other rna processing events

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other rna processing events

• Xiejiajia
• 200722040156

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Overview

• 16.1 rRNA Processing
• 16.2 tRNA Processing
• 16.3 Trans-Splicing
• 16.4 RNA Editing
• 16.5 Posttranscriptional Control of Gene
• Expression
• 16.6 Posttranscriptional Gene Silencing
• (RNA Interference)

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Brief introduction

• rRNA Processing

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rRNA Processing

• Ribosomal RNAs are made in eukaryotic nucleoli as
  precursors that must be processed to release the
  mature rRNA.

Figure 16.1a
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rRNA Processing

• The order of RNAs in the precursor is
  18S,5.8S,28S
• snoRNAs play vital roles in these processing
  steps.

Figure 16.2
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Brief introduction

• tRNA Processing

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tRNA Processing
Figure 16.9

• Cutting Apart Polycistronic Precursors the tRNAs
  have to be released from the precursors which
  they embedded in.
• Forming Mature 5-Ends maturation of the 5-end
  tRNA involved a single cut at the point that will
  be the 5-end of the mature tRNA. the enzyme that
  catalyzes this cleavage is RNase P.
• Forming Mature 3-endSix RNases involved .RNase
  D, RNase BN, RNase T, RNase PH. RNase II,
  PNPase(polynucleotide phosphorylase)

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Brief introduction

• Posttranscriptional Control of Gene Expression

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Posttranscriptional Control of Gene Expression

• It makes sense to control gene expression by
  blocking the first step-transcription. That is
  the least wasteful method .
• what if the transcription is finished ?
• special sequences in the 3'-UTR of an mRNA,called
  cytoplasmic polyadenylation elements
  (CPEs),govern the efficiency of polyadenylation
  of material messages during oocyte maturation.In
  this way,these CPEs serve as controllers of gene
  expression.

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Posttranscriptional Control of Gene Expression

• An even more important posttranscriptional
  control of gene expression is control of mRNA
  stability.
• 1. When mammary gland tissue is stimulated by
  prolactin , the synthesis of casein protein
  increase dramatically. However, most of this
  increase in casein is not due to an increase in
  the rate of transcription of the casein gene.
  Instead, it is caused by an increase in the
  half-life of casein mRNA.
• 2. When the iron concentration is high,the
  transferrin receptor---TfR mRNA decays
  rapidly.When the iron concentration is low,the
  TfR mRNA decays much more slowly.This difference
  is about 20-fold.This loss of TfR is largely due
  to decreased stability of the TfR mRNA.This
  response to iron depends on the 3'-UTR of the
  mRNA,Which contains five stem loops called iron
  response elements (IREs).If removing all of the
  IREs,or either one of two non-IRE stem loops
  renders the TfR mRNA constitutively stable.

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Brief introduction

• Posttranscriptional Gene Silencing (RNA
  Interference)

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Posttranscriptional Gene Silencing (RNA
Interference)
back

• dsRNA actually blocked gene expression.When
  placing transgenes into various organisms
  sometimes will cause undesired effect . Instead
  of turning on the transgene, organisms turn off,
  not only the transgene , but the normal cellular
  copy of the gene as well.
• The RNase could attack the target RNA
  specifically by employing an ATP-dependent RNA
  helicase to unwind the dsRNA. Then it could
  remain bound to the antisense strand, which could
  hybridize to mRNA, bringing the RNase to its
  target.

Figure 16.39
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Trans-Splicing
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Trans-Splicing findings

• First discovered in trypanosome , a protozoa,
  which causes African sleeping sickness.
• 1982, Piet Borst and his colleagues sequenced the
  5-ends of both the mRNA and the gene that
  encoded coat protein ,but they did not match.
• The mRNA had 35 extra nucleotides
• And they discovered many trypanosome mRNAs had
  the same 35-nt leader , called splice leader(SL)

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Trans-Splicing findings

• How can we explain this?

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Trans-Splicing hypotheses

• Two hypotheses for joining the SL to the coding
  region of an mRNA.
• (a) cis-splicing.SL serves as a primer .then ,the
  transcript contains 4 parts ,2 harf-introns ,SL
  and the coding region. At last ,it can be
  processed by cis-splicing.
• (b)Trans-splicing. They transcribed respectively.
  Then these two separate RNAs undergo
  trans-splicing to produce the mature mRNA.

Figure 16.11
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Trans-Splicing hypotheses

• The key is to find the intermediate
• Lariat-shaped ? cis-splicing
• Y-shaped ? trans-splicing

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Trans-Splicing evidences

• To identify the shape of the intermediate ,we use
  debranching enzyme to treat it.

Figure 16.13
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Trans-Splicing evidences
back

• Agabian and colleagues labeled trypanosome RNA
  with 32p and treated total RNA,or
  poly(A)RNA,with debranching enzyme (DBrEz).
• Then they electrophoresed the products,blotted
  them,and probed the blot with an oligonucleotide
  specific for the l00-nt SL half-intron which is
  clearly detectable in both enzyme-treated RNA
  samples.

Figure 16.14
100-nt SL half-intron
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RNA Editing
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RNA Editingfindings
Figure 16.17

• In 1986,Rob Benne and his colleagues discovered
  that several Us were added in RNA compared with
  DNA(not T)

Us were added
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RNA Editingdirection
Wild type
3-edited cDNA
Lack mitochondrial DNA
Figure 16.19

• 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, the 3-edited
  transcript should be detected, but not the
  5-edited transcripts.

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RNA EditinggRNA-model

• gRNA-1 hybridizes through its 5-end to unedited
  region of pre-mRNA.the 3-end also hybridizes to
  the farther upstream.
• The red portion is edited
• Another gRNA displaces 1 by hybridizing to the
  5-end of the newly edited region.
• It continues to editing .and this course will be
  repeated until the editing is over.

Figure 16.20
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RNA EditinggRNA-evidence

• Simpson and colleagues electrophoresed
  kinetoplastid RNA ,Northern blotted it , and
  hybridized it to labeled oligonucleotide probes
  designed to detect gRNAs.
• Result gRNAs were showed in the red frame.

Figure 16.22
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RNA EditinggRNA-mechanism

• 3 steps
• 1.Make a nick with nuclease
• 2.Delete or insert with 3-exonulease
  or TUTase (terminal uridylyl transferase)
• 3.ligase repairs the nick

Figure 16.23
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Than u!