA rearrangements create the active site for the second phosphoryl-transferase reaction, which then completes the splice. Each splicing event removes one intron through two sequential phosphoryl-transfer reactions known as transesterifications. These join two exons together while removing the intron between them as a “lariat”
- 37. RNA splicing catalyzes by an assembly of snRNPs plus other Proteins, which together constitute the spliceosome. The Spliceosome recognizes the splicing signals on a pre- mRNA molecules, brings the Two ends of the introns together, and provides the enzymatic activity For the two reaction steps required. As indicated, a set of proteins called the exon junction complex (EJC) Remains on the spliced mRNA molecule; its subsequent role will be to assemble