醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾課件

1、醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾1 李李 希希 分子醫(yī)學(xué)教育部重點(diǎn)實(shí)驗(yàn)室分子醫(yī)學(xué)教育部重點(diǎn)實(shí)驗(yàn)室 Transcription and Post-transcription Modification 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾2 Post-transcriptional Processing of RNA Making ends of RNA RNA splicing 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾3 Primary Transcript Primary Transcript-the initial molecule of RNA produced- hnRNA （heterogenous n

2、uclear RNA ） In prokaryotes, DNA RNA protein in cytoplasm concurrently In eukaryotes nuclear RNA Cp RNA 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾4 Processing of eukaryotic pre-mRNA Human dystrophin gene has 79 exons, spans over 2,300-Kb and requires over 16 hours to be transcribed! For primary transcripts containing multiple

3、 exons and introns, splicing occurs before transcription of the gene is complete-co- transcriptional splicing. 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾5 Types of RNA processing A) Cutting and trimming to generate ends: rRNA, tRNA and mRNA B) Covalent modification: Add a cap and a polyA tail to mRNA Add a methyl group to 2-O

4、H of ribose in mRNA and rRNA Extensive changes of bases in tRNA C) Splicing pre-rRNA, pre-mRNA, pre-tRNA by different mechanisms. 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾6 The RNA Pol II CTD is required for the coupling of transcription with mRNA capping, polyadenylation and splicing The coupling allows the processing facto

5、rs to present at high local concentrations when splice sites and poly(A) signals are transcribed by Pol II, enhancing the rate and specificity of RNA processing. The association of splicing factors with phosphorylated CTD also stimulates Pol II elongation. Thus, a pre-mRNA is not synthesized unless

6、the machinery for processing it is properly positioned. 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾7 Time course of RNA processing 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾8 5 and 3 ends of eukaryotic mRNA Add a GMP Methylate it and 1st few nucleotides Cut the pre-mRNA and add As 5-UTR3-UTR 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾9 Capping of pre-mRNAs Cap=modified guanine nuc

7、leotide Capping= first mRNA processing event - occurs during transcription CTD recruits capping enzyme as soon as it is phosphorylated Pre-mRNA modified with 7-methyl-guanosine triphosphate (cap) when RNA is only 25-30 bp long Cap structure is recognized by CBC（cap-binding complex） stablize the tran

8、script prevent degradation by exonucleases stimulate splicing and processing 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾10 Sometimes methylated Sometimes methylated The cap is added after the nascent RNA molecules produced by RNA polymerase II reach a length of 25- 30 nucleotides. Guanylyltransferase is recruited and activated

9、 through binding to the Ser5-phosphorylated Pol II CTD. The methyl groups are derived from S- adenosylmethionine. Capping helps stabilize mRNA and enhances translation, splicing and export into the cytoplasm. Capping of the 5 end of nascent RNA transcripts with m7G Existing in a single complex 醫(yī)用分子遺

10、傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾11 Consensus sequence for 3 process AAUAAA: CstF (cleavage stimulation factor F) GU-rich sequence: CPSF (cleavage and polyadenylation specificity factor) 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾12 Polyadenylation of mRNA at the 3 end CPSF: cleavage and polyadenylation specificity factor. CStF: cleavage stimulatory

11、 factor. CFI & CFII: cleavage factor I & II. PAP: poly(A) polymerase. PABPII: poly(A)-binding protein II. Poly(A) tail stabilizes mRNA and enhances translation and export into the cytoplasm. RNA is cleaved 1035-nt 3 to A2UA3. The binding of PAP prior to cleavage ensures that the free 3 end generated

12、 is rapidly polyadenylated. PAP adds the first 12A residues to 3-OH slowly. Binding of PABPII to the initial short poly(A) tail accelerates polyadenylation by PAP. The polyadenylation complex is associated with the CTD of Pol II following initiation. 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾13 Functions of 5 cap and 3 polyA

13、Need 5 cap for efficient translation: Eukaryotic translation initiation factor 4 (eIF4) recognizes and binds to the cap as part of initiation. Both cap and polyA contribute to stability of mRNA: Most mRNAs without a cap or polyA are degraded rapidly. Shortening of the polyA tail and decapping are pa

14、rt of one pathway for RNA degradation in yeast. 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾14 mRNA Half-life t seconds if seldom needed t several cell generations (i.e. 48-72 h) for houskeeping gene avg 3 h in eukaryotes avg 1.5 min in bacteria 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾15 Poly(A)+ RNA can be separated from other RNAs by fractionation on

15、 Sepharose-oligo(dT). 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾16 Split gene and mRNA splicing 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾17 Background: Adenovirus has a DNA genome and makes many mRNAs. Can we determine which part of the genome encodes for each mRNA by making a DNA:RNA hybrid? Experiment: Isolate Adenovirus genomic DNA, isolate one ade

16、novirus mRNA, hybridize and then look by EM at where the RNA hybridizes (binds) to the genomic DNA. Surprise: The RNA is generated from 4 different regions of the DNA! How can we explain this? Splicing! The discovery of split genes (1977) 1993 Noble Prize in Medicine To Dr. Richard Robert and Dr. Ph

17、illip Sharp 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾18 The matured mRNAs are much shorter than the DNA templates. DNA mRNA 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾19 Exon and Intron Exon is any segment of an interrupted gene that is represented in the mature RNA product. Intron is a segment of DNA that is transcribed, but removed from within the tr

18、anscript by splicing together the sequences (exons) on either side of it. 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾20 Exons are similar in size Introns are highly variable in size 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾21 GT-AG rule GT-AG rule describes the presence of these constant dinucleotides at the first two and last two positions of introns

19、of nuclear genes. Splice sites are the sequences immediately surrounding the exon-intron boundaries Splicing junctions are recognized only in the correct pairwise combinations 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾22 The sequence of steps in the production of mature eukaryotic mRNA as shown for the chicken ovalbumin gene.

20、 The consensus sequence at the exonintron junctions of vertebrate pre-mRNAs. 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾23 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾24 4 major types of introns 4 classes of introns can be distinguished on the basis of their mechanism of splicing and/or characterisitic sequences: Group I introns in fungal mitochondria, pl

21、astids, and in pre-rRNA in Tetrahymena (self-splicing) Group II introns in fungal mitochondria and plastids (self-splicing) Introns in pre-mRNA (spliceosome mediated) Introns in pre-tRNA 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾25 Group I and II introns 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾26 The sequence of transesterification reactions that spl

22、ice together the exons of eukaryotic pre-mRNAs. 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾27 Splicing of Group I and II introns Introns in fungal mitochondria, plastids, Tetrahymena pre- rRNA Group I Self-splicing Initiate splicing with a G nucleotide Uses a phosphoester transfer mechanism Does not require ATP hydrolysis. Gro

23、up II self-splicing Initiate splicing with an internal A Uses a phosphoester transfer mechanism Does not require ATP hydrolysis 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾28 Self-splicing in pre-rRNA in Tetrahymena : T. Cech et al. 1981 Exon 1Exon 2Intron 1Exon 1 Exon 2Intron 1 + pre-rRNA Spliced exon Intron circle Intron line

24、ar pre-rRNA Nuclear extract GTP + -+-+ -+- Products of splicing were resolved by gel electrophoresis: Additional proteins are NOT needed for splicing of this pre- rRNA! Do need a G nucleotide (GMP, GDP, GTP or Guanosine). 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾29 The sequence of reactions in the self-splicing of Tetrahymen

25、a group I intron. 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾30 Where is the catalytic activity in RNase P? RNase P is composed of a 375 nucleotide RNA and a 20 kDa protein. The protein component will NOT catalyze cleavage on its own. The RNA WILL catalyze cleavage by itself ! The protein component aids in the reaction but is

26、not required for catalysis. Thus RNA can be an enzyme. Enzymes composed of RNA are called ribozymes. 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾31 Hammerhead ribozymes A 58 nt structure is used in self-cleavage The sequence CUGA adjacent to stem- loops is sufficient for cleavage C U G A G A CCGG GGCC AA A A C U C G A G U C ACC

27、AC UGGUG U Bond that is cleaved. 5 3 CUGA is required for catalysis 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾32 Mechanism of hammerhead ribozyme The folded RNA forms an active site for binding a metal hydroxide Attracts a proton from the 2 OH of the nucleotide at the cleavage site. This is now a nucleophile for attack on the

28、 3 phosphate and cleavage of the phosphodiester bond. 1989 Nobel Prize in chemistry, Sidney Altman, and Thomas Cech 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾33 Distribution of Group I introns Prokaryotes eubacteria (tRNA & rRNA), phage Eukaryotes lower (algae, protists, & fungi) nuclear rRNA genes, organellar genes, Chlorell

29、a viruses higher plants: organellar genes lower animals (Anthozoans): mitochondrial 1800 known, classified into 12 subgroups, based on secondary structure 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾34 Splicing of pre-mRNA The introns begin and end with almost invariant sequences: 5 GUAG 3 Use ATP hydrolysis to assemble a large

30、 spliceosome (45S particle, 5 snRNAs and 65 proteins, same size and complexity as ribosome) Mechanism is similar to that of the Group II fungal introns: Initiate splicing with an internal A Uses a phosphoester transfer mechanism for splicing 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾35 Initiation of phosphoester transfers in

31、pre-mRNA Uses 2 OH of an A internal to the intron Forms a branch point by attacking the 5 phosphate on the first nucleotide of the intron Forms a lariat structure in the intron Exons are joined and intron is excised as a lariat A debranching enzyme cleaves the lariat at the branch to generate a line

32、ar intron Linear intron is degraded 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾36 Involvement of snRNAs and snRNPs snRNAs = small nuclear RNAs snRNPs = small nuclear ribonucleoproteins particles (snRNA complex with protein) Addition of these antibodies to an in vitro pre- mRNA splicing reaction blocked splicing. Thus the snRNP

33、s were implicated in splicing 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾37 Recognizing the 5 splice site and the branch site. Bringing those sites together. Catalyzing (or helping to catalyze) the RNA cleavage. Role of snRNPs in RNA splicing RNA-RNA, RNA-protein and protein-protein interactions are all important during splici

34、ng 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾38 snRNPs U1, U2, U4/U6, and U5 snRNPs Have snRNA in each: U1, U2, U4/U6, U5 Conserved from yeast to human Assemble into spliceosome Catalyze splicing 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾39 Splicing of pre-mRNA occurs in a “spliceosome” an RNA-protein complex” an RNA-protein complex pre-mRNAspliced mRN

35、A spliceosome (100 proteins + 5 small RNAs) The spliceosome is a large protein-RNA complex in which splicing of pre-mRNAs occurs. 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾40 Assembly of spliceosome snRNPs are assembled progressively into the spliceosome. U1 snRNP binds (and base pairs) to the 5 splice site BBP (branch-point

36、binding protein) binds to the branch site U2 snRNP binds (and base pairs) to the branch point, BBP dissociates U4U5U6 snRNP binds, and U1 snRNP dissociates U4 snRNP dissociates Assembly requires ATP hydrolysis Assembly is aided by various auxiliary factors and splicing factors. 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾41 Som

37、e RNA-RNA hybrids formed during the splicing reaction Steps of the spliceosome- mediated splicing reaction 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾42 A schematic diagram of six rearrangements that the spliceosome undergoes in mediating the first transesterification reaction in pre-mRNA splicing. Assembly of spliceosome 醫(yī)用分子

38、遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾43 The spliceosome cycle 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾44 The Significance of Gene Splicing The introns are rare in prokaryotic structural genes The introns are uncommon in lower eukaryote (yeast), 239 introns in 6000 genes, only one intron / polypeptide The introns are abundant in higher eukaryotes (la

39、cking introns are histons and interferons) Unexpressed sequences constitute 80% of a typical vertebrate structural gene 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾45 Errors produced by mistakes in splice-site selection 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾46 Mechanisms prevent splicing error Co-transcriptional loading process SR proteins recruit sp

40、liceosome components to the 5 and 3 splice sites SR protein = Serine Arginine rich protein ESE = exonic splicing enhancers SR protein regulates alternative splicing 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾47 Alternative splicing Alternative splicing occurs in all metazoa and is especially prevalent in vertebrate Five ways t

41、o splice an RNA 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾48 Regulated alternative splicing Different signals in the pre-mRNA and different proteins cause spliceosomes to form in particular positions to give alternative splicing 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾49 765 75 657 Fas pre-mRNA APOPTOSIS Alternative splicing can generate mRNAs encodi

42、ng proteins with different, even opposite functions (programmed cell death) Fas ligand Soluble Fas (membrane) Fas Fas ligand (membrane- associated) (+) (-) 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾50 Alternative possibilities for 4 exons leave a total number of possible mRNA variations at 38,016. The protein variants are imp

43、ortant for wiring of the nervous system and for immune response. Drosophila Dscam gene contains thousands of possible splice variants 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾51 Cis- and Trans-Splicing Cis-: Splicing in single RNA Trans-: Splicing in two different RNAs Y-shaped excised introns (cis-: lariat) Occur in C. eleg

44、ance and higher eukaryotes but it does in only a few mRNAs and at a very low level 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾52 pre-mRNA splicingtrans-mRNA splicing spliced leader Same splicing mechanism is employed in trans-splicing Spliced leader contains the cap structure! 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾53 RNA editing RNA editing is the p

45、rocess of changing the sequence of RNA after transcription. In some RNAs, as much as 55% of the nucleotide sequence is not encoded in the (primary) gene, but is added after transcription. Examples: mitochondrial genes in Trypanosomes （錐蟲） Can add, delete or change nucleotides by editing 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄

46、后修飾54 Two mechanisms mediate editing Guide RNA-directed uridine insertion or deletion Site-specific deamination 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾55 Insertion and deletion of nucleotides by editing Uses a guide RNA (in 20S RNP = editosome) that is encoded elsewhere in the genome Part of the guide RNA is complementary

47、to the mRNA in vicinity of editing Trypanosomal RNA editing pathways. (a) Insertion. (b) Deletion. 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾56 Mammalian example of editing The C is converted to U in intestine by a specific deaminating enzyme, not by a guide RNA. 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾57 Cutting and Trimming RNA Can use endonuclease

48、s to cut at specific sites within a longer precursor RNA Can use exonucleases to trim back from the new ends to make the mature product This general process is seen in prokaryotes and eukaryotes for all types of RNA 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾58 The posttranscriptional processing of E. coli rRNA. 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修

49、飾59 RNase III cuts in stems of stem-loops 16S rRNA23S rRNA RNase III No apparent primary sequence specificity - perhaps RNase III recognizes a particular stem structure. 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾60 Eukaryotic rRNA Processing The primary rRNA transcript (7500nt, 45S RNA) contains 18S, 5.8S and 28S Methylation

50、occur mostly in rRNA sequence 80%: O2-methylribose, 20%: bases (A or G) peudouridine 95 U in rRNA in human are converted to Ys may contribute rRNA tertiary stability 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾61 Transfer RNA Processing Cloverleaf structure CCA: amino acid binding site Anticodon 60 tRNA genes in E. coli A schem

51、atic diagram of the tRNA cloverleaf secondary structure. 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾62 Endo- and exonucleases to generate ends of tRNA Endonuclease RNase P cleaves to generate the 5 end. Endonuclease RNase F cleaves 3 nucleotides past the mature 3 end. Exonuclease RNase D trims 3 to 5, leaving the mature 3 end.

52、 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾63 Splicing of pre-tRNA Introns in pre-tRNA are very short (about 10-20 nucleotides) Have no consensus sequences Are removed by a series of enzymatic steps: Cleavage by an endonuclease Phosphodiesterase to open a cyclic intermediate and provide a 3OH Activation of one end by a kinase

53、 (with ATP hydrolysis) Ligation of the ends (with ATP hydrolysis) Phosphatase to remove the extra phosphate on the 2OH (remaining after phosphodiesterase ) 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾64 Steps in splicing of pre-tRNA P OH 5 2,3 cyclic phosphate Excised intron Intron of 10-20 nucleotides 1. Endo- nuclease 2. Phos

54、pho- diesterase 3. Kinase (ATP) 4. Ligase (ATP) 5. Phosphatase + + Spliced tRNA 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾65 CCA at 3 end of tRNAs All tRNAs end in the sequence CCA. Amino acids are added to the CCA end during “charging” of tRNAs for translation. For most eukaryotic tRNAs, the CCA is added after transcription,

55、 in a reaction catalyzed by tRNA nucleotidyl transferase. 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾66 All of the four bases in tRNA can be modified 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾67 Pathologies resulting from aberrant splicing can be grouped in two major categories Mutations affecting proteins that are involved in splicing Examples:Spinal M

56、uscular Atrophy Retinitis Pigmentosa Myotonic Dystrophy Mutations affecting a specific messenger RNA and disturbing its normal splicing pattern Examples:-Thalassemia Duchenne Muscular Dystrophy Cystic Fibrosis Frasier Syndrome Frontotemporal Dementia and Parkinsonism 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾68 Intron Advanta

57、ge? One benefit of genes with introns is a phenomenon called alternative splicing A pre-mRNA with multiple introns can be spliced in different ways This will generate mature mRNAs with different combinations of exons This variation in splicing can occur in different cell types or during different stages of development 醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾69 Intron Advantage? The biological advantage of alternative splicing is that two (or more) polypeptides can be derived from a single gene This allows an organism to carry fewer gen