Other RNA Processing Events

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Chapter 16

• Other RNA Processing
  Events
• Presented by Tang Zijian

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• In the previous two chapters, we examined
  splicing, capping, and polyadenylation.
• However ,in a few organisms, other specialized
  pre-mRNA processing events occur.

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• 16.1 Ribosomal RNA Processing
• 16.2 Transfer RNA Processing
• 16.3 Trans-Splicing
• 16.4 RNA Editing
• 16.5 Posttranscription Control of Gene
• Expression

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16.1 Ribosomal RNA Processing

• The rRNA genes of both eukaryotes and prokaryotes
  are transcribed as larger precursors that must be
  processed to yield rRNA of mature size.
• However, processing means not only removing
  unwanted material at either end of an overly long
  molecule, but also cutting out several different
  rRNA molecule which imbedded in a long precursor.

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Eukaryotic rRNA Processing

• Ribosomal RNAs are made in eukaryotic nucleoli as
  precursors that must be processed to release the
  mature rRNA.
• The order of RNAs in the precursor is
  18S,5.8S,28S in all eukaryotes,although the exact
  sizes of the mature rRNAs vary from one spices to
  another.

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• In human cells, the precursor is 45S, and the
  processing scheme create 41S,32S and 20S
  intermediates.

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Intermediates are too short-lived to be detected
labeled RNA in virus-infected cells with
32pphosphate and with 3Hmethionine. The
mobilities of the RNA were compared with those of
markers of known sedimentation coeffecients.
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Prokaryotic rRNA Processing

• Prokaryotic rRNA precursors contain tRNAs as well
  as three rRNAs.
• The rRNAs are released from their precursors by
  RNase III and RNase E

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a. Structure of the rrnD operon ,which contains
both tRNA-coding regions (red) and rRNA-coding
regions (orange), transcribed spacers
(yellow). b. Spacers in 23S RNA can form a
hairpin. RNases III worked in stem.
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16.2 Transfer RNA Processing

• Transfer RNAs are made in all cells as overly
  long precursors that must be processed by
  removing RNA at both ends.
• In eukaryotes, these precursors contain a single
  tRNA.
• In prokaryotes, a precursor may contain one or
  more tRNAs, and sometimes a mixture of rRNAs and
  tRNAs.
• However,tRNA processing in eukaryotes and
  prokaryotes are similar, so we consider them
  together.

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First step Cutting Apart Polycistronic Precursors

• The first step in tRNA processing is to cut
  between tRNAs in precursors that have two or more
  tRNAs, or cut between tRNAs and rRNAs.
• The enzyme that performs both these chores seems
  to be RNase III

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Second step Forming Mature 5-Ends

• Extra nucleotides are removed from the 5-ends
  of pre-tRNAs in one step by an endonucleolytic
  cleavage catalyzed by RNase P.

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RNase P

• RNase P is a fascinating enzyme.
• It contains two subunits, one is RNA (the M1
  RNA), which has a molecular mass of about 125KD,
  another protein subunit is only 14KD.

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The M1 RNA of RNase P has enzymatic activity
Magnesium concentration effects the enzymatic
activity of M1 RNA. M1 RNA has no influence on
p4.5. RNase P can cleave both substrates.
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Last step Forming Mature 3end
Cleavage of the precursor with RNase T1,which
cuts only after Gs ,will yield a unique
oligonucleotide 32nt long. After correct
processing of the 3-end,the CCA is the new
3-terminus, and RNase T1 digestion will yield a
new 19nt fragment instead of the 32nt fragment.
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Assay of maturation of tRNA
a. wild-type ,lane1 and 6 are markers,lane2-5
shows formed mature 3-end. b. Mutant only has
RNase PH , lane 3 shows mature 3-end appeared,
lane 4 shows phosphate is a key ingredient.
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16.3 Trans-Splicing

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• Trans-Splicing unlike cis-splicing ,in
  trans-splicing, the exons are not part of the
  same gene at all and many not even be found on
  the same chromosome

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Discovery of Trans-Splicing

• In 1982, Piet Borst sequenced the 5-end of an
  mRNA encoding a trypanosome surface coat protein
  and 5-end of the gene encodes this protein and
  found they did not match.
• The mRNA had 35 extra nucleotides (SL) that
  were missing from the gene.

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Splicing leader(SL)

• The trypanosome mRNA all have the same 35nt
  leader,called the splicing leader (SL) .
• But none of genes encoded the SL.
• Instead, the SL is encoded by a gene that is
  repeated about 200 times in the trypanosome
  genome.
• This gene encodes only the SL,plus a 100nt
  sequence that is jointed to the leader through a
  consensus 5-splice sequence.

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• How to explain the production of an mRNA
  derived from two widely separated DNA regions
  that are sometimes even found on separate
  chromosomes?
• two classes of explanations are provided.

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a.SL (blue), with its attached half-intron (red),
is transcribed to yield a 135-nt RNA. This RNA
then serves as a primer for transcription of a
coding region (yellow), including its attached
half-intron (black). Then the whole intron can
be spliced out to yield mature mRNA. b.The SL
with its attached half-intron and the coding
region with its half-intron are transcribed
independently, then these two separate RNAs
undergo tans-splicing to produce the mature mRNA.
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Detailed trans-splicing scheme for a trypanosome
mRNA
Mature mRNA and the Y-shaped intron were produced
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According to the previous hypothesis, unlike
lariat in cis-splicing a Y-shaped structure will
be produced. So, when treated with
debranching enzyme, a 100-nt fragment will appear
to corroborate the trans-splicing hypothesis.
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Polycistronic Arrangement of Coding Region in
Trypanosomes

• Trypanosome coding regions, including genes
  encoding rRNAs and tRNAs, are arranged in long
  ,polycistronic transciption units governed by a
  single promoter.

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• How to prove the whole region is transcribed
  as a unit?

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• Using increasing doses of UV radiation to
  introduce pyrimidine dimers.
• Allow transcription going on
• If the hypotheses is true , transcription of the
  3end of a long transcription unit will be much
  sensitive to UV irradiation than transcription of
  the 5end.

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(No Transcript)
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16.4 RNA Editing

• Trypanosomatid mitochondria encode incomplete
  mRNAs that must be edited before they can be
  translated.

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Discovery of RNA editing

• In 1986,Rob Benne and his colleagues
  discovered that the sequence of the cytochrome
  oxidase(COII) mRNA from trypanosomes does not
  match the sequence of the COII genethe mRNA
  contains four nucleotides that are missing from
  the genes.

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• By 1988,a number of trypanosomatid kinetoplast
  genes and corresponding mRNAs had been sequenced
• A 731nt stretch of the COIII mRNA of Trypanosoma
  brucei contains 407 UMPs added by editing
  ,whereas 19 UMPs are deleted.

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Mechanism of Editing

• Editing is a posttranscriptional event because
  unedited transcripts can be found along with
  edited versions of the same mRNA .
• Moreover, editing occurs in the poly A tails of
  mRNAs, which are added posttranscriptionally.

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Editing proceeds in a 3-5 direction.
Use an unedited 5-primer and an edited
3-primer to detect 3-edited transcripts ,or an
edited 5-primer and an unedited 3-primer to
detect 5-edited transcripts. If editing goes
from 3 to 5 in the transcript, then 3-edited
transcript, but not 5-edited transcripts, should
be detected.
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Guide RNAs (gRNAs)

• Found in 1990 by Larry Simpson
• can hybridize to the unedited region of the
  mRNA and provide As and Gs as templates for the
  incorporation of Us missing from the mRNA .

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Model for the role of gRNAs in editing
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Evidence for gRNAs
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16.5 posttranscriptional control of gene
expression

• It makes sense to control gene expression by
  blocking the first step-transcription. That is
  the least wasteful method because the cell
  expends no energy making an mRNA for a protein
  that is not needed.
• An even more important posttranscriptional
  control of gene expression is control of mRNA
  stability.
• Joe Harford has point out that cellular mRNA
  levels often correlate more closely with
  transcript stability than with transcription
  rate.

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• A common form of posttranscriptional control
  of gene expression is control of mRNA stability.

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Casein mRNA Stability
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Transferrin Receptor mRNA Stability

• One of the best studied examples of
  posttranscriptional control concerns iron
  homeostasis (control of iron concentration) in
  mammalian cells.
• Consequently, cells have to regulate the
  intracellular iron concentration carefully.
• Mammalian cells do this by regulating the amounts
  of two proteinsan iron import protein called the
  transferrin receptor, and an iron storage protein
  called ferritin.

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transferrin

• Transferrin is an ironbearing protein that can
  get into a cell via the transferrin receotor on
  the cell surface. Once the cell imports
  transferrin, it passes the iron to celluar
  proteins, such as cytochromes, that need iron.
  Alternatively, if the cell receives too much
  iron, it stores the iron in the form of ferritin.
  
• It employs posttranscriptional strategies to do
  both these things It regulates the rate of
  translation of ferritin mRNA, and it regulates
  the stability of the transferrin receptor mRNA.

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Identifying iron response elements
TfR Promoter was not responsible for iron
responsiveness. The part of the 3-UTR deleted in
this experiment apparently included the iron
response element.
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IRE in the TfR mRNA
These five loops are mediators of the
responsiveness of TfR expression to iron.
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Evidents of proteins in human cell which bind
specifically to the human TfR IREs
Labeled RNA mixed with a cytoplasmic
extract. Lane1,no competitor.lane2, TfR mRNA
competitor. lane 3, ferritin mRNA competitor.
lane 4, globin mRNA competitor. Arrow points to a
specific protein-RNA complex. Results suggest the
similarity between the ferritin and TfR IREs,
which they may bind to the same proteins.
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The Rapid Turnover Determinant

• Knowing that iron regulates the TfR gene by
  controling mRNA stability, and knowing that a
  protein binds to one or more IREs in the 3-UTR
  of TfR mRNA , we assume that the IRE-binding
  protein protects the mRNA from degradation.
• This kind of regulation demands that the TfR mRNA
  be inherently unstable
• The instability is caused by a rapid turnover
  determination that also lies in the 3-UTR

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Structure of human and chicken IRE regions in the
3-UTR of the TfR mRNA
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the IRE A,IRE E, and central loop could be
deleted without altering iron regulation.
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TRS-1, removing IRE A and E, and the large
central loop TRS-3,removing the remaining three
IREs TRS-4,deleting a single C at the 5-end of
each IRE loop Removing all of the IREs, or either
one of two non-IRE stem loops renders the TfR
mRNA constitutively stable. Thus, each of the
non-IRE stem loops ,and at least one of IREs B-D,
are all parts of the rapid turnover
determinant. Removing a C from IREs B-D renders
the TfR mRNA constitutively unstable and unable
to bind the DRE-binding protein.
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TfR mRNA Stability

• So far we have seen that the level of TfR mRNA
  responds to intracelluar iron concentration and
  that this responsiveness is determined by the
  3-UTR, rather than by the promoter.
• This strongly suggested that iron regulated the
  TfR mRNA half-life,rather than the rate of mRNA
  synthesis
• Ernst found that the TfR mRNA was very stable
  when the iron concentration was low. On the other
  hand, at high iron concentration the TfR mRNA
  decayed much faster.

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Effect of iron on stability of wild-type and
mutant TfR mRNA
The TRS-3 mRNA ,with no rapid turnover
determinant, is constitutively stable (stable in
both high and low iron concentrations). The
TRS-4 mRNA ,with no ability to bind the
IRE-binding proteinm is constitutively unstable
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• Another form of posttranscriptional control of
  gene expression is posttranscriptional gene
  silencing (RNA Interference).

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Posttranscriptional Gene Silencing(RNA
Interference)

• This phenomenon is called by several names
  cosuppression and posttranscriptional gene
  silencing (PTGS) in plants, RNA interference
  (RNAi) in animals .

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Mechanism of RNAi

• A nuclease digests the dsRNA that initiates
  RNAi into fragments about 25nt long, and these
  fragments then associate with the nuclease and
  provide guide sequences that allow the nuclease
  to target the corresponding mRNA.

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Generation of 21-23-nt RNA fragments
Label one strand of the dsRNA at a time (or
both). The appearance of the siRNAs did not
require the presence of mRNA, so these short RNA
apparently derived from dsRNA, not mRNA. When
capped antisense RNA was labeled, a small mount
of siRNA appeared.
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Dicer and Argonaute

• Dicer a member of the RNase III family, dices
  double-stranded RNA up into uniform-sized pieces.
  Dicer also has RNA helicase activity, so it can
  separate the two strands of the siRNA it creates.
• Argonaute, presumably associated in the RISC with
  an RNase that uses the small RNAs created by
  Dicer as a template to find and degrade the
  corresponding mRNA.

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A model for RNAi
First, dsRNA is cleaved into siRNA fragments
21-23nt long. Then ,two strands separate as well
as by the help of Dicers. Last ,single strand
targets to the mRNA by the help of Argonaute.
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Function of RNAi

• One important function of RNAi may be to
  inhibit the replication of viruses by degrading
  their mRNA.
• Some genes required for RNAi are also required
  to prevent certain transposons from transposing
  within the genome. Thus RNAi may have utility
  even in cells that are not infected by a virus

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• How to explain RNAis great sensititvity?
• How to explain the principle that a few molecules
  of dsRNA can set in motion a process that totally
  silences a gene, not only in one cell ,but in a
  whole organism-and even the descendants of that
  organism?

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Amplification of siRNA
The cells employ an enzymeRNA-directed RNA
polymerase (RdRP) that uses antisense siRNAs as
primers to make many copies of siRNA.
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