PHAR2811 Dale

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PHAR2811 Dales lecture 7The Transcriptome

• Synopsis If protein-coding portions of the human
  genome make up only 1.5 what is the rest doing?

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Definitions

• Genome the total amount of genetic material,
  stored as DNA.
• The nuclear genome refers to the DNA in the
  chromosomes contained in the nucleus in the case
  of humans the DNA in the 46 chromosomes. It is
  the nuclear genome that defines a multicellular
  organism it will be the same for all (almost)
  cells of the organism.

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Genome

• You can have organelle genomes such as the
  mitochondrial genome.
• When you want to identify or distinguish one
  organism from another, such as in forensic
  testing, you investigate the genome.

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Transcriptome

• The total amount of genetic information which has
  been transcribed by the cell. This information
  will be stored as RNA.
• This represents some 90 of the total genomic
  sequences
• There is 5X more RNA than DNA in a cell, most of
  it rRNA (80) and tRNA (15)

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Transcriptome

• The transcriptome is unique to a cell type and is
  a measure of the gene expression.
• Different cells within an organism will have
  different transcriptomes. Cell types can be
  identified by their transcriptome.

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Proteome

• The cells complete protein output. This reflects
  all the mRNA sequences translated by the cell.
• Cell types have different proteomes and these can
  be used to identify a particular cell.
• Only 1 2 of the genome codes for the proteome

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Non-coding RNA

• Only 1-2 of the genome codes for proteins
• BUT a large amount of it is transcribed some
  estimates have it as high as 98.

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• How can the disparity between the number of
  sequences transcribed and translated be explained?

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Non-coding RNA

• The difference is the RNA which is an end in
  itself.
• This non-coding RNA (ncRNA) consists of
• the introns of protein coding genes,
• non coding genes (what are these??)
• Sequences antisense to or overlapping protein
  coding genes.

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Non-coding RNA

• Ribosomal RNA (rRNA)
• Transfer RNA (tRNA)
• Small nuclear RNA (snRNA)
• Small nucleolar RNA (snoRNA)
• MicroRNA (miRNA)
• Short interfering RNA (siRNA)

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RNA polymerases

• There are 3 RNA polymerases in eukaryotes RNA
  pol I, II III
• RNA pol I transcribes rRNA, localised to
  nucleolus (insensitive to alpha amanitin)
• RNA pol II transcribes mRNA (very sensitive to
  alpha amanitin)
• RNA pol III transcribes tRNA and other small RNAs
  (less sensitive to alpha amanitin)

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RNA polymerases

• All three polymerases have gt10 subunits 500
  700 kD BIG!!!
• Some of the subunits are unique to each
  polymerase
• All have 2 large subunits (gt140 kD) similar in
  sequence to the b and b subunits of bacterial
  RNA polymerase (fundamental catalytic site
  between the 2 faces conserved throughout life)

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Lets start with the most complex!

• RNA polymerase II which transcribes mRNA.
• The primary transcript is a direct copy of the
  gene.
• It includes the introns, 5 and 3UTRs but NOT
  the promoter region
• This process is really complicated

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RNA polymerase II abbreviations

• TATA box
• TBP TATA binding protein
• TAFs TBP associated factors
• TFII transcription factor (RNA pol II) there
  are A, B. D, E, F and H
• CTD C terminal Domain (of RNA pol II)

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RNA polymerase II
This is the basal transcription apparatus!!
TFIID
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RNA polymerase II
TFIIH is the only transcription factor with
enzymic activity.
2 subunits of TFIIH unwind the DNA
C-terminal Domain CTD of RNA pol II
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RNA polymerase II elongation
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Gene Expression
Acts on the basal machinery
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Other RNA polymerases

• The regulation of eukaryotic gene expression is
  the subject of later lectures
• Lets consider the other polymerases

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Infrastructural RNA

• Ribosomal RNA in eukaryotes is actually 4
  separate RNA species 28S RNA, 18S RNA, 5.8S RNA
  and 5S RNA.
• The 28S, 18S and 5.8S rRNA are transcribed as a
  long precursor pre-rRNA of 45S.
• The bacterial rRNAs (23S, 16S and 5S) are also
  transcribed as one long molecule.

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Processing pre-r RNA

• The 5.8S 28S fragment is cleaved from the 18S
  then the 5.8S species is released, although it
  remains hydrogen bonded to the 28S rRNA.

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Processing pre-r RNA

• Initially the 45S pre-rRNA is modified by 2
  O-ribose methylation at many sites (humans have
  106 sites) and the uracils are converted to
  pseudouracils.
• This process is guided by snoRNAs (we will meet
  them later).

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Ribosomal RNA

• The rRNA is then modified by methylation at some
  sites.
• There are many copies of the ribosomal RNA
  sequences in the genome (as well as the histone
  proteins).
• Some sequences are required by all cells in such
  large quantities that they have multiple copies
  in the genome.

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Infrastructural RNA

• Transfer RNA is also transcribed as a long
  precursor containing several tRNAs joined
  together.
• Promoter lies within the coding region
• RNase P releases the separate tRNAs by cleavage
  at the 5 end of the tRNAs.

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RNase P

• RNase P is an interesting enzyme because it
  contains both RNA and protein and it is the RNA
  component that is capable of the RNase activity.
• It was this enzyme that led scientists to the
  discovery of ribozymes the RNA species capable
  of catalytic activity.

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Infrastructural RNA

• The 3 end of the tRNAs all have a CCA, some of
  which are attached after cleavage (some have the
  sequence encoded in the DNA). The attachment is
  done by a special enzyme.
• The CCA is important as this is where the amino
  acid is attached.
• Several of the bases e.g. pseudouracils in tRNA
  molecules are modified at this stage.

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Other non-coding RNAs.

• Small nuclear RNAs (snRNAs) form part of the
  spliceosome which cleaves the introns out of mRNA
  precursors.
• There are 5 snRNAs U1, U2, U4, U5 and you
  guessed it U6. I have no idea what happened to
  U3???

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Other non-coding RNAs.

• These RNA species are between 50 and 200
  nucleotides long and complex with proteins to
  form snRNPs (small nuclear ribonucleoprotein
  particles..snurps).
• These small RNAs contribute to the recognition of
  splice sites in the mRNA and in catalysing the
  breaking and joining of the mRNA.

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Splicing

• Process where the introns are removed from the
  pre-mRNA
• Occurs in the nucleus
• Capping (meG at 5 head) and polyA tailing at 3
  end carried out first
• Splice sites are defined by a sequence
• Formation of a lariat by the spliceosome (U1,
  U2, U4, U5 U6 and 10 proteins)

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Splicing
Branch site
Exon 1
Exon 2
AGGUAAGU
YNYRAY
YYYNCAGG
5
Lariat formed when 5 p of the intron G attaches
to 2 OH of A
Y pyrimidine R purine N any nuc
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snoRNA

• snoRNA are small nucleolar RNAs between 60 and
  300 nucleotides in length.
• RNA editing function
• They recognise their target sequence by base
  pairing and then recruit specialised proteins to
  perform nucleotide modifications to these RNAs
• 2 O-ribose methylation,
• base deaminations such as adenine to inosine
  conversions
• addition of pseudouridines.

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snoRNA

• These modifications are crucial to ribosome
  biogenesis.
• snoRNAs are derived from introns.
• sno RNAs in conjunction with snRNAs have been
  suggested as regulators for alternative splice
  sites.

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Alternative splicing

• A typical eukaryotic gene consists of introns and
  exons.
• The introns are removed by the spliceosome.
• The exons are joined in the same order as they
  appear in the gene sequence.
• In about 60 of human genes certain exons are
  missed.

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Typical Human Genome

• Human genes typically contain around 10 exons
  (each of on average about 300bp in length, with
  the final exon often being considerably longer)
  spanning 9 introns (which may vary from a few
  hundred bps to many kilobases or 100s of
  kilobases in length).

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Alternative splicing

• This leads to alternative splicing.
• There are some genes with many different
  potential exons and these genes have the
  potential to form multiple different mature mRNAs
  and proteins.

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Alternative splicing
introns
exons
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Alternative splicing
introns
Spliceosome, made up of 5 snRNPs and 150 proteins
exons
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Alternative splicing
introns
Spliceosome, made up of 5 snRNPs and 150 proteins
exons
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OR
introns
exons
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OR
introns
exons
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snoRNA

• snoRNAs are derived from the introns of pre-mRNA
  transcripts, suggesting that introns are not
  junk DNA.

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miRNA and siRNA

• microRNA (miRNA) and short interfering RNA
  (siRNA) are very small RNA molecules, ranging
  between 21 to 25 nucleotides long.
• These are the hot molecules! They are seen as the
  next anti-viral agents, cures for cancer etc even
  a replacement for fossil fuels!!!

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miRNA and siRNA

• The 2 species are quite similar, the variations
  come from their source or origin.
• MicroRNA comes from short endogenous hairpin loop
  structures, synthesised by RNA pol II, often from
  within introns.
• The hairpin structures are cleaved in the
  nucleus, exported to the cytoplasm and further
  processed to 22 nt duplexes.

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Pre-miRNA in the nucleus
Synthesised by RNA pol II
exon
intron
3
5
Drosha
65 75 nt stem loop structure ready for export
to cytoplasm
3
5
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Pre-miRNA in the cytoplasm
dicer
dicer
Translational inhibition of partially
complementary mRNA
Degradation of complementary mRNA
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miRNA

• It cuts off the hairpin loop and the 65 75 nt
  pre-miRNAs are exported to the cytoplasm by
  exportin 5
• It is further processed by another RNase III
  endonuclease system, Dicer.
• The mature miRNA s are 22 nt duplexes and act
  usually to repress translation of target mRNA
  sequences.

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siRNA

• siRNAs are similar but are produced from long
  double stranded RNA molecules or giant hairpin
  molecules, often of exogenous origin.
• This whole process is thought to be part of the
  cells antiviral defense.

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siRNA

• Researchers can also introduce their own double
  stranded RNA.
• The double stranded molecules are processed by
  Dicer, the cytoplasmic RNase III endonuclease
  system.

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siRNA

• The processed interfering RNA (RNAi) can catalyse
  the destruction of endogenous mRNAs of the same
  sequence and this process has been used very
  successfully by scientists to silence genes or
  knock them down.

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How does miRNA and siRNA regulate gene expression?

• Translation repression of target sequences
• mRNA destruction of target sequences
• Silencing chromatin

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Translational Repression
5UTR
AAAAAAAAAAAAAAAA
3UTR
Protein that binds to 5UTR
RNA
Recruited proteins
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mRNA destruction sequence specific targetting
siRNA and miRNA
5UTR
RNA targets sequence for destruction
AAAAAAAAAAAAAAAA
3UTR
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Pharmaceutical Applications

• Use of modified anti-miRNA oligonucleotides
  (AMOs)
• Complementary to miRNA
• Inhibit a particular miRNA activity
• Example is inhibition of miR-122
• Cholesterol conjugated AMO injected
  intraperitoneally (X2 weekly)

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Pharmaceutical Applications

• miR-122 is a liver specific miRNA
• Its target gene mRNAs are sequences involved in
  cholesterol regulation
• Increasing the level of the target mRNAs lowers
  cholesterol

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Pharmaceutical Applications

• The AMO lowered the miR-122 which increased the
  target mRNA levels
• This resulted in significantly reduced plasma
  cholesterol levels after 4 weeks

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AMO to miR-122
miR-122
Inhibits translation of target mRNAs involved in
cholesterol regulation in liver
miR-122 target mRNAs increase ? lower plasma
cholesterol