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Download Chapt 16: Other RNA Processing 16.1 Ribosomal RNA Processing
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3/30/2011 Chapt 16: Other RNA Processing Student learning outcomes: • Explain how rRNA precursors are cleaved to give final products • Explain how tRNA precursors are trimmed, modified • Describe how trans-splicing and RNA editing occur in some protists or parasitic worms • Describe how RNA interference (RNAi) uses ds RNA to degrade specific mRNA • Figures: 1, 2*, 3, 4*, 5*, 7, 10, 13, 14, 17, 20, 29, 31, 33*, 36*, 37*, 38, 40, 45 • Problems: 1, 2, 3, 5, 16, 17, 20, 22, 23; AQ 1 16-1 16.1 Ribosomal RNA Processing • mRNA in eukaryotes frequently requires splicing, but does not undergo any trimming from ends • rRNA genes of both eukaryotes and bacteria are transcribed as larger precursors; must be processed to yield rRNAs of mature size • Several different rRNA molecules are embedded in a long, precursor; each must be cut out • No splicing occurs, only cutting – (except Tetrahymena) 16-2 Eukaryotic rRNA Processing • Ribosomal RNAs made by pol I in nucleoli are precursors: process to release mature rRNAs • Processing uses snoRNAs (snoRNPs) • Order of RNAs in precursor: – 18S – 5.8S – 28S in all eukaryotes Exact sizes of mature rRNAs vary among species Fig. 1 transcription by pol I of Newt rRNA genes; Many rRNA from 1 gene 16-3 1 3/30/2011 Processing of rRNA in Human Cells 1. 5’-end of 45S precursor RNA is removed to 41S 2. 41S precursor is cut in 2: – – 20S precursor of 18S rRNA 32S precursor of 5.8S, 28S 3. 3’-end of 20S precursor removed, yielding mature 18S rRNA 4. 32S precursor is cut to give 5.8S and 28S rRNA 5. 5.8S and 28S rRNA associate by base-pairing Fig. 2 16-4 Bacterial rRNA Processing • Multiple copies of genes for rRNAs • rRNA precursors contain tRNA, the 3 rRNAs • rRNAs are released by RNase III and RNase E: – RNase III performs at least the initial cleavages that separate individual large rRNAs – RNase E removes 5S rRNA from precursor 16-5 16.2 Transfer RNA Processing • tRNAs made as long precursors in all cells – processed by removing RNA at both ends • Nuclei of eukaryotes have precursors of single tRNA – Made by pol III • Bacteria, precursor may contain one or more tRNA molecules or even rRNA – RNase III cleaves out individuals 16-6 2 3/30/2011 RNase P Forms Mature 5’-Ends of tRNA • Extra nucleotides removed from 5’-ends of pre-tRNA by endonucleolytic cleavage catalyzed by RNase P • RNase P has catalytic RNA subunit - M1 RNA – (bacteria and eukaryotic nuclei) Norm Pace, Sid Altman • Catalysis requires Mg++ • RNase P also has protein 16-7 Fig. 5 RNases Form Mature 3’-Ends of tRNA • 6 RNases contribute to final trimming: – Including RNase D, RNase BN • RNase II and polynucleotide phosphorylase (PNPase) remove most extra nucleotides to +2 • RNases PH and T remove last 2 nucleotides Fig. 7 16-8 16.3 Trans-Splicing • Most splicing is cis-splicing: 2 or more exons from same gene • trans-splicing - exons not part of same gene; may not even be on same chromosome – Trypanosome mRNA: trans-splicing between leader exon (splice leader, SL) and one of many independent exons • Trans-splicing in several organisms – Parasitic and free-living worms (C. elegans) – First discovered in trypanosomes (5’ end of mRNA not match gene sequence; extra 35 nt shared with other mRNAs) 16-9 3 3/30/2011 Trans-Splicing Scheme • Branchpoint A within half-intron attached to coding exon attacks junction between leader exon and its half-intron • Creates Y-shaped intron-exon intermediate analogous to lariat intermediate Trypanosome and red blood cell Fig. 10 16-10 16.4 RNA Editing • Pseudogenes - duplicate copies of genes, mutated, no longer function or not used • Cryptogenes - incomplete genes • Trypanosomatid mitochondria cryptogenes for COX II encode incomplete mRNA - must be edited before translated • Editing occurs 3’→ →5’ direction by successive actions of guide RNAs to insert/ delete Us Fig. 13 minicircles regulate; maxicircles are mitochondrial genes 16-11 Mechanism of Editing • Unedited transcripts found with edited versions of same mRNAs • Editing occurs in poly(A) tails of mRNAs, that were added posttranscriptionally • Partially edited transcripts isolated, always edited at their 3’-ends but not at 5’-ends Fig. 14 example of edited section of Cox II 16-12 4 3/30/2011 Role of gRNA in Editing • Guide RNAs (gRNA) could direct insertion and deletion of UMPs in mRNA • 5’-end of gRNA hybridizes to unedited region at 3’border of editing pre-mRNA • When editing is done, another gRNA could hybridize near 5’-end of newly edited region Fig. 17 16-13 RNA Editing • gRNAs provide A’s and G’s as templates for incorporation of U’s missing from mRNA; • Some G-U bp are used Figs. 18, 20; mechanisms of insertion, deletion 16-14 Mechanism of Removing, Adding U’s • If gRNA is missing A or G to pair with U in mRNA – Then U is removed • Mechanism of removing U’s involves – Cutting pre-mRNA just beyond U to be removed – Removal of U by exonuclease – Ligating two pieces of pre-mRNA together • Adding U’s uses same first and last step • Middle step involves addition of one or more U’s from UTP by TUTase (terminal uridyl transferase) 16-15 5 3/30/2011 16.5 Posttranscriptional Control of Gene Expression • Common form of posttranscriptional control of gene expression is control of mRNA stability • Example: mammary gland tissue stimulated by prolactin -> increase synthesis of casein protein – Most increase in casein not due to increased rate of transcription of the casein gene – Is increase in half-life of casein mRNA Example: transferrin receptor mRNA stability and response to iron concentration 16-16 RNA Interference: posttranscriptional control • RNAi - selective inhibition of expression of genes • Originally antisense RNA thought to block translation by binding mRNA; sense strand also works, and dsRNA is best Fig. 29 C. elegans. Inject antisense or ds RNA to mex-3; In situ hybridize embryos to mex-3 probe. a) Negative; No probe; b) Positive hybridize, no RNAi c) Antisense mex-3 d) ds RNA to mex-3 16-17 RNA Interference • RNA interference - cell encounters dsRNA from: – Virus, Transposon, ds RNA • Trigger dsRNA degraded to 21-23 nt fragments (siRNAs, short interfering RNA) by Dicer, RNase III-like • ds siRNA, with Dicer, Dicer-associated protein R2D2 form Complex B • Complex B delivers siRNA to RISC loading complex (RLC) – Separates 2 strands of siRNA – Transfers guide strand to RNAinduced silencing complex (RISC) that Fig. 33 Model includes protein Argonaute2 (Ago2) 16-18 6 3/30/2011 • Guide strand of siRNA base-pairs with target mRNA in active site of PIWI domain of Ago2 – Ago2 is RNase H-like enzyme known as Slicer – Slicer cleaves target mRNA in middle of region of base-pairing with siRNA – Ago2 purified from an Archaeaon ATP-dependent step -> cleaved RNA ejected from RISC, RISC then accepts new molecule of mRNA for degradation 16-19 Specificity of RNAi, Complex B, RLC, RISC (Ago2) Fig. 36 Ago2 + siRNAs for 2 sites; RNAi requires Mg++ Fig. 37 Assembly of the complexes 16-20 Physiological role, usefulness of RNAi Possible physiological role • Fight ds viruses • Prevent endogenous transposons • Silence transgenes Usefulness to experimenters: • Tool to study basic principles –affect phenotype • Potential to silence oncogenes • ShRNAs provided long-lived research tool (short hairpin RNAs) 16-21 7 3/30/2011 Amplification of siRNA • siRNA is amplified during RNAi when antisense siRNAs hybridize to target mRNA and prime synthesis of full-length antisense RNA by RdRP (RNA-dependent RNA polymerase) • New dsRNA is digested by Dicer into new pieces of siRNA Fig. 38 16-22 RNAi is involved in Heterochromatin Formation in yeast • Genes at yeast centromeres are heterochromatin and silenced; also silent mating-type regions • Yeast mutant in genes for Dicer, Ago and RdR are defective in silencing genes near centromere • Fission yeast Schizosaccharomyces pombe has active transcription of reverse strand at outermost regions of centromere – Rare forward transcripts can base-pair with reverse transcript to trigger RNAi – Recruits histone methyltransferase, methylates Lys-9 of H3 – This recruits Swi6, causing heterochromatization 16-23 RNAi is involved in Heterochromatin Formation in fission yeast near centromere Rare forward transcript -> ds RNA, Dicer, Ago1 and RITS complex, methylation of histones, heterochromatin Fig. 40 16-24 8 3/30/2011 Heterochromatin Formation in Plants and Mammals • Formation of heterochromatin aided by DNA methylation • methylation of C of CpG sequences attracts heterochromatization machinery • Individual genes silenced in mammals by RNAi that targets gene’s control region rather than coding region (ex. X-inactivation) • Silencing involves DNA methylation rather than mRNA destruction 16-25 MicroRNAs (miRNAs) and Gene Silencing • MicroRNAs - 18-25 nt RNAs produced by cleavage from 75-nt stem-loop precursor RNA • Dicer RNase cleaves ds stem part of precursor to yield miRNA in ds form • Single-stranded form of miRNAs joins Argonaute protein in RISC to control gene expression by basepairing to mRNAs – In animals, miRNAs tend to base-pair imperfectly to 3’UTRs of target mRNAs -> inhibition of protein product accumulation of such mRNA 16-26 Pathways to Gene Silencing by miRNAs Fig. 45 16-27 9 3/30/2011 Review questions 1. Draw structure of mammalian rRNA precursor, showing locations of 3 mature rRNAs. 2. What is function of RNAseP? What is unusual about the enzyme? 3. What is the difference between cis- and trans-splicing? 5. Describe RNA editing. What is a cryptogene? 16. Present a model for mechanism of RNA interference 16-28 10