1. PHAR2811 Dale’s lecture 7 The Transcriptome
Synopsis: If protein-coding portions of the human genome make up only 1.5% what is the rest doing?

2. 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.

3. 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.

4. 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%)

5. 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.

6. Proteome:
The cell’s 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

7. 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%.

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

9. 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.

10. Non-coding RNA Ribosomal RNA (rRNA) Transfer RNA (tRNA)
Small nuclear RNA (snRNA)
Small nucleolar RNA (snoRNA)
MicroRNA (miRNA)
Short interfering RNA (siRNA)

11. 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)

12. RNA polymerases
All three polymerases have >10 subunits; 500 – 700 kD BIG!!!
Some of the subunits are unique to each polymerase
All have 2 large subunits (>140 kD) similar in sequence to the b and b’ subunits of bacterial RNA polymerase (fundamental catalytic site between the 2 faces conserved throughout life)

13. Let’s 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 3’UTRs but NOT the promoter region
This process is really complicated

14. 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)

15. RNA polymerase II This is the basal transcription apparatus!! TFIID
TAFs
TFIIE
RNA polymerase II
TFIIF
DNA
TATA
Start site
TFIIA
TFIIB
TFIIH
TBP

16. RNA polymerase II TFIID TAFs
TFIIH is the only transcription factor with enzymic activity.
DNA
TATA
Start site
TBP
TAFs
TFIID
TFIIA
TFIIB
RNA polymerase II
TFIIF
TFIIE
TFIIH
The CTD is phosphorylated by protein kinases; one is a subunit of TFIIH
2 subunits of TFIIH unwind the DNA
C-terminal Domain CTD of RNA pol II

17. RNA polymerase II: elongation
TBP
DNA
TAFs
TFIID
RNA polymerase II
TFIIF
TATA
RNA

18. Gene Expression Mediator TAFs enhancer Transcriptional activator
RNA pol II
TAFs
TBP
nucleosomes
Transcriptional activator
Translational coactivators and corepressors
Chromatin remodelling complex
Mediator
Histone modification complex
Acts on the basal machinery

19. Other RNA polymerases
The regulation of eukaryotic gene expression is the subject of later lectures
Let’s consider the other polymerases

20. 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.

21. 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.

22. 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).

23. 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.

24. 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.

25. 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.

26. 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.

27. 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???

28. 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.

29. 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)

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

31. 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.

32. 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.

33. 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.

34. 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).

35. 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.

36. Alternative splicing
introns
exons

37. Alternative splicing introns
Spliceosome, made up of 5 snRNPs and ~150 proteins
exons

38. Alternative splicing introns
Spliceosome, made up of 5 snRNPs and ~150 proteins
exons

39. OR
introns
exons

40. OR
introns
exons

41. snoRNA
snoRNAs are derived from the introns of pre-mRNA transcripts, suggesting that introns are not “junk” DNA.

42. 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!!!

43. 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.

44. 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’

45. Pre-miRNA in the cytoplasm
dicer 3’ 5’
RISC
21 – 26 ds RNA 5’ 3’
dicer
siRNA
Translational inhibition of partially complementary mRNA
Degradation of complementary mRNA

46. miRNA
It cuts off the hairpin loop and the 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.

47. 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 cell’s antiviral defense.

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

49. 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.

50. How does miRNA and siRNA regulate gene expression?
Translation repression of target sequences
mRNA destruction of target sequences
Silencing chromatin

51. Translational Repression
5’UTR
AAAAAAAAAAAAAAAA
3’UTR
Protein that binds to 5’UTR
RNA
Recruited proteins

52. mRNA destruction: sequence specific targetting siRNA and miRNA
5’UTR
RNA targets sequence for destruction
AAAAAAAAAAAAAAAA
3’UTR

53. 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)

54. 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

55. 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

56. AMO to miR-122
miR-122
Inhibits translation of target mRNAs: involved in cholesterol regulation in liver
Introduce the AMO, a stabilised complementary oligonucleotide to miR-122, given intraperitoneally X2 weekly
Inactivation of miR-122
miR-122 target mRNAs increase  lower plasma cholesterol