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8 Kontrol pasca transkripsi 261114.ppt
- 1. 8. Kontrol pasca transkripsi 8.1 Pemrosesan pre-mRNA eukariot 8.2 Regulasi pemrosesan pre-mRNA 8.3 Transport mRNA 8.4 Kontrol pasca transkripsi di sitoplasma 8.5 Pemrosesan rRNA dan t RNA 1
- 2. RNA yang dibahas dalam bab 8 • mRNA • pre-mRNA • hnRNA • snRNA • pre-tRNA • snoRNA • siRNA • miRNA • Fully processed messenger RNA • mRNA precursor • Heterogeneous nuclear RNAs • small nuclear RNAs: removal of introns • tRNA precursor • Small nucleolar RNA: rRNA processing • Short interfering RNA: cleavage of the "target" mRNA • Micro RNA: inhibits translation of the "target" mRNA 2
- 4. 8.1 Pemrosesan pre- mRNA eukariot 4
- 6. • Terikat pada CTD RNA pol II yang terfosforilasi: • Capping enzyme • Fosfatase • Guanil transferase • Metil transferase • Cap dibentuk setelah RNA pol II mensintesis 25 nt • Negative Elongation Factor terfosforilasi dan lepas • Laju elongasi transkripsi meningkat 6 Penambahan“cap”padaujung5’ mRNA
- 7. Protein hnRNP • Protein hnRNP • terlibat pemrosesan RNA • Mempunyai domain pengikat RNA dan pengikat protein lain • Masing-masing terikat pada bagian RNA yang berbeda • Protein hnRNP A1, C dan D terikat daerah kaya pirimidin dekat ujung 3’ intron Sel Xenopus sel manusia. Sel fusi hnRNP C manusia Remains in nucleus hnRNP A1 manusia Can travel through cytoplasm Sintesis protein dihentikan setelah fusi sel
- 8. hnRNP proteins have RNA binding domains (RBD) • RNA Recognition motif (RRM) • 4 untai sheet dan 2 heliks • hnRNP K protein • KH motif sepanjang 45 asam amino • Struktur lebih kecil dari RRM domain: 3 untai sheet dan 2 heliks • RGG box • 5 pengulangan Arg-Gly-Gly diselingi asam amino aromatik 8
- 9. Biru : muatan positif Merah: muatan negatif RNP1 dan RNP2 • Sequence lestari dari ragi sampai ke manusia • Interaksi dengan RNA
- 10. Splicing pre-mRNA • Splicing sudah dimulai sebelum transkripsi selesai Hibrid mRNA-DNA
- 11. Pre-mRNA vertebrata consensus sequence pada sisi splicing Posisi intron dapat ditentukan dengan membandingkan sequence DNA dengan sequence mRNA/cDNA
- 12. Splicing: reaksi transesterifikasi Proses transesterifikasi tidak memerlukan energi
- 13. Perpasangan antara pre-mRNA dengan snRNA • Proses splicing memerlukan 5 snRNA dan ~ 170 protein • snRNA berpasangan dengan splice sites
- 15. Proses splicing pre- mRNA 15 SF1 = splicing factor 1 U2AF = U2 associated factors Perubahan perpasangan basa
- 16. CTD RNA polimerase II ragi • CTD berasosiasi dengan faktor-faktor pemrosesan pre-mRNA • Mempercepat pemrosesan dan elongasi pre-mRNA • CTD RNA polimerase II mamalia dua kali lebih panjang 16
- 17. Pengenalan exon melalui pengikatan protein SR dan faktor splicing pada pre-mRNA • Protein SR (SR = Serine Arginine) • protein pengikat RNA • Mempunyai beberapa RRM • Mempunyai domain interaksi protein-protein yang kaya dengan Ser dan Arg • ESE = exonic splicing enhancers, sisi pengikatan protein SR 17 Permits precise specification of exons
- 18. Intron self splicing • Grup I ditemukan di gen-gen rRNA inti di protozoa • Grup II ditemukan di gen-gen pengkode protein, rRNA, tRNA di mitokondria pada tumbuhan dan fungi dan kloroplast tumbuhan 18
- 19. Intron Grup II : self splicing 19 Spliceosome structure resemble group II intron Maturase (protein) binds to RNA of group II intron, stabilize 3D structure of intron rapid self-splicing
- 20. Pemotongan dan poliadenilasi ujung 3’ pre-mRNA 20 CPSF = cleavage & polyadenilation specificity factor CStF = cleavage stimulatory factor PABPII = Poly(A) binding protein CFI & CFII stabilize complex
- 21. Exosome • Complex of: • 11 exonucleases 3’ 5’ • Predominant nuclear decay pathway of RNA • Degrade intron and pre-mRNA that has not been properly spliced • RNA Helicases • Disrupt base pairing and RNA-protein interactions • 5’ cap terikat nuclear cap-binding complex • Melindungi dari exosome • Berperan dalam transport mRNA ke sitoplasma • Ekor poly(A) terlindungi dari exosome karena terikat protein PABPII (Poly A Binding Protein II)
- 22. 8.2 Regulasi pemrosesan pre- mRNA Splicing alternatif Editing RNA 22
- 23. Regulasi splicing 23 • Cascade of regulated splicing that controls sex determination in Drosophila • Splicing repressors & activators stop kodon
- 24. Aktivasi splicing pre-mRNA dsx Drosophila 24 • Pada Drosophila betina, exon 4 dikenali sebagai exon. • Dsx repress transcription of genes required for sexual differentiation of the opposite sex SR protein
- 25. Peranan alternative splicing dalam pendengaran 25 • Ca2+ activated K+ channel • Cytosolic domain regulates opening of channel • Different isoforms open at different Ca2+ concentrations • Respond to different frequencies • Panah merah menunjukkan posisi splicing alternatif pada pre-mRNA yang menyebabkan perubahan urutan asam amino yang menyebabkan terbentuknya isofom berbeda Epithelium of chicken cochlea
- 26. RNA editing of apo-B pre-mRNA 26 • Apo-B is a component of large lipoprotein complexes that transport lipids in the serum • Green: domain associates with lipid • Orange: domain binds to LDL receptors on cell membranes • RNA editing wide spread in the mitochondria of protozoan & plants , & chloroplasts • Addition & deletion of U follows templates provided by guide RNAs
- 28. Struktur NPC (Nuclear Pore Complex) 28
- 29. Domain Fg dari Fg-nukleoporin 29 • FG domain hydrophobic • Associate with each other reversibly & rapidly • Allows diffusion of small water soluble molecules • Nuclear transporters have hydrophobic regions on their surface • Bind reversibly with FG-domain • Can diffuse in and out of the nucleus Fg-nukleoporin
- 30. Protein-protein mRNP pada transport mRNA ke luar inti 30 • mRNP exporter is a heterodimer • Nuclear export factor 1 (NXF1) or TAP • Nuclear export transporter 1 (Nxt1) • RNA export factor (REF) • Component of exon junction complex mRNP remodelling: • CPC eIF4E • PABPII PABPI
- 31. Fosforilasi dan transport mRNP 31 • Npl3 = SR protein • Dephospho rylated: promote binding of mRNP exporter • Phosphoryl ated: dissociate from mRNP
- 32. Pembentukan hnRNP dan mRNP 32 • Pre-mRNA berikatan dengan protein membentuk hnRNP • Setelah pemrosesan, pre-mRNA menjadi mRNP, mRNP keluar ke sitoplasma
- 33. Eksport mRNP ke luar nukleus 33 • mRNP • uncoil during passage through the nuclear pore • Bind to ribosome as they enter cytoplasm • Pre-mRNA associated with snRNP in spliceosomes are prevented from being transported into cytoplasm
- 34. Transport mRNA HIV ke luar nukleus 34 RRE = Rev Response Element
- 35. 8.4 Kontrol pasca transkripsi di sitoplasma 35
- 36. Regulasi ekspresi gen dengan RNA pendek 36 Translasi di hambat dalam P body
- 37. Pemrosesan miRNA 37 Drosha: • endonuklease spesifik dsRNA • Memotong pri-miRNA DGCR8/Pasha= dsRNA binding protein Dicer: • memotong pre-miRNA • Ujung 3’: 2 nt untai tunggal TRBP= dsRNA binding protein Satu rantai terikat protein Argonaute di RISC (RNA Induced Silencing Complex)
- 38. Fungsi miRNA dalam perkembangan kaki 38
- 39. miRNA • a class of 19-24 nucleotide long non-coding RNAs derived from hairpin precursors • mediate the post-transcriptional silencing of an estimated 30% of protein-coding genes in mammals • differentiation, apoptosis, proliferation, the immune response, and the maintenance of cell and tissue identity • mammalian miRNAs exist stably in the sera and plasma of humans and animals • Microvesicles (MVs) from human plasma are a mixture of microparticles, exosomes, and other vesicular structures and that many types of MVs in human plasma contain miRNAs • miRNAs could be selectively packaged into MVs and actively delivered into recipient cells where the exogenous miRNAs can regulate target gene expression and recipient cell function Zhang et al. (2011)
- 40. miRNAdari makanandapatlolos dari sistem pencernaandan masukke dalam darah dan jaringantubuh • Zhang et al. (2011) Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross- kingdom regulation by microRNA, Cell Research :1-20.
- 41. Kontrol poliadenilasi dan inisiasi translasi 41 CPE= cytoplasmic polyadenylation element CPEB= CPE Binding protein • Punya RRM dan Zn finger domain Maskin menghambat interaksi eIF-4E dengan eIF yang lain maupun subunit ribosom 40S Immature oocyte + hormon CPSF= cleavage and polyadenylation specificity factor PAP= poly(A) polymerase PABPI= cytoplasmic poly(A) binding protein CPEB terfosfrilasi, maskin lepas P
- 42. Jalur degradasi mRNA eukariot 42
- 43. TOR (target of rapamycin) • Rapamycin • antibiotik yang dihasilkan Streptomyces • dapat menghambat respons imun dari pasien transplantasi organ • TOR (target of rapamycin) • Protein kinase ~ 2400 asam amino • Regulasi proses seluler pada ragi sebagai respons terhadap keadaan nutrisi • mTOR (metazoan TOR) • Responds to multiple signals from cell-surface-signaling proteins to coordinate cell growth with developmental programs as well as nutritional status. 43
- 44. Jalur mTOR untuk regulasi translasi 44 • TSC1/TSC2= GTPase activating protein (GAP) untuk Rheb • AMPK= AMP kinase • mendeteksi penurunan rasio ATP/AMP • TSC1/TSC2 difosforilasi (jadi aktif) • Rheb= monomeric small G protein • S6K memfosforilasi S6 (protein ribosom) dan meningkatkan translasi • 4E-BP menghambat interaksi eIF4E dengan cap mRNA • GEF= guanine nucleotide exchange factor
- 45. Regulasi translasi dan degradasi mRNA yang tergantung Fe 45 IRE= iron response element IRE-BP= IRE-Binding Protein TfR= transferrin receptor
- 46. Nonsense mediated mRNA decay (NMD) (1) 46 • In all properly spliced mRNAs, the stop codon is in the last exon • Akibat adanya delesi, muncul stop kodon di exon 1 atau 2, mRNA didegradasi • mRNA globin dideteksi dengan metode S1 nuclease protection • Actinimycin D menghambat transkripsi
- 47. Nonsense mediated mRNA decay (1) 47 EJC • exon-exon junction complex • EJC lepas saat ribosom pertama lewat PTC= premature termination codon Norm ter= normal termination codon Waktu kompleks SURF terbentuk: • UPF1 terfosforilasi • UPF1 berikatan dengan kompleks UPF2/UPF3 • mRNA berasosiasi dengan P- bodies, ekor poli(A) lepas, degradasi mRNA
- 48. Perubahan mating type ragi 48 • Ash1 (asymetric synthesis of HO) • Represi transkripsi HO • Protein Ash1 hanya dihasilkan oleh daughter cell
- 49. Mekanisme perubahan mating type terbatas pada mother cell 49 • She2 terikat 3’UTR mRNA • She2 terikat She3 yang terikat Myo4 (myocin motor) • Myo4 bergerak sepanjang actin ke arah sel anak • mRNa Ash1 ditransport ke arah daughter cel (tunas)
- 50. Percobaan untuk melihat transport mRNA dari mother cell ke daughter cell (1) 50 Protein fusi GFP- dan RFP-MS2 diberi Nuclear Localization Signal. Protein yang tidak terikat mRNA akan masuk ke dalam inti sel
- 51. Percobaan untuk melihat transport mRNA dari mother cell ke daughter cell (2) 51 Waktu 0.00 46.80 85.17 131.22 168.75 215.65 (detik) RNP di sel induk RNP pindah ke sel anak
- 52. Lokalisasi mRNA neuron pada sinaps 52 • GFP-VAMP expressed in sensory neuron, marks the location of synapses formed between sensory and motor neuron processes • In situ hybridization of an antisensorin mRNA probe (red fluorescence label) • Sensorin = neurotransmitter expressed by the sensory neuron only
- 53. 8.5 Pemrosesan rRNA dan tRNA 53
- 54. Total RNA in rapidly growing mammalian cells • 80% rRNA • 15% tRNA • 5% other RNAs, including mRNA
- 55. Synthesis of ribosomes • Nucleolus • Synthesis of components • RNA pol I synthesizes • 28S & 5.8S rRNA (large subunit) • 18S rRNA (small subunit) • RNA pol III synthesizes • 5S rRNA (large subunit) • RNA pol II synthesizes • mRNA that codes for ~ 70 ribosomal proteins • Processing of pre-rRNA • Assembly • 150 other RNAs and proteins interact transiently with the two ribosomal subunits • Nucleoplasm: • Quality control step occurs before nuclear export • Cytoplasm: • Final steps of ribosome maturation
- 59. Modifikasipre-rRNAyang diarahkanoleh Sno-RNP 59 Box C+D snoRNA: Ribose 2’-O-methylation Box H+ACA snoRNA: pseudouridylation
- 60. Expression of snoRNAs • From own promoters by RNA polymerase II or III • From spliced out introns of • Genes encoding proteins involved in ribosome synthesis or translation • Non-functional mRNAs
- 62. Self-splicing oleh intron Grup I dan Grup II 62 • First discovered in pre-rRNA genes of protozoa Tetrahymena thermophila G= guanosine cofactor
- 63. Pemrosesan tRNA 63 • 5’ end removed by RNAse P • Splicing of pre-tRNA introns • catalyzed by proteins • One step • Require GTP & ATP • UU pada ujung 3’ diganti dengan CCA D = dihydrouridine; = pseudouridine
- 64. Nuclear bodies (Badan Inti) • Specialized nuclear domains • Not surrounded by membranes • Regions of high concentrations of specific proteins and RNAs that form distinct, roughly spherical structures within the nucleus • Proteins associated with nuclear bodies can diffuse in and out of the nuclear bodies 64
- 65. Examples of nuclear bodies • Nucleoli • Site of ribosomal subunit synthesis and assembly • Assembly of immature SRP ribonucleoprotein complexes involved in protein secretion and ER membrane insertion • Storage region for Cdc14 protein phosphatase that regulates processes in the final stages of mitosis and tumor suppressor protein called ARF • Cajal bodies • ~0.2-11µm spherical structures • centers of RNP-complex assembly for snRNPs and telomerase RNP • post-transcriptional modifications of snRNP directed by a guide RNA molecules called scaRNAs (small Cajal body-associated RNAs). • Nuclear Speckles • 25-50 irregular, amorphous structures 0.5-2 1 µm in diameter • storage regions for snRNPs and proteins involved in pre-mRNA splicing • Promyelocytic leukemia (PML) Nuclear Bodies • -10-30 roughly spherical regions 0.3-1 µm in diameter • Located in the nuclei of mammalian cells 65
- 66. Badan-badan inti yang semi-permeabel terhadap berbagai molekul 66 • Xenopus oocyte nucleus injected with fluorescent dextran of different molecular weight • Top: intensity of fluorescence is a measure of dextran concentration (dark: no dextran) Open arrowheads: nucleoli; Closed arrowheads: Cajal bodies with attached nuclear speckles
- 67. Promyelocytic Leukemia (PML) nuclear bodies • Assembly and modification of protein complexes involved in DNA repair and induction of apoptosis • required for cellular defenses against DNA • Protein post-translational modification through the addition of SUMO1 protein (small ubiquitin like moiety-1) • Control the activity and sub-cellular locations of modified proteins (e.g. transcriptional repressors)
Editor's Notes
- Gen sxl punya 2 promoter. Pada awal perkembangan, promoter I hanya aktif pada embrio betina menghasilkan protein Sxl. Selanjutnya,promoter 1 inaktif, promoter 2 aktif di kedua seks.