Download Lecture 12 - U of L Class Index

RNA Processing and
Editing
Ribosomal RNA Processing
– rRNA genes of both eukaryotes and bacteria are
transcribed as larger precursors and must be
processed to yield rRNAs of mature size
– Several different rRNA molecules are embedded
in a long, precursor and each must be cut out
Ribosomal RNA Processing
rRNA genes are organized in clusters separated by
nontranscribed spacers (NTS)
Eukaryotic rRNA Processing
• Ribosomal RNAs are made in eukaryotic nucleoli as
precursors that must be processed to release mature
rRNAs
• Order of RNAs in the precursor is
– 18S
– 5.8S
– 28S in all eukaryotes
– Exact sizes of the mature rRNAs vary from one
species to another
• rRNA processing requires complex of small
nucleolar RNAs (snoRNAs) and small nucleolar
ribonucleoproteins (snoRNPs)
Human Cells Processing
1. 5’-end of 45S precursor RNA
is removed yielding the 41S
2. 41S precursor is cut into 2
parts:
1. 20S precursor of 18S
2. 32S precursor of 5.8S and
28S rRNA
3. 3’-end of 20S precursor is
removed, yielding mature 18S
rRNA
4. 32S precursor is cut to liberate
5.8S and 28S rRNA
5. 5.8S and 28S rRNA associate
by base-pairing
Bacterial rRNA Processing
• Bacterial rRNA precursors contain tRNAs and all
3 rRNA
• rRNA are released from their precursors by
RNase III and RNase E
– RNase III is the enzyme that performs at least the
initial cleavages that separate the individual large
rRNAs
– RNase E is another ribonuclease that is responsible
for removing the 5S rRNA from the precursor
Processing Bacterial rRNA
Spacers surrounding individual rRNAs genes are
complementary and can form an extended hairpin; the double
stranded region will serve as a target for RNAase III
Transfer RNA Processing
• Transfer RNAs are made in all cells as long
precursors
– These must be processed by removing RNA at both
ends
• Nuclei of eukaryotes contain precursors of a
single tRNA
• In bacteria, precursor may contain one or more
tRNA, sometimes in combination with rRNAs
Polycistronic Precursors
• In processing bacterial RNA that contain more
than one tRNA
– First step is to cut precursor up into fragments with
just one tRNA each
• Cutting between tRNAs in precursors having two or
more tRNA
• Cutting between tRNAs and rRNAs in precursors
– Enzyme that performs both chores is the RNase III
5’-Ends Maturation
– Extra nucleotides are removed from the 5’-ends
of pre-tRNA in one step by an endonucleolytic
cleavage catalyzed by RNase P
– RNase P from bacteria and eukaryotic nuclei
have a catalytic RNA subunit called M1 RNA
– Spinach chloroplast RNase P appears to lack an
RNA subunit
5’-Ends Maturation
– RNase P makes a cut
at the site that becomes
mature 5’-end of a
tRNA
– This enzyme is all that
is needed to form
mature 5’-ends
3’-Ends Maturation
• Six RNases participate in 3’ ends processing: RNase D,
RNase BN, RNase T, RNase PH, RNase II and
polynucleotide phosphorylase (PNPase)
• RNase II and polynucleotide phosphorylase cooperate
in removing most of 3’-trailer from the end of a tRNA
precursor
– Stop at the +2 stage, with 2 extra nucleotides remaining at
the 3’ end of tRNA
• RNases PH and T remove the last two nucleotides from
RNA
– RNase T is the major participant in removing very last
nucleotide
3’-Ends Maturation
Trans-Splicing
– Splicing that occurs in all eukaryotic species is
called cis-splicing because it involves two or
more exons that exist together in the same gene
– Alternatively, trans-splicing has exons that are
not part of the same gene at all, may not even be
on the same chromosome
Trans-Splicing
– First discovered in trypanosomes
– Trypanosome mRNA has 35 extra nucleotides
that are missing from the gene
– All Trypanosome mRNA have the same 35bp
sequence, spliced leader (SL) on the 5’ end
– The SL is encoded by a separate gene that is
repeated over 200 times in a genome
– The SL gene is composed of a short SL exon
followed by 5’ part of intron
Joining the SL to the Coding Region
of an mRNA
Priming
Trans-splicing
Trans-Splicing Scheme
– Branchpoint adenosine
within the half-intron
attached to the coding
exon attacks the junction
between the leader exon
and its half-intron
– Creates a Y-shaped intronexon intermediate
analogous to the lariat
intermediate
9 Formation of Y-shaped
intermediate confirms the second,
trans-splicing hypothesis
Posttranscriptional RNA Modification –
Editing
RNA editing – is a posttranscriptional changes
of RNA nucleotide sequence involving:
– Insertions
– Deletions
– Substitutions (conversions)
Versus
Capping, splicing, 3’ end formation, trimming of
ends, modification of bases (e.g. methylation) etc.
are RNA processing.
Two Mechanisms of RNA Editing
Substitution Editing:
– chemical alteration of individual nucleotides (the
equivalent of point mutations)
– catalyzed by enzymes that recognize a specific
target sequence of nucleotides:
– cytidine deaminase that converts a C in the RNA to
uracil (U)
– adenosine deaminase that converts an A to inosine
(I), which the ribosome translates as a G (the CAG
codon (for Gln) can be converted to a CGG codon
(for Arg)
Two Mechanisms of RNA Editing
Insertion/Deletion Editing:
– insertion or deletion of nucleotides in RNA
– mediated by guide RNA molecules that serve
as a template for the addition and/or removal of
nucleotides in the target RNA
RNA Editing
– Cryptogenes are incomplete genes
– Trypanosomatid mitochondria, kinetoplasts,
encode incomplete mRNA that must be edited
before being translated
– Editing occurs in the 3’→5’ direction by
successive action of one or more guide RNAs
RNA Editing
¾ Trypanosomatid kinetoplasts contain two types of
circular DNA linked together
– Maxicircles (contain
mitochondrial genes,
encode gRNAs)
– Minicircles
(involved in control
of mitochondrial
gene expression)
RNA Editing
– The U’s added by editing are
shown in grey; the T’s present
in a gene but absent in the
mRNA are shown in blue
– 731-nt fragment of COIII
mRNA contains 407 UMPs
added by editing; 19 UPMs
originally coded by gene were
removed from mRNA.
gRNAs
The gRNAs have three
main regions
–Anchor region – a 5' sequence
that is complementary to the premRNA just 3' of the editing site
–Guiding region – 3’ sequence
that provides information needed
for editing of pre-mRNA; the G is
allowed to pair with U as well as
with C nucleotides
–3' oligo[U] tail – non-encoded
sequence of U nucleotides
9 gRNA would form a
perfect duplex with edited
mRNA except for the oligo[U]
tail.
Mechanism of Editing
• Unedited transcripts can be found along with
edited versions of the same mRNAs
• Editing occurs in the poly(A) tails of mRNAs that
are added posttranscriptionally
– Editing is a posttranscriptional modification
• Partially edited transcripts have been isolated;
they were always edited at their 3’-ends but not
at their 5’-ends
– Editing goes in the 3’→5’ direction
gRNA and Editing
– Guide RNAs (gRNA) could direct the
insertion and deletion of UMPs over a
stretch of nucleotides in the mRNA
– 5’-end of gRNA is complementary to the
region of pre-mRNA that requires no
editing; 3’-end directs the editing of premRNA. Editing starts at 3’-end of premRNA
– When editing is done, the new gRNA may
hybridize near the 5’-end of newly edited
region and continue the editing
– The editing progressively continues in the
direction of the 5’-end of pre-mRNA
RNA Editing
– gRNAs provide A’s and G’s as
templates for the incorporation
of U’s missing from the mRNA
– If gRNA is missing an A or G to
pair with a U in the pre-mRNA,
then the U is removed
¾ Formation of weak G-U pairs enables displacement of 3’
end of an old gRNA by 5’ end of a new gRNA that base
pairs with the newly edited region
Removing and Adding U’s
• Mechanism of removing U’s involves
– Cutting pre-mRNA just beyond U to be removed
– Removal of U by exonuclease
– Ligating the two pieces of pre-mRNA together
• Mechanism of adding U’s uses same first and last
step
– Middle step involves addition of one or more U’s from
UTP by terminal uridylyl transferase (TUTase)
Examples of RNA Editting
Type of RNA editing
Posttransciptional
insertion and deletion
of nucleotides.
Cotranscriptional
insertion of nucleotides
Modification –
substitution of
Base
Mechanism
Genes and Organism
U
Guide RNAs
Mitochondrial genes of Trypanosomes
C
?
Mitochondrial genes of Physarum
polycephalum
A
Polyadenylation Mitochondrial genes of vertebrates
G
Polymerase
stuttering
Phosphoprotein gene of
Paramyxoviruses
C
Deamination
(to U)
Apolipoprotein gene of mammals.
Mitochondrial and chloroplast genes of
plants
U
(to C)
Wilm’s tumor susceptibility gene, WT1
of mammals
A
(to I)
Mammalian glutamate receptor genes,
matrix gene of Measles virus.
nucleotides
Conversions
Insertion/deletions
Examples of RNA Editting
1. Transcipts of
trypanosomes
2. Mitochondria of P.
polycephalum
3. Paramyxoviruses (P
gene)
4. Mitochondria of
vertebrates
5. Apolipoprotein B gene
of mammals
6. Mitochondria of higher
plants
7. Chloroplasts of higher
plants
8. Glutamate-gated ion
channels of mammals
..AAUUUAUGUUGUCUUU
..AUCUCUAAGGGUUUAACCGG
Frameshift
..AUUAAAAAGGGGCACAC…
Frameshift
..CAGUAAAAAAAAAA…
Stop
C
..GAUAUAAUUUGAUCAGUAUA…
C
Stop
C
..UUUUUCAUUGUGGUUUAC…
PHE
TYR
C
..AAUAAUAUGGCGAAACAU…
Start
A
..UUUAUGCGGCAAGGA…
ARG
Reading:
R. Weaver, Molecular Biology, 4th ed.
Chapter 16: pages 481-494.