1 / 33

RNA PROCESSING EUKARYOTES

BIOCHEMISTRY

mprasadnaidu
Download Presentation

RNA PROCESSING EUKARYOTES

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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. Over all Processes • 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

  5. Modified guanine nucleotide added to the 5 end TRANSCRIPTION DNA Protein-coding segment Pre-mRNA RNA PROCESSING 5 3 mRNA G P P P AAUAAA Ribosome TRANSLATION Start codon Stop codon 5 Cap 5 UTR 3 UTR Polypeptide 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

  7. 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

  8. 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

  9. 50 to 250 adenine nucleotides added to the 3 end TRANSCRIPTION DNA Polyadenylation signal Protein-coding segment Pre-mRNA RNA PROCESSING 5 3 mRNA G P P AAA…AAA P AAUAAA Ribosome TRANSLATION Start codon Stop codon Poly-A tail 5 Cap 5 UTR 3 UTR Polypeptide Cleavage site

  10. Assembly of the cleavage/polyadenylation complex • 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

  11. Cleavage site (PABP) Cleavage and polyadenylation Specificity Factor Cleavage Stimualtory Factor

  12. (i)

  13. (ii) CPSF PAP

  14. (iii)

  15. Intron Exon 5 Exon Intron Exon 3 5 Cap Poly-A tail Pre-mRNA TRANSCRIPTION DNA 30 1 31 104 105 146 Pre-mRNA RNA PROCESSING Introns cut out and exons spliced together mRNA Coding segment Ribosome TRANSLATION 5 Cap Poly-A tail mRNA Polypeptide 1 146 3 UTR 5 UTR c) Splicing out Introns

  16. 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

  17. 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

  18. (U1, U2, U4, U5 and U6)

  19. 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

  20. Exception: RIBOZYME Thomas Cech (1981) Nobel prize in 1989

  21. 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

  22. Comparison

  23. Alternative Splicing Patterns 1, 2B, 3 1, 2A, 3 1, 2A, 2B, 3 1, 3

  24. Two predominant Poly(A) sites in Rats Cell type specific RNA splicing (Calcitonin-gene related protein)

  25. Processing of pre-rRNA transcripts

  26. 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

  27. RNA Editing

  28. 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?

  29. Gene DNA Exon 1 Exon 2 Intron Exon 3 Intron Transcription RNA processing Translation Domain 3 Domain 2 Domain 1 Polypeptide Correspondence between exons and protein domains

  30. Conclusions • 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 asSplicing • Splicing is mediated by a particle: Spliceosome • A spliceosome is made ofsnRNAandprotein • There are several snRNAs in a spliceosome, U1 to U6 • Some introns have self-splicing sequences: Ribozymes

More Related