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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
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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)
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Eukaryotic rRNA Processing
Fig. 1 transcription by pol I of Newt rRNA genes;Many rRNA from 1 gene
• 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
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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
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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
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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
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RNase P Forms Mature 5’-Ends of tRNA
Fig. 5
• 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
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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
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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)
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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
Fig. 10
Trypanosome and red blood cell
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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
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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
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Role of gRNA in Editing
Fig. 17
• 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
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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
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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)
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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
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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 RNAic) Antisense mex-3d) ds RNA to mex-3
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RNA Interference
Fig. 33 Model
• 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 RNA-
induced silencing complex (RISC) that includes protein Argonaute2 (Ago2)
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• 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
16-20Fig. 36 Ago2 + siRNAs for 2 sites; RNAi requires Mg++Fig. 37 Assembly of the complexes
Specificity of RNAi, Complex B, RLC, RISC (Ago2)
Physiological role, usefulness of RNAiPossible 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
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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
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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
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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
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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
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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 base-pairing to mRNAs– In animals, miRNAs tend to base-pair imperfectly to 3’-
UTRs of target mRNAs -> inhibition of protein product accumulation of such mRNA
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Pathways to Gene Silencing by miRNAs
Fig. 45
Review questions1. 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
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