RNA的轉錄后加工分子生物學

1、1977年Robert J and Sharp P A分別發(fā)現(xiàn)斷裂基因（interrupted gene），1993年獲得諾貝爾生理學和醫(yī)學獎當用RNA與其轉錄的模板DNA分子雜交時，RNA鏈取代DNA雙鏈中對應的鏈，形成R-突環(huán)(R-loop)3.7 真核生物RNA的轉錄后加工3.7.1 RNA中的內(nèi)含子真核生物的基因往往是斷裂的基因，其轉錄所形成的RNA前體要經(jīng)過剪切，將內(nèi)含子切除后，將外顯子拼接起來才能形成成熟的mRNA。存在于真核生物基因中無編碼意義而被切除的序列。在前體RNA中的內(nèi)含子也常被稱作“間插序列”。 真核基因平均含810個內(nèi)含子，前體分子一般比成熟mRNA大410倍。3.7

2、.2 tRNA前體的加工加工tRNA前體3端的核酸內(nèi)切酶是RNase F負責修剪的核酸外切酶可能主要是RNase DThe 5 end of tRNA is generated by a cleavage action catalyzed by the enzymeRNase P細菌的tRNA前體存在兩類不同的3端序列。一類其自身具有CCA三核苷酸，位于成熟tRNA序列與3端附加序列之間，當附加序列被切除后即顯露出該末端結構，另一類其自身并無CCA序列，當前體切除3端附加序列后，必須外加CCA。添加CCA是在tRNA 核苷酰轉移酶(nucleotidyl transferase)催化下進行，由

3、CTP和ATP提供胞苷酸和腺苷酸原核生物真核生物tRNA基因的數(shù)目比原核生物tRNA基因的數(shù)目要大得多。真核生物的tRNA也成簇排列，并且被間隔區(qū)所分開真核生物tRNA前體的3端不含CCA序列，成熟tRNA 3端的CCA是后加上去的，由核苷酸轉移酶催化此反應。tRNA的修飾成分由特異的修飾酶所催化。真核生物的tRNA除含有修飾堿基外，還有2-O-甲基核糖，其含量約為核苷酸的百分之一。有些tRNA還具有居間序列，需要進一步拼接真核生物tRNA前體的加工3.7.3 原核生物rRNA的加工Processing of pre-rRNA transcripts in bacteria. 1 Before

4、 cleavage,the 30S RNA precursor is methylated at specific bases. 2 Cleavage liberates precursors of rRNAs and tRNA(s). Cleavage at the points labeled 1, 2, and 3 is carried out by the enzymes RNase III, RNase P, and RNase E, respectively. RNase P is a ribozyme. 3 The final 16S, 23S, and 5S rRNA prod

5、ucts result from the action of a variety of specific nucleases. The seven copies of the gene for pre-rRNA in the E. coli chromosome differ in the number, location, and identity of tRNAs included in the primary transcript. Some copies of the gene have additional tRNA gene segments between the 16S and

6、 23S rRNA segments and at the far 3 end of the primary transcript.6真核生物rRNA前體的加工哺乳動物轉錄產(chǎn)生45S rRNA前體。果蠅是38S，酵母是37S在核仁內(nèi)進行RNA的拼接共有4種方式：核mRNA的拼接體的拼接(nuclear mRNA spliceosomal splicing)類型I自我拼接(group I self-splicing)類型II自我拼接(group II self-splicing)核tRNA的酶促拼接（nuclear tRNA enzymatic splicing）3.7.4 真核生物mRNA前體

7、的加工細胞內(nèi)已發(fā)現(xiàn)的RNA剪接有3種（不包括tRNA的加工）。hnRNA的拼接GT-AG原則（GT-AG rule, GU-AG rule）此規(guī)則不適合于線粒體和葉綠體基因的內(nèi)含子，也不適合于tRNA和rRNA的內(nèi)含子The branch site lies 18-40 nucleotides upstream of the 3 splice site.GU-AG指的是內(nèi)含子的兩端序列在內(nèi)含子內(nèi)部部分序列也可能參與內(nèi)含子的剪接。他們可能是pre-mRNA剪接過程中各種核糖核蛋白剪接調節(jié)因子的結合位點，對于有效和準確的剪接非常重要。剪接的普遍性任何單個mRNA前體的剪接點是通用的，無特異性；剪接

8、裝置無組織特異性，一個RNA 分子在任何細胞均可被正確地剪接。剪接的特異性比較同源基因的進化過程發(fā)現(xiàn)內(nèi)含子的異化大于外顯子，特定的內(nèi)含子還可能在進化過程中丟失，因此內(nèi)含子的功能及其在生物進化中的地位是一個引人注目的問題，另外，許多人類疾病是內(nèi)含子剪接異常引起的，如地中海貧血患者的珠蛋白基因中，大約有1/4的核苷酸圖標發(fā)生在內(nèi)含子的邊界保守序列上，或者雖然位于內(nèi)含子中間但干擾了前體mRNA的正常剪接。13 pre-mRNA剪接的機制和套索結構第一階段，內(nèi)含子的5端切開，左側的外顯子呈線狀，右側的內(nèi)含子-外顯子分子形成一個套索(Lariat)結構。內(nèi)含子游離的5端通過5-2磷酸二酯鍵與分支位點的A

9、相連。第二階段，內(nèi)含子的3剪接點被切斷然后以套索狀釋放，與此同時兩側的外顯子連在一起。 兩階段同時發(fā)生。腺苷酸原來已有3,5-磷酸二酯鍵依然存在，加上此2,5-磷酸二酯鍵連接后，在腺苷酸處出現(xiàn)了一個套索。RNA 剪接由剪接體（splicesome）執(zhí)行轉酯反應是由一個稱為剪接體的大復合體介導的。剪接體中包含約150種蛋白和5種小RNA，大小類似核糖體。RNA 和蛋白質都參與共有序列的識別。剪接體的多數(shù)功能是由RNA分子執(zhí)行的； RNA分子識別內(nèi)含子與外顯子交界序列，親自催化剪接。5 種核內(nèi)小RNA（ snRNA） ：U1，U2，U4，U5，U6 （序列中富含U）與蛋白質形成 RNA-prote

10、in 復合物稱為小的核內(nèi)核糖核蛋白 (snRNP）。 AG前一位核苷酸可以影響剪切效率：CAG=UAGAAGGAGThe snRNPs 在剪接中有3個功能：識別5 剪接位點和分支點; 把這2個位點集結到一起; 催化或協(xié)助催化剪接和連接反應。U2AF (U2 auxiliary factor), recognizes thepolypyrimidine (Py) tract/3 splice site,snRNA pairing is important in splicingU1 snRNP initiates splicing以堿基互補的方式識別pre-mRNA的5剪接點20U2 pairs

11、 with the branch site21U6 pairs with the 5-splicing site22 西北農(nóng)林科技大學 郭澤坤U2AF (U2 auxiliary factor), recognizes thepolypyrimidine (Py) tract/3 splice site,早期復合體EA 復合體B1 復合體B2 復合體C 復合體23U4的釋放，促進U6和U2配對24U5 snRNP helps to bring the two exons together25U2, U5, U6 bring the reaction group close 26一個矛盾（par

12、adox）In principle any 5splice site may be able to react with any 3splice site. But in the usual circumstances splicing occurs only between the 5and 3sites of the same intron. What rules ensure that recognition of splice sites is restricted so that only the 5and 3 sites of the same intron are spliced

13、?27外顯子遺漏28The first guard mechanismThis co-transcriptional loading process greatly diminishes the likelihood of exon skipping.29The second guard mechanism SR (Serine Argenine rich) proteins bind to sequences called exonic splicing enhancers (外顯子剪接增強子ESEs) within the exons. SR proteins bound to these

14、 sites interact with components of the splicing machinery SR proteins recruit the U2AF proteins to the 3 splice site and U1 snRNP to the 5 site3031how does alternative splicing occur so often? The basic answer is that some splice sites are used only some of the time, leading to the production of dif

15、ferent versions of the RNA from different transcripts of the same gene. Alternative splicing can be either constitutive or regulated. In the former case, more than one product is always made from the transcribed gene.In the case of regulated splicing, different forms are generated at different times

16、, under different conditions, or in different cell or tissue types.32正常情況外顯子遺漏外顯子延伸內(nèi)含子保留可變剪接33肌鈣蛋白T34兩種抗原的比例因剪接相關蛋白SF2/ASF的表達水平而不同。SF2/ASF is an SR protein, when abundant, this protein directs the machinery to favor use of the closest 5 splice site.35Alternative Splicing Is Regulated by Activators a

17、nd RepressorsESE: exonic splicing enhancerISE: intronic splicing enhancerESS: exonic splicing silencerISS: intronic splicing silencer36Most of activators are recognized by SR proteinThe SR protein family-which is large and diverse-has specific roles in regulated alternative splicing as well, by dire

18、cting the splicing machinery to different splice sites under different conditionsEach SR protein has another domain, rich in arginine and serine, called an RS domain.The RS domain, found at the C-terminal end of the protein, mediates interactions between the SR protein and proteins within the splici

19、ng machinery.SR蛋白家族數(shù)量龐大種類多樣，在不同的條件下引導剪接體到不同的剪接位點發(fā)揮作用。在發(fā)育的某個階段，或者在某種類型的細胞中，一種特定的SR蛋白的存在與否或者活性高低，就可以決定某一特定的剪接位點是否得到利用。37Most silencers are recognized by members of the heterogeneous nuclear ribonucleoprotein (核不均一核糖核蛋白hnRNP) family. These bind RNA but lack the RS domains and so cannot recruit the spli

20、cing machinery. Instead, by blocking specific splice sites, they repress the use of those sites.38有時hnRNP1結合到多聚嘧啶區(qū)，全面阻斷剪接體的結合；有時它結合到某個外顯子兩外側的序列上，使該外顯子不能進入成熟的mRNA。單個外顯子的剔除有兩種可能的原因：外顯子兩端的hnRNPI通過相互作用，使外顯子突起成一個環(huán)，當剪接體經(jīng)過時把它漏掉?；蛘咴撏怙@子兩端的hnRNPI與其他hnRNPI協(xié)同結合，將外顯子所處的一段RNA覆蓋起來，使得剪接體“看不到”這個外顯子。39RNA剪接的調控某些可變剪接的

21、外顯子總是在成熟mRNA出現(xiàn)，除非受到某種抑制蛋白的阻止（見a),另外一些則相反，只有某種激活因子發(fā)揮作用，才能包含在成熟的mRNA中（見b)。這種剪接調控使得某一特定的外顯子出現(xiàn)在一種類型細胞的成熟mRNA中，而不會出現(xiàn)在另一種類型細胞的成熟mRNA中。與mRNA前體中主要（GU-AG類）和次要（AU-AC類）內(nèi)含子剪接方式不同，I、II類內(nèi)含子能進行自我剪接。內(nèi)含子類型細胞內(nèi)定位GU-AG細胞核，pre-mRNA（真核）AU-AC細胞核，pre-mRNA（真核）I類內(nèi)含子細胞核，pre-mRNA（真核），細胞器RNA，少數(shù)細菌RNAII類內(nèi)含子細胞器RNA，部分細菌RNAIII類內(nèi)含子細胞

22、器RNA雙內(nèi)含子細胞器RNAPre-tRNA內(nèi)含子細胞核，pre-tRNA（真核）生物體內(nèi)各種內(nèi)含子5. I類和II類自剪接內(nèi)含子類型I自我拼接Autosplicing (Self-splicing) describes the ability of an intron to excise itself from an RNA by a catalytic action that depends only on the sequence of RNA in the intron.Group I and group II introns are found in organelles and i

23、n bacteria. (Group I introns are found also in the nucleus in lower eukaryotes.) Group I and group II introns are classified according to their internal organization.Each can be folded into a typical type of secondary structure.The reaction requires only a monovalent cation（單價陽離子）, a divalent cation

24、, and a guanine nucleotide cofactor. No other base can be substituted for G; but a triphosphate is notneeded; GTP, GDP, GMP, and guanosine itself all can be used, so there is no net energy requirement. The guanine nucleotide must have a 3 OH group In vivo, self-splicing intron is complexed with a nu

25、mber of proteins。The proteins help stabilize the correct structure-partly by shielding屏蔽 the negative charges provided by the phosphates in those backbone regionsRNA通過折疊形成一個鳥苷結合口袋，結合游離的GL19-RNA同樣具有催化活性The L-19 RNA is generated by opening the circular intron Figure 26.8 illustrates the mechanism by w

26、hich the oligonucleotide C5 is extended to generate a C6 chain. The C5 oligonucleotide binds in the substrate-binding site, while G414 occupies the G-binding site. By transesterification reactions, a C is transferred from C5 to the 3terminal G, and then back to a new C5molecule. Further transfer rea

27、ctions lead to the accumulation of longer cytosine oligonucleotides. The reaction is a true catalysis, because the L-19 RNA remains unchanged, and is available to catalyze multiple cycles.線型L-19 RNA可催化寡聚胞苷酸（C5）延長，使兩個C5轉化為一個C4和一個C6，稱為polyC聚合酶活性或核苷酸轉移酶活性Group I introns form a characteristicsecondary s

28、tructureGroup I introns form a secondary structure with 9 duplex regions.The core of regions P3, P4, P6, P7 has catalytic activity.Regions P4 and P7 are both formed by pairing between conserved consensus sequences.A sequence adjacent to P7 base pairs with the sequence that contains the reactive GIGS

29、: internal guide sequence.內(nèi)部引導序列酶活性和底物的多樣性核酶具有多種催化活性序列特異性內(nèi)切核酶RNA連接酶磷酸酯酶類似于第一步轉酯反應類型II自我拼接Group II introns excise themselves from RNA by an autocatalytic splicing event.The splice junctions and mechanism of splicing of group II introns are similar to splicing of nuclear introns.A group II intron fold

30、s into a secondary structure that generates a catalytic site resembling the structure of U6-U2-nuclear intron.In the first reaction, the 5 exon-intron junction is attacked by a free hydroxyl group (providedby an internal 2 OH position in group II introns). In the second reaction, the free 3 OH at th

31、e end of the released exon in turn attacks the 3 intron-exon junction主要存在于真核生物的線粒體和葉綠體rRNA基因中。完成第一次轉酯反應的催化部位，其結構在II類自剪接內(nèi)含子和pre-mRNA/snRNP復合體中高度相似 西北農(nóng)林科技大學 郭澤坤54II類自剪接的化學過程與剪接體介導的剪接反應過程基本相同，由內(nèi)含子內(nèi)高度活潑的腺苷酸啟動剪接過程，并形成套索狀產(chǎn)物。I類自剪接內(nèi)含子的RNA通過折疊形成鳥苷結合口袋，從而結合一個游離的鳥苷用以啟動剪接過程。雖然這類內(nèi)含子在體外無需蛋白質協(xié)助就能夠進行自身剪接，但在體內(nèi)環(huán)境下，它們

32、通常需要蛋白質組分來激活剪接反應。3.8 RNA的編輯、再編碼及化學修飾3.8.1 RNA的編輯 RNA的編輯是某些RNA，特別是mRNA前體的一種加工方式，如插入、刪除或取代一些核苷酸殘基，導致DNA編碼的遺傳信息的改變。 介導RNA編輯的機制有兩種： 位點特異性脫氨基作用； 引導RNA指導的尿嘧啶插入或刪除。DNA正鏈序列： GA G A AmRNA 序列： GAU UGU AUA蛋白質 序列： Asp Cys Ile人載脂蛋白B基因位點特異性脫氨基作用site-specific deamination 位點特異性脫氨基作用site-specific deamination 載脂蛋白CU導

33、致提前終止由脫氨基酶催化RNA的編輯不是很普遍。脫氨基是由脫氨酶催化，在RNA編輯時脫氨酶亞基復合體能識別特異性靶位點。RNA editing can be directed by guide RNAs指導RNAA guide RNA is a small RNA whose sequence is complementary to the sequence of an RNA that has been edited. It is used as a template for changing the sequence of the pre-edited RNA by inserting o

34、r deleting nucleotides.Extensive RNA editing in trypanosome（錐蟲） mitochondria occurs by insertions or deletions of uridine.The substrate RNA base pairs with a guide RNA on both sides of the region to be edited.The guide RNA provides the template for addition (or less often deletion) of uridines.Editi

35、ng is catalyzed by a complex of endonuclease, terminal uridyltransferase （尿苷酰轉移酶）activity, and RNA ligase.In 1986, Rob Benne and his colleagues discovered that the sequence of the cytochrome oxidase(細胞色素氧化酶COXII) mRNA from trypanosomes does not match the sequence of the COII gene.Part of the edited

36、sequence of the COXIII mRNA of T.Brucei布魯氏菌尿苷酸的缺失和添加a model for gRNA action in the cytochrome b gene of Leishmania（利什曼蟲）Editing of uridines is catalyzed by a 20S enzyme complex that contains an endonuclease, a terminal uridyltransferase (TUTase), and an RNA ligase。It binds the guide RNA and uses it to pair with the pre-ed