Download Forming mature 5`ends

Post-transcriptional events III: others
1. Processing of rRNA (eucaryotic and procaryotic)
2. Processing of tRNA
3. Trans-splicing
4. RNA editing
5. 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)
Eukaryotic rRNA processing
Processing scheme of 45S human rRNA precusor
1964, R. Perry, pulse-chase experiment
• Weinberg and Penman (1970),
32P-phosphate and 3Hmethionine, gel electrophoresis,
slice,
Isolation of 45S rRNA processing intermediates from poliovirus-infected
Hela cells
Electron microscopy of human rRNA processing
intermediates, P. Wellauer and I. Dawid (1973)
Methyl groups as signal
for processing
Methylation at 2’OH;
110 CH3-group in 45 S;
all preserved in final
products
Processing bacterial rRNA precursors
tRNA
30 S
Mutation of the RNase III, 30S accumulates
• 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
Altman, Pace and others
RNase P: protein + M1 RNA
5‘-Tyr
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
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.
RNase D, RNase BN,
RNase T, RNase PH,
RNase II, RNPase
(Polynucleotide
phosphorylase)
Li and Deutscher
(1994)
Substrate for in vitro assay of tRNA 3’end maturation
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+
RNase T,
RNase PH
Effect of RNase mutation on maturation of tRNAtyr su3+ 3’end.
Effect of RNase II and PNPase
mutations 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
Schistosoma mansoni
trypanosome
Ascaris lumbricoides
Some organisms that trans-splice
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
Agabian et al
5‘3’
artifact
SL half-itron is
associated with
poly (A) RNA
Treating hypothetical splicing
intermediates with debranching
enzyme
Release of the SL half
intron from a larger RNA
by 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 the
splicing site in an
intergenic region from
Leishmania
Ullu and colleagues
Alteration of the pyrimidine-rich region of the intergenic region
affects both splicing of the down stream gene and polyadenylation
of the upstream gene
Summary
• 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