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RNA editing 2011

2012-02-14 19页 pdf 1MB 23阅读

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RNA editing 2011 1 RNA editing - process by which nucleotides not coded by a gene are introduced at specific positions in an RNA molecule after (or during) transcription – discovered 1986 (Rob Benne) some mRNAs in African trypanosomes differed from their genes – insertions...
RNA editing 2011
1 RNA editing - process by which nucleotides not coded by a gene are introduced at specific positions in an RNA molecule after (or during) transcription – discovered 1986 (Rob Benne) some mRNAs in African trypanosomes differed from their genes – insertions changed coding properties - occurs in wide range of organisms – only in eukaryotes, diverse mechanisms 1. Subsitution editing - chemical modification of individual nucleotides catalyzed by enzymes that recognize a specific target sequence of nucleotides occurs in both pre-mRNAs and tRNA - cytidine deaminases that convert a C in the RNA to uracil (U); -adenosine deaminases that convert an A to inosine (I), which the ribosome translates as a G. 2. Insertion/Deletion editing: Insertion or deletion of nucleotides in the RNA - alterations are mediated by guide RNA molecules that - base-pair as best they can with the RNA to be edited (pre-mRNA) and - serve as a template for the addition (or removal) of nucleotides in the target Possible Consequences: Insertion/Deletion editing Substitution editing changes in splice site selection 2 Subsitution editing - chemical modification of individual nucleotides catalyzed by enzymes that recognize a specific target sequence of nucleotides cytidine deaminases convert a C in the RNA to uracil (U) Factors required: APOBEC protein family human: APOBEC-1, hAID, APOBEC-2, APOBEC3A-3H,APOBEC-4 // C // C // AAA-3’ Cis-element C // AAA-3’ U // AAA-3’ Specificity factor transaminase Transcription Mechanism of RNA editing in mitochondria and plants – in organelles Pre-edited RNA Gene Assembly of multicomponent complex Editing Edited mRNA Specificity factor binds to cis-element and recruits transaminase Dissociation of editing complex 3 Lipoprotein metabolism Mammalian apolipoprotein B Two forms of apolipoprotein B in mammalian serum - both transcribed from same gene In vivo - need spliced and polyadenylated transcript, occurs in nucleus introns suppress editing Apo-B100 - transport of cholesterol in blood Apo-B48 - absorption of lipids from intestines 4563 aa 2351 aa Cytosine deaminase with RNA-binding specificity Glu stop editosome APOBEC – apolipoprotein B mRNA-editing catalytic polypeptide 4 The mooring sequence and the 3′ efficiency element form a double- stranded (ds) stem that is predicted to position the edited cytosine in a favorable configuration for deamination. Model for the apolipoprotein B editing site Original Transcript > 14,000 residues minimal sequence within ~30 nts flanking edited base* secondary structure important Mooring sequence - 11 nts located 4-6 nt downstream of edited base Editosome apobec-1 (apob editing catalytic complex)- catalytic deaminase (CDAR-cytidine deaminase acting on RNA) ZDD- zinc-dependent deaminase domain binds to AU-rich sequences binds to AU-rich elements in 3’ UTR of RNAs and regulate mRNA stability additional proteins ACF apobec-1 complementation factor - adaptor protein binds deaminase and RNA substrate, 5 Subsitution editing - chemical modification of individual nucleotides catalyzed by enzymes that recognize a specific target sequence of nucleotides - adenosine deaminases that convert an A to inosine (I) Can lead to codon change • Most widespread in higher eukaryotes •Complexity of machinery and number of targets increases from lower to higher organisms • occurs in ds RNAs – mostly in 3’UTRs or in non-coding regions Adenosine deaminases that act on RNA (ADARs) - recognize the adenosine to be edited not by a surrounding consensus sequence but by the structure of the duplex that is formed between the editing site and an editing site complementary sequence (ECS) that is usually located in a downstream intron. Adenosine deaminases that act on RNA (ADARs) dsRAD - double stranded RNA adenine deaminase Target recognition basepaired region with mispairing A to I editing All ADARS contain 1-3 double stranded binding motifs (dsRBDs) mediate the binding to the duplex. Found in wide range of organisms from yeast to mammals mRNAs, tRNAs, viral RNAs, non-coding RNAs are substrates for ADAR Subsitution editing - adenosine deaminases that convert an A to inosine (I), which the ribosome translates as a G Substrate - partially ds RNA structure involving exonic and intronic sequences 6 translated exon untranslated exon Experimentally validated structure Re-coding editing Single amino acid changes Types of RNA subject to editing by ADARS glutamate receptor subunit GRIA2 exon 11 Q/R site More than 98% of all pre-mRNAs subject to Alu mediated RNA editing Most Alu repeats in introns and non-translated exons - editing will not directly influence protein function but can indirectly alter protein expression or function • can create cryptic splice donor or acceptor site to induce alternative pre-mRNA splicing • can modify splicing enhancer or inhibitor sequences to modulate alternative splicing efficiency • may lead to the nuclear binding, storage, degradation or release of these I-containing RNA s. 7 Can modulate miRNA structure or miRNA binding sites Can modulate rate of processing by RNase Drosha or can prevent the further maturation and expression of the miRNA miRNA – small regulatory RNA molecules having diverse roles in development, differentiation and cell cycle regulation 16% of all human miRNA genes are subject to A-to-I modification. miRNA excised from longer, hairpin- structured precursors through the sequential action of the RNases Drosha and Dicer A to I conversions in tRNAs I present at positions 34 and 37 in all eukaryotic tRNA Ala of higher eukaryotes Conclusion- A34 and A37 modification may influence translation at different levels Functional signifcance --can influence the stability and structure of tRNAs and improve the fidelity and efficiency of tRNAs in decoding the genetic message -- can improve reading frame maintenance -- can alter cellular distribution and/or function in protein synthesis 8 ADAR - adenine deaminase that acts on RNA Undergoes self-editing In mice - ADAR null mutation is lethal Highly conserved in vertebrates, insects, worms, absent in protozoa, yeasts, plants ADAT - adenine deaminase that acts on tRNA Z-DNA binding motif In A to I editing - splicing occurs AFTER editing because the double- stranded RNA must be formed between the upstream Exon and the downstream Intron 9 RNA editing can regulate splicing by targeting the adenosines involved in splicing Rat ADAR2 edits its own pre-mRNA such that a 3′ splice site is generated, the use of which adds 47 nucleotides to rat ADAR2 and changes the predicted open reading frame. Internal translation initiation then leads to the production of an active enzyme, but lower protein levels are expressed, because the internal translation initiation is relatively inefficient. Self-editing results in a lower ADAR2 concentration, so this process can be thought of as a negative autoregulatory mechanism whereby rat ADAR2 can regulate protein expression by changing a downstream splice site. AUG Factors affecting A to I pre-mRNA editing Likely that splicing and editing components interact with each other Relative localization of methylation and editing machineries important in determining kind and rate of modification made Stability of dsRNA structure sensitive to temperature, availability of RNA helicases, splicing rate regulate editing levels 10 Examples of mammalian A to I editing Most of genes in mammals, Drosophila, Caenorhabditis found to undergo AI editing are expressed in nervous system Cytoplasmic RNase activity specifically cleaves I-dsRNA - may be part of cellular mechanism to process hypermodified dsRNA (viral or cellular) molecules Disruption of the RNA editing balance Direct causal relationship Correlations Possible cross-connections 11 APOBEC and HIV infection APOBEC3G is a cellular restriction factor which inhibits infection with HIV, but to HIV has evolved a protein Vif – viral infection factor which targets APOBEC3G for ubiquitination and degradation. Retrovirus production in a nonpermissive cell expressing APOBEC3G results in the incorporation of APOBEC3G into viral progeny which are able to invade host cells and undergo the early steps of reverse transcription the APOBEC3G cytidine deaminase induces rapid accumulation of deoxyuridine residues HIV has evolved a protein Vif – viral infection factor which targets APOBEC3G for ubiquitination and degradation 12 1. Subsitution editing - chemical modification of individual nucleotides catalyzed by enzymes that recognize a specific target sequence of nucleotides - cytidine deaminases that convert a C in the RNA to uracil (U); - adenosine deaminases that convert an A to inosine (I), which the ribosome translates as a G. 2. Insertion/Deletion editing: Insertion or deletion of nucleotides in the RNA - alterations are mediated by guide RNA molecules that - base-pair as best they can with the RNA to be edited and - serve as a template for the addition (or removal) of nucleotides in the target First found in Trypanosome kinetoplast a single large mitochondrion Trypanosome kinetoplast a single large mitochondrion extends the full length of cell mRNAs synthesized in kinetoplast can be extensively edited - usually through addition of U - to increase their size by as much as 50% Insertion/Deletion editing: Insertion or deletion of nucleotides in the RNA Mitochondrial genome is compartmentalized in kinetoplast – consists of large network of catenated circular DNA molecules Consists of about 50 maxicircles (between 15kbp and 80 kbp) and 10,000 minicircles (between 0.9 kbp and 2.5 kbp) 13 DNA: Maxicircles (22 kb in T. brucei), contains most of the genes - encodes apocytochrome b, subunits 1 and 2 of cytochrome c oxidase (cox), and unassigned reading frames (URFs). Minicircles (1-3 kb), heterogenous Sequencing of genomic Mt DNA (Maxicircles) revealed apparent pseudogenes: Full of Stop codons Deletions of important amino acids Sequences of cDNA clones of some of the kinetoplast mRNAs were partially complementary to pseudogenes on maxicircle DNA cytochrome oxidase subunit II – the COXII DNA sequence is missing 4 Us found in the mRNA Sequencing of other mitochondrial cDNAs and their comparison to the genomic sequence showed not only the addition of U’s but also their deletion 1988 - T. brucei coxIII gene shown to be derived from one of the maxicircle URFs through extensive editing – insertion of 547 U’s and deletion of 41 U’s, doubling the size of the RNA. 1990 - shown that the editing information was provided by small guide RNA’s encoded mainly by minicircles 1996 - in vitro systems to examine the mechanisms of editing were developed extensive editing – e.g. in T. brucei there is a combined total of 3583 inserted and 322 deleted U’s to generate the repertoire of mitochondrial mRNAs. 14 Editing Mechanism -Post-transcriptional -Guide RNAs (gRNAs) direct editing -gRNAs are small and complementary to portions of the edited mRNA -Base-pairing of gRNA with unedited RNA gives mismatched regions, which are recognized by the editing machinery -Machinery includes an Endonuclease, a Terminal UridylylTransferase (TUTase), and a RNA ligase -Editing is directional, from 3’ to 5’ Three regions: 1. Anchor - can base-pair with region of message immediately beside (3’ to) region to be edited 2. Region that directs editing - stretch complementary to message to be edited but containing additional As 3. 3’ polyU stretch - role unclear 15 Formation of duplex looped out single-stranded regions where Us to be inserted Endonuclease recruited to target mRNA cuts opposite loops Tutase (3’ terminal uridylyl transferase) transfers Us into gap RNA ligase closes gap Editing of trypanosome coxII gene Pre-mRNA gRNA The RNA editing process - proceeds 3’ to 5’ respect to mRNA Number of Us added dictated by guide RNA 16 RNA editing proceeds 3’ to 5’ Pre-edited mRNA 3’UTR 5’ - -3’ 3’ UUUU 5’ Guide RNA (1) Pre-edited mRNA 3’UTR 5’ - -3’ 3’ UUUU 5’ Guide RNA (2) Edited mRNA Pre-edited mRNA 3’UTR 5’ - -3’ 3’ UUUU 5’ Guide RNA (3) Edited mRNA Pre-edited mRNA 3’UTR 5’ - -3’ 3’ UUUU 5’ Guide RNA (4) Edited mRNA 3’UTR 5’ - -3’ Fully-edited mRNA Pan-editing of the L. tarentolae A6 mRNA 17 The final pan-edited A6 mRNA has a single open reading frame creation of methionine for translation initiation ribosome-binding site? Editosome contains proteins and RNAs encoded by mitochondrial and nuclear genomes within the Trypanosome Kinetoplast DNA – network of maxicircles and minicircles Nuclear encoded, imported into mitochondria 18 Kinetoplastid RNA editing proteins Model for editosome - multiprotein complex in mitochondrion 20S-40S Separate catalytic sectors for insertion and deletion ligase Tutase exoUase RNase RNA recognition Few enzymes required for catalytic steps in editing but numerous specific dynamic molecular interactions must occur 19 The editing cycle Binding of premRNA and gRNA to editosome Factors stabilize complex Several sites edited as specified by gRNA First gRNA replaced by second Each gRNA cycle entails formation of an anchor duplex with edited sequence formed by previous gRNA each gRNA remains associated with the editosome until decoded and then replaced by subsequent gRNA RNA binding proteins may participate to stabilize association of RNAs with editosome
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