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RNA PROCESSING AND RNPs

Molecular Biology Course. RNA PROCESSING AND RNPs. Section O. RNA Processing. Very few RNA molecules are transcribed directly into the final mature RNA . Most newly transcribed RNA molecules ( primary transcripts ) undergo various alterations to yield the mature product

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RNA PROCESSING AND RNPs

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  1. Molecular Biology Course RNA PROCESSING AND RNPs Section O

  2. RNA Processing • Very few RNA molecules are transcribed directly into the final mature RNA. • Most newly transcribed RNA molecules (primary transcripts) undergo various alterations to yield the mature product • RNA processing is the collective term used to describe the molecular events allowing the primary transcripts to become the mature RNA.

  3. Cytoplasm Nucleus or Nucleolus primary transcript RNA processing Romoval of nucleotides addition of nucleotides to the 5’- or 3’- ends modification of certain nucleotides mature RNA.

  4. (1) Removal of nucleotides by both endonucleases and exonucleases • endonucleases to cut at specific sites withina precursor RNA • exonucleases to trim the ends of a precursor RNA • This general process is seen in prokaryotes and eukaryotes for all types of RNA

  5. (2) Addition of nucleotides to 5’-or 3’-ends of the primary transcripts or their cleavage products. Add a cap and a poly(A) tail to pre-mRNA AAAAAA

  6. (3) Modification of certain nucleotides on either the base or the sugar moiety. • Add a methyl group to 2’-OH of ribose in mRNA (A) and rRNA • Extensive changes of bases in tRNA

  7. RNPs Ribonucleoproteins = RNA protein complexes • The RNA molecules in cells usually exist complexed with proteins • specific proteins attach to specific RNAs • Ribosomes are the largest and most complex RNPs

  8. 3-D structure

  9. Digital cryo-electron micrography RNP颗粒的低温电镜图

  10. Section O: RNA processing and RNPs O1: rRNA PROCESSING AND RIBOSOMES • rRNA processing in prokaryotes • rRNA processing in eukaryotes • Prokaryotic ribosomes • Eukaryotic ribosomes

  11. O1-1:rRNAprocessing inprokaryotes • There are 7 different operons for rRNA that are dispersed throughout the genome. • Each operon contains one copy of each of the 5S,the 16S and the 23S rRNA sequences. About 1~4 coding sequences for tRNA molecules are also present in these rRNA operons.

  12. pre-5S rRNA Pre-16S rRNA Pre-tRNA Pre-23S rRNA Pre-tRNA Promoters Terminators O1-1: rRNA processing in prokaryotes 3. The initial transcript has a sedimentation coefficient of 30s (6000 nt) and is normally quite short-lived(fig1) rRNA operon

  13. O1-1: rRNA processing in prokaryotes • RNase III, involved in the first step of rRNA processing • RNase M5, M16 and M23 are involved in the second step of rRNA processing

  14. pre-5S rRNA Pre-16S rRNA Pre-tRNA Pre-23S rRNA Pre-tRNA Promoters Terminators O1-1: rRNA processing in prokaryotes Transcription 30S pre-rRNA Processing steps

  15. Step 1: Following or during the primary transcription, the RNA folds up into a number of stem-loop structures by base pairing between complementary sequences RNA folding

  16. Step 2: The formation of this secondary structure of stems and loops allows some proteins to bind to form a RNP complex which remain attached to the RNA and become part of the ribosome RNP complex formation

  17. Step 3: After the binding of proteins, nucleotide modifications take place. Example: methylation of adenine by methylating agent S-Adenosylmethonine (SAM) Step 4: RNA cleavage

  18. pre-5S rRNA Pre-16SrRNA Pre-tRNA Pre-23S rRNA Pre-tRNA Promoters Terminators Transcription 30S pre-rRNA: RNase III III P F III III P F P E Cleavage at RNase M16 M16 M23 M23 M5 16S rRNA tRNA 23S rRNA 5S rRNA tRNA O1-1: rRNA processing in prokaryotes: CLEAVAGE rRNA operon

  19. O1-2: rRNA processing in eukaryotes • rRNA in eukaryotes is also generated from a single, long precursor molecule by specific modification and cleavage steps • The processes are not so well understood

  20. O1-2: rRNA processing in Eukaryotes: rRNA features The rRNA genes are present in a tandemly repeated cluster containing 100 or more copies of the transcription unit, and are transcribed in nucleolus by RNA Pol I Precursor sizes are different among organisms (yeast: 7000 nt; mammalian 13500 nt), and pre-mRNA processing is also slightly different among organism.

  21. O1-2: rRNA processing in Eukaryotes: rRNA features 3. The precursor contains • one copy of the 18S coding region and • one copy each of the 5.8S and 28S coding regions, which together are the equivalent of the 23S rRNA in prokaryote 4. The large precursor RNA undergoes a number of cleavages to yield mature RNA and ribosome.

  22. O1-2: rRNA processing in Eukaryotes: rRNA features 5.The eukaryotic 5S rRNA • is transcribed by RNA Pol III from unlinked genes to give a 121nt transcript • the transcript undergoes little or no processing

  23. 18S 5.8S 28S 47S ETS1 ITS1 ITS2 ETS2 45S 41S 20S and 32S Mature rRNAs 18S rRNA 5.8S rRNA 28S rRNA Mammalian pre-rRNA processing Indicates RNase cleavage

  24. O1-2: rRNA processing in Eukaryotes: rRNA processing • The 5.8S region must base-pair to the 28S rRNA before the mature molecules are produced. • Mature rRNAs complex with protein to form RNPs(nucleolus) • Methylation occurs at over 100 sites to give 2’-O-methylribose, which is known to be carried out by snRNPs (nucleolus)

  25. O1-2: rRNA processing in Eukaryotes: rRNA processing • Introns (group I) in rRNA genes of some lower eukarytes (Tetrahymena thermophila) must be spliced outto generate mature rRNAs. • Many group I introns are found to catalyze the splicing reaction by itself in vitro, therefore called ribozyme

  26. O1-3: Ribosomes • Protein biosynthetic machinery • Made of 2 subunits (bacterial 30S and 50S, & Eukaryotes 40S and 60S) • Intact ribosome referred to as 70S ribosome in Prokaryotes and 80S ribosome in Eukaryotes • In bacteria, 20,000 ribosomes per cell, 25% of cell's mass.

  27. Prokaryotic Ribosome E. coli ribosome is 25 nm in diameter, 2750 kD in mass, and consists of two unequal subunits that dissociate at < 1mM Mg2+

  28. Prokaryotic Ribosome Prokaryotic Ribosomal proteins Size varied from 46 aa to 557 aa. Most are basic proteins which bind to RNA. rRNA chaperons to assist the folding of rRNA to catalytic structure. Ribosomal rRNA Responsible for peptide bond formation.

  29. Features of the E.coli ribosome Cleft Platform Central protuberance Stalk Small

  30. Prokaryotic Ribosome Ribosome Assembly • Assembly is coupled w/ transcription and pre-rRNA processing

  31. Prokaryotic Ribosome Ribosome Structure (1)

  32. Ribosome Structure (2) • mRNA is associated with the 30S subunit • Two tRNA binding sites (P and A sites) are located in the cavity formed by the association of the 2 subunits. • The growing peptide chain threads through a “tunnel” that passes through the 30S subunit.

  33. Eukaryotic Ribosome • larger and more complex than prokaryotic ribosomes, but with similar structural and functional properties

  34. Section O: RNA processing and RNPs O2: tRNA PROCESSING, RNaseP AND RIBOZYMES • tRNA processing in prokaryotes • tRNA processing in eukaryotes • RNase P • Ribozymes

  35. tRNA 3-D structure

  36. tRNA processing in prokaryotes Mature tRNAs are generated by processing longer pre-tRNA transcripts, which involves • specific exo- and endonucleolytic cleavage by RNases D, E, F and P (general) followed by • base modifications which are unique to each particular tRNA type.

  37. tRNA processingin prokaryotes Primary transcripts RNase D,E,F and P (See your text book) tRNA with mature ends Base modifications mature tRNAs

  38. tRNA processing in eukaryotes The pre-tRNA is synthesized with a • 16 nt 5’-leader, • a 14 nt intron and • two extra 3’-nucleotides.

  39. tRNA processingin Eukaryotes • Primary transcripts forms secondary structures recognized by endonucleases • 5’ leader and 3’ extra nucleotide removal • tRNA nucleptidyl transferase adds 5’-CCA-3’ to the 3’-end to generate the mature 3’-end • Intron removal

  40. RNase P • Ribonuclease P (RNase P) is an enzyme involved in tRNA processing that removes the 5' leader sequences from tRNA precursors

  41. RNase P (2) • RNase P enzymes are found in both prokaryotes and eukaryotes, being located in the nucleus of the latter where they are therefore small nuclear RNPs (snRNPs) • In E. coli, the endonuclease is composed of a 377 nt RNA and a small basic protein of 13.7kDa.

  42. RNase P (1) • RNA component can catalyze pre-tRNA in vitro in the absence of protein. Thus RNase P RNA is a catalytic RNA, or ribozyme.

  43. Ribozyme (1) • Ribozymes are catalytic RNA molecules that can catalyze particular biochemical reactions. • RNase P RNA is a ribozyme. • RNase P RNA from bacteria is more catalytically active in vitro than those from eukaryotic and archaebacterial cells. All RNase P RNAs share common sequences and structures.

  44. Ribozyme (2) • Self-splicing introns: the intervening RNA that catalyze the splicing of themselves from their precursor RNA, and the joining of the exon sequences • Group I introns, such as Tetrahymena intron • Group II introns.

  45. Ribozyme (3) • Self-cleaving RNA encoded by viral genome to resolve the concatameric molecules of the viral genomic RNA • HDV ribozyme • Hairpin ribozyme • Hammer head ribozyme

  46. Ribozyme (4) Ribozymes can be used as therapeutic agents in • correcting mutant mRNA in human cells • inhibiting unwanted gene expression • Kill cancer cells • Prevent virus replication

  47. Section O: RNA processing and RNPs O3: mRNAPROCESSING, hnRNPsANDsnRNPs • Processing of mRNA • hnRNP • snRNP particles • 5’Capping • 3’Cleavage and polyadenylation • Splicing • Pre-mRNA methylation

  48. Processing of mRNA: prokaryotes • 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

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