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Chapter 6 RNA post-transcriptional processing

Chapter 6 RNA post-transcriptional processing. 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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Chapter 6 RNA post-transcriptional processing

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  1. Chapter 6RNA post-transcriptional processing

  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(核糖核蛋白) 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. 6.1: rRNA PROCESSING • rRNA processing in prokaryotes • rRNA processing in eukaryotes

  11. 6.1-1: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 3. The initial transcript has a sedimentation coefficient of 30s (6000 nt) and is normally quite short-lived rRNA operon

  13. 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 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 rRNA operon

  19. 6.1-2: 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. 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. 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. 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. rRNA前体加工的基本步骤 ①在转录的45S前体rRNA上发生甲基化作用； ②在5’端切除非编码序列生成41S rRNA； ③41S rRNA再被切割成两段，一段为32S，含有28S和5.8S rRNA，另一段为20S，含有18S rRNA； ④32S被剪切成28S和5.8S rRNA， 28S和5.8S rRNA中的部分序列互补，配对； ⑤20S被剪切成18S rRNA；

  24. 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

  25. 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)

  26. 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

  27. 6.2: tRNA PROCESSING, RNaseP AND RIBOZYMES • tRNA processing in prokaryotes • tRNA processing in eukaryotes • RNase P • Ribozymes

  28. tRNA 3-D structure

  29. 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.

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

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

  32. 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

  33. tRNA加工修饰过程包括： ①RNA内切核酸酶在tRNA5’端切断，使5’端逐步成熟； ②RNA内切核酸酶在tRNA3’端切断，再由RNA外切核酸酶从3’端逐个切去附加序列； ③在tRNA3’端添加－CCAOH； ④核酸的修饰和异构化。

  34. 1、tRNA 3’端的成熟 1 由RNaseP切割成tRNA片段，但是3’和5’端仍需要修饰； 2 RNaseF从3’端切割，由RNaseD外切酶对3’端进一步加工， RNaseD是3’端的成熟酶； 3 在tRNA核苷酰转移酶的作用下，添加3’端CCAOH 4 tRNA分子中存在许多修饰碱基，包括甲基化修饰，假尿嘧啶 修饰等。

  35. 2、tRNA 5’端的成熟 RNaseIII切割tRNA的片段中，5’端含有多余的核苷酸，通过RNaseP切除。因此， RNaseP是tRNA 5’端的成熟酶。

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

  37. 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.

  38. 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.

  39. 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.

  40. 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.

  41. 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

  42. 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

  43. 6.3: mRNAPROCESSING, hnRNPsANDsnRNPs • Processing of mRNA • hnRNP • snRNP particles • 5’Capping • 3’Cleavage and polyadenylation • Splicing • Pre-mRNA methylation

  44. 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

  45. Processing of mRNA in eukaryotes • 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

  46. Pre-mRNA molecules are processed to mature mRNAs by 5’-capping, 3’-cleavage and polyadenylation, splicing and methylation.

  47. Eukaryotic mRNA processing: overview

  48. hnRNP: hnRNA + proteins • 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

  49. snRNP particles: snRNA + proteins • snRNAs are rich in the base uracil, which complex with specific proteins to form snRNPs. • The most abundant snRNP are involved in pre-mRNA splicing, U1,U2,U4,U5 and U6. • A large number of snRNP define methylation sites in pre-rRNA.

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