hn RNA processing


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hn RNA processing

  1. 1. M.Prasad Naidu MSc Medical Biochemistry, Ph.D.Research Scholar
  2. 2.  Processing of mRNA  hnRNP  snRNP particles  5’Capping  3’Cleavage and polyadenylation  Splicing  Pre-mRNA methylation
  3. 3.  There is essentially no processing of prokaryotic mRNA, it can start to be translated before it has finished being transcribed.  Prokaryotic mRNA is degraded rapidly from the 5’ end
  4. 4.  In eukaryotes, mRNA is synthesized by RNA Pol II as longer precursors (pre- mRNA), the population of different RNA Pol II transcripts are called heterogeneous nuclear RNA (hnRNA).  Among hnRNA, those processed to give mature mRNAs are called pre-mRNAs
  5. 5. Pre-mRNA molecules are processed to mature mRNAs by 5’-capping, 3’- cleavage and polyadenylation, splicing and methylation.
  6. 6.  The hnRNA synthesized by RNA Pol II is mainly pre-mRNA and rapidly becomes covered by proteins to form heterogeneous nuclear ribonucleoprotein (hnRNP)  The hnRNP proteins are though to help keep the hnRNA in a single-stranded form and to assist in the various RNA processing reactions
  7. 7. 1. snRNAs are rich in the base uracil, which complex with specific proteins to form snRNPs. 2. The most abundant snRNP are involved in pre-mRNA splicing, U1,U2,U4,U5 and U6. 3. A large number of snRNP define methylation sites in pre-rRNA.
  8. 8.  snRNAs are synthesized in the nucleus by RNA Pol II and have a normal 5’-cap.  Exported to the cytoplasm where they associate with the common core proteins and with other specific proteins.  Their 5’-cap gains two methyl groups and then imported back into the nucleus where they function in splicing.
  9. 9.  Very soon after RNA Pol II starts making a transcript, and before the RNA chain is more then 20 -30 nt long, the 5’-end is chemically modified.  7-methylguanosine is covalently to the 5´ end of pre-mRNA.  Linked 5´  5´  Occurs shortly after initiation
  10. 10. 7-methylguanosine (m7G)
  11. 11.  Protection from degradation  Increased translational efficiency  Transport to cytoplasm  Splicing of first exon
  12. 12.  In most pre-mRNAs, the mature 3’-end of the molecule is generated by cleavage followed by the addition of a run, or tail, of A residues which is called the poly(A) tail.
  13. 13.  RNA polymerase II does not usually terminate at distinct site  Pre-mRNA is cleaved ~20 nucleotides downstream of polyadenylation signal (AAUAAA)  ~250 AMPs are then added to the 3´ end  Almost all mRNAs have poly(A) tail
  14. 14.  Increased mRNA stability  Increased translational efficiency  Splicing of last intron
  15. 15.  the process of cutting the pre-mRNA to remove the introns and joining together of the exons is called splicing.  it takes place in the nucleus before the mature mRNA can be exported to the cytoplasm.
  16. 16.  Introns: non-coding sequences  Exons: coding sequences  RNA splicing: removal of introns and joining of exons  Splicing mechanism must be precise to maintain open reading frame  Catalyzed by spliceosome (RNA + protein)
  17. 17. Biochemical steps of pre- mRNA splicing Step 1: a cut is made at the 5′splice site, separating the left exon and the right intron-exon molecule. The right intron-exon molecule forms a lariat, in which the 5′terminus of the intron becomes linked by a 5′-2′ bond to a base within the intron. The target base is an A in a sequence that is called the branch site Step 2: cutting at the 3′ splice site releases the free intron in lariat form, while the right exon is ligated (spliced) to the left exon.
  18. 18. C U R A Y Lariat
  19. 19. Nuclear splicing occurs by two transesterification reactions in which a free OH end attacks a phosphodiester bond.
  20. 20.  Catalyzes pre-mRNA splicing in nucleus  Composed of five snRNPs (U1, U2, U4, U5 and U6), other splicing factors, and the pre-mRNA being assembled  U1 binds to the 5’ splice site, then U2 to the branchpoint, then the tri-snRNP complex of U4, U5 and U6. As a result, the intron is looped out and the 5’- and 3’ exon are brought into close proximity.  U2 and U6 snRNA are able to catalyze the splicing reaction.
  21. 21.  The final modification or processing event that many pre-mRNAs undergo is specific methylation of certain bases.  The methylations seem to be largely conserved in the mature mRNA.
  22. 22.  Alternative processing  Alternative poly(A) sites  Alternative splicing  RNA editing
  23. 23.  Alternative mRNA processing is the conversion of pre-mRNA species into more than one type of mature mRNA.  Types of alternative RNA processing include alternative (or differential) splicing and alternative (or differential) poly(A) processing.
  24. 24.  Some pre-mRNAs contain more than one poly(A) site and these may be used under different circumstances to generate different mature mRNAs.  In one cell the stronger poly(A) site is used by default, but in other cell a factor may prevent stronger site from being used.
  25. 25.  The generation of different mature mRNAs from a particular type of gene transcript can occur by varying the use of 5’- and 3’- splice sites in four ways: (i) By using different promoters (ii) By using different poly(A) sites (iii) By retaining certain introns (iv) By retaining or removing certain exons
  26. 26. Alternative splicing
  27. 27. (A) A cassette exon can be either included in the mRNA or excluded.
  28. 28. (B) Mutually exclusive exons occur when two or more adjacent cassette exons are spliced such that only one exon in the group is included at a time.
  29. 29. (C, D) Alternative 5’ and 3’ splice sites allow the lengthening or shortening of a particular exon.
  30. 30. (E, F) Alternative promoters and alternative poly(A) sites switch the 59- or 39-most exons of a transcript.
  31. 31. (G) A retained intron can be excised from the pre-mRNA or can be retained in the translated mRNA.
  32. 32. (H) A single pre-mRNA can exhibit multiple sites of alternative splicing using different patterns of inclusion.