post-transcriptional events iii: others 1. processing of rrna (eucaryotic and procaryotic) 2....
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Post-transcriptional events III: others
1. Processing of rRNA (eucaryotic and procaryotic)2. Processing of tRNA3. Trans-splicing4. RNA editing5. Post-transcriptional control of gene expression
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• Ribosomal RNA processing– gene repeat, cluster;
nucleolus
– non-transcribed spacer (NTS)
– transcribed spacers
– Oscar Miller et al.; newt nucleolus, Christmas tree
transcription of rRNA precusor genes (cluster)
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Processing scheme of 45S human rRNA precusor1964, R. Perry, pulse-chase experiment
Eukaryotic rRNA processing
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Isolation of 45S rRNA processing intermediates from poliovirus-infected Hela cells
• Weinberg and Penman (1970), 32P-phosphate and 3H-methionine, gel electrophoresis, slice,
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Electron microscopy of human rRNA processing intermediates, P. Wellauer and I. Dawid (1973)
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Methyl groups as signal for processingMethylation at 2’OH; 110 CH3-group in 45 S; all preserved in final products
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Processing bacterial rRNA precursors
tRNA
Mutation of the RNase III, 30S accumulates
30 S
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• How does the processing apparatus determine what to remove and what to save?
– Pattern of methylation, 2’OH
– 110 methyl groups in 45S rRNA (Hela cells), preserved in mature rRNA
• rRNAs are made in eukaryotic cells as precursors that must be processed to release the mature rRNAs. The order of RNAs in precusor is 18S, 5.8S, 28S in all eukaryotes.
• Prokarytoic rRNA precursors contain tRNAs as well as all three rRNAs. The rRNAs are released from their precuosrs by RNase III and RNase E
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Transfer RNA processing
• Forming mature 5’ends
RNase P action
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The M1 RNA of E.coli RNase P has enzymatic activity
Tyr: mature tRNA; 5’-Tyr: cleaved 5’ fragment;RNase P has no effect on 4.5 S RNA precursor
Altman, Pace and others RNase P: protein + M1 RNA
5‘-Tyr
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Eucaryotic RNase P also has an RNA part and it has the enzymatic activity.Spinach chloroplast RNase P appears not to have an RNA part.
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Forming mature 3’ends.
Li and Deutscher(1994)
Substrate for in vitro assay of tRNA 3’end maturation
RNase D, RNase BN, RNase T, RNase PH, RNase II, RNPase (Polynucleotide phosphorylase)
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Role of RNase D, RNase BN, RNase T, RNase PH, RNase II, polynucleotide phosphorylase (PNPase)
Assay for maturation of tRNATyr su3+
Wild-type RNase PH+,PNPase+
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Effect of RNase mutation on maturation of tRNAtyr su3+ 3’end.
RNase T, RNase PH
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Effect of RNase II and PNPasemutations on maturation of tRNAtyr su3+ 3’end.
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• RNase II and polynucleotide phosphorylase (PNP) cooperate to remove most of the extra nucleotides at the ends of a tRNA precursor, but stop at the +2 state with two extra nucleotides remaining;
• RNase PH and T are most active in removing the last two nucleotides from the tRNA with RNase T being the major participants in removing the very last nucleotide.
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Trans-splicing vs. Cis-splicing
Some organisms that trans-splice
trypanosome
Schistosoma mansoni
Ascaris lumbricoides
Euglena
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• Piet Borst and coworkers (1982), trypanosome a surface coat protein mRNA and gene 5’end no match, extra 35 nt in mRNA.
• More mRNAs discovered to have the extra 35 nt, called the spliced leader (SL)
• none of the genes encode the SL
• SL is encoded by a gene repeat 200X, The gene encodes SL plus 100 nt (an intron -like; with 5’ splice sequence)
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• Two hypothesis for joining the SL to the coding region of an mRNA
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Trans-splicing scheme for a trypanosome
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SL half-itron is associated withpoly (A) RNA
Agabian et al
artifact5‘3’
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Treating hypothetical splicing intermediates with debranching enzyme
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Release of the SL halfintron from a larger RNAby debranching enzyme
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Trypanosome coding regions, including genes encoding rRNAs and tRNAs, are arranged in long, multicistronic transcription units governs by a single promoter
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Trypanosome mRNAs are formed by trans-splicing between a short leader exon and any one of many independent coding exon
1. mRNAs of Trypanosomes have poly(A) tails.2. However, the genes of the parasites lack of polyadenylation signals.
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LeBowitz et al.
Deletions around thesplicing site in anintergenic region fromLeishmania
Ullu and colleaguesAlteration of the pyrimidine-rich region of the intergenic region affects both splicing of the down stream gene and polyadenylation of the upstream gene
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• Polyadenylation in trypanosomes depends on trans-splicing of the downstream coding region to an SL.
• The pyrimidine-rich tract just upstream of the splice site governs both splicing of the downstream gene and polyadenylation of the gene just upstream.
• All the genes in a transcription unit are transcribed equally, yet the amounts of the various mRNAs derived from the transcription unit vary. Control at splicing and polyadenylation level
Summary