Lecture9 JF

Information about Lecture9 JF

Published on October 16, 2007

Author: Lassie

Source: authorstream.com

Content

Slide1:  RNA processing DNA RNA PROTEIN CENTRAL DOGMA one gene, one enzyme: Beadle and Tatum, 1940s (Nobel in 1958) later modified to one gene, one polypeptide DNA is the genetic material: Watson and Crick, 1953 (Nobel in 1962) Slide2:  RNA processing DNA RNA PROTEIN discovery of reverse transcriptase: RNA can be back-transcribed into DNA Baltimore, Temin – 1970 (Nobel in 1975) Slide3:  RNA processing DNA MATURE RNA PROTEIN pre-mRNA discovery of pre-mRNA splicing Sharp, Roberts in 1977 (Nobel in 1993) Slide4:  RNA processing DNA MATURE RNA PROTEIN PRECURSOR RNAs pre-rRNA pre-tRNA pre-mRNA Cleavage Nucleotide addition Nucleotide insertion Nucleotide removal Sequence addition Sequence removal Base modification Sugar modification Other RNA-related factors affecting expression abundance (combination of transcription and degradation) localization recruitment to ribosomes Slide5:  RNAs that function in RNA processing rRNA snoRNAs form complexes with protein, direct nt modifications snoRNAs also modify tRNAs, and likely other RNAs tRNA RNase P has both RNA and protein components snoRNAs mRNA snRNPs U1,2,4,5,6 form spliceosomes with many proteins gRNAs provide sequence information for RNA editing miRNAs important for regulating gene expression* siRNAs important for regulating gene expression* *Fire, Mello---Nobel in 2006 Slide6:  RNAs that function in RNA processing RNA functions in RNA processing based on complementary basepairing to direct site of action action is usually catalyzed by protein some RNAs—ribozymes—have catalytic activity self-splicing intron in Tetrahymena rRNA—Cech 1982 (Nobel in 1989) ‘hammerhead’ ribozymes are self-cleaving another RNA with catalytic function is LSU rRNA Telomerase RNA for telomere replication RNA primer for mitochondrial replication Slide7:  rRNA processing Cleavage: Pre-rRNA is cleaved to 18S, 5.8S, 28S rRNAs; cleavage order is precise (within species). Modification: Bases and sugars are modified prior to assembly into ribosomes. 5S rRNA encoded separately, elsewhere in genome Slide8:  rRNA processing in nucleolus Modifications of nucleotides: rRNAs ~100 riboses are 2’O-methylated 10 bases methylated 95 Us isomerized to pseudoUs (ψs) tRNAs ~100 kinds of modified nucleotides some incorporated during transcription some chemically modified post-transcription Slide9:  RNA modification snoRNAs modify rRNAs, tRNAs, miRNAs, siRNAs, and mRNAs number variable between organisms; more being found size range ~60 to ~300 nt encoded individually, in polycistronic clusters, or in introns C/D snoRNAs direct methylation H/ACA snoRNAs direct pseudouridylation Most C/D snoRNAs (and snRNAs) have a 5’ trimethylguanosine (TMG) cap. Patients with motor neuron degeneration diseases often develop antibodies that recognize TMG caps. Slide10:  tRNA processing Removal of 5’ leader and 3’ trailer; order not absolute CCA may be encoded (prok.) or added post-transcriptionally (euk.) Acceptor stem sometimes edited Some tRNAs have introns in the anticodon loop Many nucleotide modifications editing intron Slide11:  mRNA processing Capping Splicing Polyadenylation Editing Export Localization Translation Turnover Aguilera 2005 From birth to death, an mRNA associates with a variety of proteins and other RNAs that modify it directly or affect its abundance and recruitment to ribosomes. mRNP (messenger ribonucleoprotein particle): mRNA + associated proteins Slide12:  mRNA processing - capping 5’ capping required for translation of eukaryotic mRNAs mediates initial ribosome binding 7-methylguanosine cap added as RNA exits RNApol II. G linked via a 5’-5’ pyrophosphate bridge to first nt of mRNA G methylated post-addition first bases in mRNA may also become methylated Aguilar 2005 Slide13:  RNA processing - splicing Removes blocks of non-coding sequence (introns), ligates the surrounding coding sequences (exons). Catalyzed by an RNA/protein complex, the spliceosome, which is composed of 5 small nuclear RNAs (snRNAs) designated U1, U2, U4, U5, and U6 plus 50+ proteins Occurs by two transesterification reactions (no energy required) cis-splicing: both exons on same RNA trans-splicing: exons on different RNAs Slide14:  2’OH attack mRNA processing - splicing 3’OH attack Pre-mRNA 5’ exon 3’ exon GU A YAG basepairing of intron with U2 snRNA causes bulged A basepairing of snRNAs with intron sequence and other snRNAs is key to positioning of nt to be spliced Slide15:  mRNA processing - snRNPs snRNP gymnastics snRNAs are packaged with proteins to form snRNPs. Protein:protein, protein:RNA, and RNA:RNA interactions are involved in splicing U1 RNA (snRNP) forms helix with 5’ splice site U2 RNA (snRNP) forms helix with branch point U4, U5, U6 RNA (snRNP) forms helix with 5’ splice site, displacing U1 then forms helix with U2, with loss of U4 first step of splicing occurs rearrangement occurs second step of splicing occurs 1st TE 2nd TE Slide16:  RNA splicing – mechanisms for diversity Alternate splicing Alternate promoters Alternate polyadenylation sites Once considered the exception, it now appears that generating more than one mRNA per gene is a common mechanism for increasing diversity without the ‘expense of maintaining additional genes. Based on ESTs, at least 50% of human genes may produce alternatively spliced mRNAs. Drosophila Dscam gene theoretically has 38,016 possible mRNAs! Slide17:  RNA PROCESSING - EJC Exon junction complex (EJC) core set of proteins and a changing cast of other proteins impacts mRNA splicing, export, localization, translation, and turnover associates with mRNA 20-25 nt upstream of exon-exon junctions. binding to mRNA is position-dependent, not sequence dependent. effect is location-dependent. EJC in ORF enhances translation. EJC in 3’ UTR enhances turnover. stays associated with mRNA until translation begins. Slide18:  mRNA splicing – polyadenylation/3’ end formation Details for transcription termination and 3’ end cleavage are debated. 3’ ends of (almost all) eukaryotic mRNAs are generated by cleavage. Same or similar endonuclease used for 3’ end of mRNAs and of snRNAs. Poly(A) tail is added following 3’ end formation and mRNP is exported to the cytoplasm. Aguilar 2005 Slide19:  RNA export mRNP export from nucleus: association with adaptors exit through NPC localization mRNAs may be translated near site of protein use mRNAs for interacting proteins may be translated near each other translation mRNAs may be translated immediately upon exit from nucleus mRNAs may be stored until needed – common developmental approach turnover nonsense-mediated decay recognizes mRNAs with premature stop codons varied mechanisms affect rate of turnover for “correct” mRNAs turnover occurs in P-bodies Slide20:  RNA localization mRNP export from nucleus: association with adaptors exit through NPC localization mRNAs may be translated near site of protein use mRNAs for destined to be associated may be translated on co-localized polysomes. translation mRNAs may be translated immediately upon exit from nucleus mRNAs may be stored until needed – common developmental approach turnover nonsense-mediated decay recognizes mRNAs with premature stop codons varied mechanisms affect rate of turnover for “correct” mRNAs turnover occurs in P-bodies PABP binds eIF-4E, eIF-4G, circularizing polysomes and increases efficiency of protein synthesis Slide21:  RNA utilization Localization within cell Storage until needed Recruitment to ribosomes RNA turnover Proteins destined to be associated may be translated on co-localized polysomes. PABP binds eIF-4E, eIF-4G, circularizing polysomes and increases efficiency of protein synthesis. Slide22:  mRNA localization a) DAPI stained S. cerevisiae; b) ASH1 mRNA in same cells; c) hairy (green) and even-skipped (red) mRNAs in Drosophila embryo; d) vasa mRNA localizing to division planes in zebrafish embryo, red is β-catenin; e) dpp mRNA (red) at centrosomes in 8 cell embryo. Blue is DAPI, green microtubules; f) β-actin in cultured neurons (red), green is tau, an axonal marker. Slide23:  RNA utilization Localization within cell Storage until needed Recruitment to ribosomes RNA turnover Translational control Slide24:  RNA turnover Steady state abundance of any molecule reflects the balance between its rate of synthesis and degradation. Fine-tuning cell functions thus requires not only transcription but mRNA turnover. Degradation occurs at discrete foci in the cytoplasm called processing bodies or P bodies. Enzymes and partially degraded mRNAs have been co-localized to P bodies. Recently siRNAs and miRNAs have been identified as exerting considerable effect on RNA degradation and translational blocking, respectively. Slide25:  siRNA, miRNA siRNA: small interfering RNA miRNA: microRNA Both are processed to 21-23 nt RNAs which associate with proteins in a RISC complex (RNA-induced silencing complex). Key roles in regulating gene expression in many eukaryotes but not universal. Slide26:  RNA editing changes the sequence of an RNA from that encoded by DNA, producing a functional transcript. First considered a bizarre relic; now recognized as widespread RNA editing has been reported in: protozoa, plants and mammals, not yet fungi or prokaryotes nuclear, mitochondrial, chloroplast, and viral RNAs mRNA, tRNA, rRNA Two general types Base modification (deaminase) A to I double-stranded mechanism, seen in viruses, human genes C to U, U to C seen in chloroplasts, plant mitochondria, human genes Insertion/deletion U insertion/deletion, seen in kinetoplastid protozoa mono/di nucleotide insertion, seen in Physarum nucleotide replacement, seen in Acanthamoeba tRNAs RNA editing Slide27:  A to I RNA editing Nishikura 2006 deamination of A yields I I preferentially pairs with C after DNA replication, A has effectively become G most common mechanism in humans Slide28:  A to I RNA editing Nishikura 2006 G-protein coupling functions of serotonin (5-HT) receptor-2C (5-HT2CR) are dramatically reduced by A→I RNA editing A to I editing is mediated by ADAR protein family. Different members recognize different sequences; multiple members may act on single mRNA. A to I editing common in Alu motifs C to U/U to C editing also involves deamination; mediated by different proteins than a to I Slide29:  RNA editing in kinetoplastid mitochondria ND8 ND9 ND7 COII MURF2 CR4 CR5 RPS12 A6 CYB COIII CR3 22kb MAXICIRCLE (~50 copies) MINICIRCLE (>1000 different molecules) 12 maxicircle mRNAs are edited degree of editing ranges from 4 Us added (COII) to hundreds of additions and dozens of deletions. >50% of COIII mRNA due to U addition and deletion. Several other mRNAs are also extensively edited. Slide30:  ...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGUGAUUUUGGAGUUAUAG... |·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGUGAUUGGAGUUAUAG... ||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGUGuuAUUGGAGUUAUAG... ·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...AGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGuuUGuuAUUGGAGUUAUAG... ·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGGuuUGuuAUUGGAGUUAUAG... |·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...GAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAuuuGGuuUGuuAUUGGAGUUAUAG... ·||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...GCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG... ||··||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...AGGAAAGGUUAGGGGGAGGAGAGAAGAAAGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG... ··|||··||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...GAAAGGUUAGGGGGAGGAGAGAAGAAAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG... ||···|||··||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...AAAGGUUAGGGGGAGGAGAGAAGAAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG... ||||···|||··||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...AGGUUAGGGGGAGGAGAGAAGAuuAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG... ||·||||···|||··||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... ...GUUAGGGGGAGGAGAGAAGuuAuuAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG... |·||||·||||···|||··||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC... 5’...UUAGGGGGAGGAGAGAuAGuuAuuAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG...3’ ····|··|·|·|||·||||·||||···|||··||||·||·||·|||||·|||||||||| 3’ UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...5’ Anchor Information U tail directed by guide RNAs (gRNAs) -- 60-70 nt RNAs with post-transcriptionally added oligo(U) tails Edited region specified by single gRNA: block Editing starts at the 3’ end of pre-edited mRNA. Editing directed by the first gRNA creates the mRNA sequence which will be recognized by the next gRNA. This creates an overall 3’ to 5’ direction for editing. RNA editing in kinetoplastid mitochondria gRNA mRNA Slide31:  Insertion editing Deletion editing RNA editing mechanism

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