Post-transcriptional events III: others 1. Processing of rRNA (eucaryotic and procaryotic) 2....

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

• 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)

Processing scheme of 45S human rRNA precusor1964, R. Perry, pulse-chase experiment

Eukaryotic rRNA processing

Isolation of 45S rRNA processing intermediates from poliovirus-infected Hela cells

• Weinberg and Penman (1970), 32P-phosphate and 3H-methionine, gel electrophoresis, slice,

Electron microscopy of human rRNA processing intermediates, P. Wellauer and I. Dawid (1973)

Methyl groups as signal for processingMethylation at 2’OH; 110 CH3-group in 45 S; all preserved in final products

Processing bacterial rRNA precursors

tRNA

Mutation of the RNase III, 30S accumulates

30 S

• 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

Transfer RNA processing

• Forming mature 5’ends

RNase P action

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

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.

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)

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+

Effect of RNase mutation on maturation of tRNAtyr su3+ 3’end.

RNase T, RNase PH

Effect of RNase II and PNPasemutations on maturation of tRNAtyr su3+ 3’end.

• 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.

Trans-splicing vs. Cis-splicing

Some organisms that trans-splice

trypanosome

Schistosoma mansoni

Ascaris lumbricoides

Euglena

• 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)

• Two hypothesis for joining the SL to the coding region of an mRNA

Trans-splicing scheme for a trypanosome

SL half-itron is associated withpoly (A) RNA

Agabian et al

artifact5‘3’

Treating hypothetical splicing intermediates with debranching enzyme

Release of the SL halfintron from a larger RNAby debranching enzyme

Trypanosome coding regions, including genes encoding rRNAs and tRNAs, are arranged in long, multicistronic transcription units governs by a single promoter

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.

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

• 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