RNA processing final eukaryotes.
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This document discusses RNA processing in eukaryotic cells. It describes how the primary transcript is modified through capping, polyadenylation, and splicing before being exported to the cytoplasm for protein synthesis. Capping adds a 5' methylguanosine cap. Polyadenylation adds a poly-A tail to the 3' end. Splicing removes introns and joins exons with the help of spliceosomes that contain snRNPs. These processing steps increase mRNA stability and translatability.
RNA processing final eukaryotes.
- 1. RNA Processing M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.
- 2. Overview of the Eukaryotic mRNA Processing
- 3. Eukaryotic cells process the RNA in the nucleus before it is moved to the cytoplasm for protein synthesis The RNA that is the direct copy of the DNA is the primary transcript Two methods are used to process primary transcripts to increase the stability of mRNA for its export to the cytoplasm RNA capping Polyadenylation
- 4. RNA capping happens at the 5’ end of the RNA, usually adds a methylgaunosine shortly after RNA polymerase makes the 5’ end of the primary transcript Splicing of introns removes the intervening sequences in RNA Polyadenylation modifies the 3’ end of the primary transcript by the addition of a string of As Over all Processes
- 5. Modified guanine nucleotide added to the 5′ end Protein-coding segment 3′ UTR Stop codonStart codon 5′ Cap 5′ UTR AAUAAA TRANSCRIPTION RNA PROCESSING DNA Pre-mRNA mRNA TRANSLATION Ribosome Polypeptide G P P P 5′ 3′ a) 5’ Capping of Transcript Modified GTP is added, backwards, on the 5’ end
- 6. After about 30 nt are added, 5’-P is almost immediately modified A phosphate (terminal) is released by hydrolysis The diphosphate 5’ end then attacks the alfa phosphate of GTP to form a very unusual 5’-5’ triphosphate linkage – this is called condensation This highly distinctive terminus is called a cap The N-7 nitrogen of the terminal G is then methylated by S-adenosyl methionine to form cap0
- 8. Uses of Capping Caps are important for subsequent splicing reactions They also contribute to the stability of mRNAs by protecting their 5’ ends from phosphatases and nucleases In addition, caps enhance the translation of mRNA by eukaryotic protein-synthesizing systems Note: tRNA and rRNA molecules do not have caps
- 9. b) Poly-Adenylation Most Eukaryotic mRNAs contain poly A tail Poly A tail is not encoded by DNA Some mRNAs contain an internal AAUAAA (AAU = Asn, AAA = Lys). This highly conserved sequence is only a part of the cleavage signal, but its context is also important The cleavage site is 11 to 30 nt away from the AAUAAA site on the 3’ side After the cleavage by an endonuclease, 50 to 250 A residues are added by Poly adenylate polymerase
- 10. 50 to 250 adenine nucleotides added to the 3′ end Protein-coding segment Polyadenylation signal Poly-A tail3′ UTR Stop codonStart codon 5′ Cap 5′ UTR AAUAAA AAA…AAA TRANSCRIPTION RNA PROCESSING DNA Pre-mRNA mRNA TRANSLATION Ribosome Polypeptide G P P P 5′ 3′ Cleavage site
- 11. Mutating the cleavage sequence in the parent DNA will result in mRNA that is not polyadenylated and not exported to the cytoplasm – instead it is rapidly degraded A second downstream signal that is a G/U rich sequence is required for efficient cleavage and polyadenylation, and is located ca. 50 nucleotides from the site of cleavage. The cleavage and polyadenylation specficity factor (CPSF), a large 4-subunit protein (ca. 360 kDa), forms an unstable complex with the AAUAAA sequence that is subsequently stabilized by the addition of at least 4 separate protein complexes that bind to the CPSF-RNA complex. CstF: Cleavage stimulatory factor interacts with G/U rich sequence CFI: Cleavage factor I and CFII help stabilize protein-RNA complex PAP: Poly(A) polymearse binds to complex before cleavage occurs PABP: Polyadenylate-binding protein binds the Poly (A ) polymerase Assembly of the cleavage/polyadenylation complex
- 12. Cleavage and polyadenylation Specificity Factor Cleavage Stimualtory Factor (PABP) Cleavage site
- 13. (i)
- 14. (ii) CPSF PAP
- 15. (iii)
- 17. TRANSCRIPTION RNA PROCESSING DNA Pre-mRNA mRNA TRANSLATION Ribosome Polypeptide 5′ Cap Exon Intron 1 5′ 30 31 Exon Intron 104 105 146 Exon 3′ Poly-A tail Poly-A tail Introns cut out and exons spliced together Coding segment 5′ Cap 1 146 3′ UTR5′ UTR Pre-mRNA mRNA c) Splicing out Introns
- 18. RNA splicing is responsible for the removal of the introns to create the mRNA Introns contain sequences that act as clues for their removal Carried out by assembly of small nuclear ribonucleoprotein particles (snRNPs) – Spliceosomes
- 19. Spliceosome Activity snRNPs come together and cut out the intron and rejoin the ends of the RNA U1 snRNP attaches to GU of the 5’ intron U2 snRNP attaches to the branch site U4, U5 and U6 snRNPs form a complex bringing together both U1 and U2 snRNPs First the donor site is cut followed by 3’ splice site cut Intron is removed as a lariat – loop of RNA like a cowboy rope
- 20. (U1, U2, U4, U5 and U6)
- 21. Mechanism of Splicing 1. The branch-point A nucleotide in the intron sequence, located close to the 3’ splice site, attacks the 5’ splice site and cleaves it. The cut 5’ end of the intron sequence becomes covalently linked to this A nucleotide 2. The 3’-OH end of the first exon sequence that was created in the first step adds to the beginning of the second exon sequence, cleaving the RNA molecule at the 3’ splice site, and the two exons are joined
- 22. Thomas Cech (1981) Nobel prize in 1989 Exception: RIBOZYME
- 23. Self-splicing of Intron Sequences Group I intron sequences bind a free G nucleotide to a specific site to initiate splicing Group II intron sequences use s specially reactive A nucleotide in the intron sequence itself for the same purpose Both are normally aided by proteins that speed up the reaction, but the reaction is mediated by the RNA in the intron sequence The mechanism used by Group II intron sequences forms a lariat and resemble the activity of spliceosomes
- 25. Comparison
- 26. Alternative Splicing Patterns 1, 2A, 3 1, 2B, 3 1, 2A, 2B, 3 1, 3
- 27. (Calcitonin-gene related protein) Two predominant Poly(A) sites in Rats Cell type specific RNA splicing
- 28. Processing of pre-rRNA transcripts
- 29. Benefits of Splicing Allows for genetic recombination Link exons from different genes together to create a new mRNA Also allows for one primary transcript to encode for multiple proteins by rearrangement of the exons
- 30. RNA Editing
- 31. How do mRNAs get to the cytosol? Why do eukaryotes have DNA within a membrane bound compartment and prokaryotes do not? Could eukaryotes function without it?
- 32. Correspondence between exons and protein domains Gene DNA Exon 1 Intron Exon 2 Intron Exon 3 Transcription RNA processing Translation Domain 3 Domain 1 Domain 2 Polypeptide
- 33. Sequences removed are called Introns Coding sequences flanking introns are called Exons Exons are not removed and are in the mRNA Intron removal is referred to as Splicing Splicing is mediated by a particle: Spliceosome A spliceosome is made of snRNA and protein There are several snRNAs in a spliceosome, U1 to U6 Some introns have self-splicing sequences: Ribozymes Conclusions