Microsoft PowerPoint - pr 18 L8 ch26 RNA metabolism [호환 모드]

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수아 백
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1 Processing of trna and rrna Bases are modified in post transcription rxs (see Fig ) Pseudouridine ( ) Thiouridine Dihydrouridine, etc. rrnas and trnas are cleaved from longer precursors

2 Modified Bases in trnas and rrnas

3 Ribozyme(RNaseP), RNase III,RNase E FIGURE Processing of pre-rrna transcripts in bacteria.

4 Small nucleolar RNA :snorna +protein =snornp Modification/cleavage 에관여 FIGURE Processing of pre-rrna transcripts in vertebrates.

5 small nuleolar RNA(snoRNA): rrna modification, cleavage, b pseudouridylylations methylation pseudouridine FIGURE The function of snornas in guiding rrna modification.

6 p1080 FIGURE Processing of trnas in bacteria and eukaryotes.

7 Small RNA a) small nuclear RNA(snRNA): splicing, b b) small nuleolar RNA(snoRNA): rrna modification, cleavage, b c) microrna(mirna): RNA 번역억제, 절단, gene regulation, 22b, genome 1%, by RNase III d) si RNA: small interfering RNA, RNA 절단 RNA interference(rnai): sirna, shrna

8 MicroRNAs Function in Gene Regulation MicroRNAs (mirnas): Short noncoding RNAs of ~22 nucleotides Bind to specific regions of mrna to alter translation Assist in cleaving the mrnas Or block the mrna from translation ~1% of the human genome may encode mirna! Synthesized from larger precursors Processed by two endoribonucleases, Drosha and Dicer

9 Steps in mirna Processing Long precursor pri mirna made in nucleus Droshaand DGCR8 cleave pri mirna to a nt precursor Exportin and Ran export this precursor to the cytoplasm Dicer cleaves the pre mirna into dsrna Complement of mirna removed by helicase mirna loaded onto protein complex such as RNAinduced silencing complex (RISC) RISC binds to target mrna

10 RISC mirna prevents translation of mrna (From previous slide): The mirna sequence in RISC binds to complement in target mrna If mirna is ~ perfect complement, target mrna is cleaved Thus, the mrna is not translated If mirna is only partial complement, translation is blocked

11 How mirnas are Processed to Prevent Translation

12 mirna repress mrnas via seed pairing mrna 3 UTR *Near perfect match :sirna, mirna( 식물 ) * Partial match : mirna Seed region mrna cleavage Translation repression

13 RNAi (RNA interference) 21-25nt 의 small size RNA 에의해상보적인염기서열을갖는 mrna 가선택적으로분해되거나 translation 이억제되는현상

14 Types of RNAi mirna : micro RNA sirna : short interfering RNA or small interfering RNA shrna : short hairpin RNA or small hairpin RNA Which type of si-/shrna should be used? Time speed : sirna > shrna Stablility : sirna < shrna - vector How long : sirna < shrna Price : sirna > shrna sirna shrna

15 Viral Vector 1) Adeno virus 광범위한세포주에비교적높은효율로 transduction 이가능하다라는장점을가지고있는바이러스벡터로 shrna 의발현을일주일이상지속시킬수있지만 stable clone 은만들수없다. 재조합 adenovirus 를제조하는데상당한경험과시간이필요하며 high-titer 의바이러스를감염시키면세포독성이나타난다는단점을가지고있다. 또한강한면역성을나타내기때문에동물모델을이용한실험에서반복주입이불가능하다는문제점도가지고있다. 2) Retro virus 면역성과독성을나타내지않으며비교적간단하게 shrna 를발현하는 stable cell line 을구축할수있는바이러스벡터시스템이다. 그러나 primary cell, lymphocyte cell lines, 그리고몇몇세포주에서는비교적낮은 transduction dfficiency 를나타내며, 더군다나 nondividing cell 에는유전자전달이불가능하다는단점이있다. 3) Lentivirus 위에서언급한 shrna 발현벡터들의장점을모두가지고있으며단점들을해결할수있는벡터시스템으로 primary cell 및 non-dividing cell 을비롯한광범위한세포로 transduction efficiency 가매우높으며, 특히동물모델에도효율적인적용이가능하다. 면역성및독성을유발하지않고높은감염효율을바탕으로 transient assay 도다양한세포주에서수행할수있게하며, shrna 를장기간발현하는 stable cell line 의구축도쉽게진행할수있다. Ex) brain, liver, muscle, retina and hematopoietic cell

16 RNAi Overview

18 Ribozymes are RNA molecules that catalytically cleave RNA Cleave themselves or another RNA 3 D structure integral to function Inactive if denatured Show Michaelis Menton kinetics Saturable, have active site, have measureable K M, can be competitively inhibited Nucleophilic attack of sugar OH on phosphate (transesterification) followed by phosphodiester bond hydrolysis

19 Examples of Well characterized Ribozymes Self splicing group I introns Example: 26S rrna precursor from protozoan Tetrahymena RNase P cleaves precursors to trnas Hammerhead ribozyme cleaves RNA of virusoids (circular RNAs that are replicated by plant viruses) So named because 2 structure looks like head of hammer

20 Hammerhead Ribozyme

21 RNA 는서로다른속도로분해됨 - 원핵세포 : mrna 반감기 1.5 분 - 진핵세포 : mrna 반감기 3 hr -by endoribonuclease, exoribonuclease -Exosome complex: 10 conserved exoribonucleases :rrna,trna,snrna,snorna 의 processing/mrna 분해 Cf)Exosome(vesicle): secreted by cell

22 Cellular mrnas are degraded at different rates RNA lifetime is one means of gene regulation Half lives vary from seconds to hours Typical vertebrate mrna ~3 hrs ~10 turnovers per cell generation Shorter(~1.5 mins) half lives for bacterial mrnas Degradation via ribonucleases Hairpin structures in mrna can extend half life

23 Reverse transcriptase(rt) 1) Central dogma DNA복제 2) Role Transcription DNA Reverse transcription RNA RNA 복제 Translation 단백질합성 1 virus RNA에상보적인 DNA가닥의합성을촉매 2 virus RNA DNA 혼성체에서 RNA 가닥을분해하여 DNA로대치 virus RNA로부터 DNA 만듦 3) retrovirus 의종류 reverse transcriptase 를갖는 RNA virus 1 gag virus 입자의내부중심을구성 2 pol β-subunit coding 3 env virus의 envelop를 coding *proofreading exonuclease 활성없음 : 10배이상 error발생 (20,000b당 1개오류 ) >>> 진화, 변종출현

24 Integrase FIGURE Retroviral infection of a mammalian cell and integration of the retrovirus into the host chromosome.

25 Structure and Gene Products of an Integrated Retroviral Genome

26 4) Catalytic reaction (RT) - RNA dependent DNA synthesis, RNA degradation, DNA- dependent DNA synthesis 5) cdna - reverse transcriptase 에의해 mrna 주형에상보적인 DNA 를합성한것 6) Retrovirus: 암, HIV(human immnodeficiency virus) 에의한 AIDS 유발 - 인간에게 후천성면역결핍증 을유발시키는 retrovirus 1 Oncogene 대부분의 retrovirus 가자신의숙주세포를죽이지않고세포내 DNA 에통합되어존재하다가세포가분열할때복제됨 ex) Sarcoma virus 2 HIV 의특징 자신이감염하는 T 세포 살해 숙주의면역체계손상 3 Protease inhibitors virus 효소억제 반복적세포감염과복제의순환발생 / 4 AIDS 치료제 AZT(azido-dideoxy thymidine) HIV 감염확산방지 ㄱ ) 기전 : T 림프구에의해흡수 AZT triphosphate 로전환 HIV reverse transcriptase 에 결합 dttp 의결합을경쟁적으로억제 virus DNA 의합성 stop!!! ㄴ ) 특징 T 림프구자체에독성 골수세포 에는독성! 빈혈현상 생존기간연장 ( 약 1 년 ) or 초기단계 ; 발병늦춰줌 *envelop protein 으로백신개발, ( 돌연변이율높아문제 ), Protease 저해 or RT 저해 >> 복합치료 (combination therapy) (Primer 로서 trna 사용 )

27 T 림프구에의해흡수 AZT triphosphate 으로전환 HIV reverse transcriptase 에결합 dttp 의결합을경쟁적으로억제 virus DNA 의합성 stop

28 Retroviruses can make DNA from RNA Retroviruses have genomes of ssrna and the enzyme reverse transcriptase Virus enters host cell Reverse transcriptase makes DNA from the RNA Then degrades the RNA from the DNA RNA hybrid and replaces it with DNA DNA can then be incorporated into host DNA

Chapter 26

Chapter 26 11 주 RNA 합성 11.1 DNA-dependent synthesis of RNA: Bacteria에서의 transcription l RNA polymerase (5 subunits로구성 : 2α, β, β, σ) l 효소에의한 RNA 합성의특징 - Complementary sequence to template DNA - RNA chain의합성방향 : 5