PHAR2811 Dale’s lecture 7The Transcriptome

Synopsis: If protein-coding portions of the human genome make up only 1.5% what is the

rest doing?

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.

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.

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%)

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.

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

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%.

How can the disparity between the number of

sequences transcribed and translated be explained?

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.

Non-coding RNA

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

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)

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 and ’ subunits of bacterial RNA polymerase (fundamental catalytic site between the 2 faces conserved throughout life)

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

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)

RNA polymerase II

DNATATA

Start site

TBP

TAFs

TFIID

TFIIATFIIB

RNA polymerase IITFIIF

TFIIE

TFIIH

This is the basal transcription apparatus!!

RNA polymerase II

DNATATA

Start site

TBP

TAFs

TFIID

TFIIATFIIB

RNA polymerase IITFIIF

TFIIE

TFIIH

TFIIH is the only transcription factor with enzymic activity.

2 subunits of TFIIH unwind the DNA

C-terminal Domain CTD of RNA pol II

The CTD is phosphorylated by protein kinases; one is a subunit of TFIIH

RNA polymerase IITFIIF

TATA

RNA

RNA polymerase II: elongation

TBPDNA

TAFs

TFIID

Gene Expression

RNA pol II

TAFs

TBPnucleosomes

enhancer

Transcriptional activator

Mediator

Acts on the basal machinery

Histone modification complex

Chromatin remodelling complex

Translational coactivators and corepressors

Other RNA polymerases

• The regulation of eukaryotic gene expression is the subject of later lectures

• Let’s consider the other polymerases

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.

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.

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).

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.

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.

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.

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.

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???

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.

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)

Splicing

AGGUAAGU YNYRAY YYYNCAGG

Branch siteExon 1 Exon 2

Y pyrimidine

R purine

N any nuc

5’

5’ AG-OH

AGpG

Lariat formed when 5’ p of the intron G attaches to 2’ OH of A

5’

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.

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.

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.

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).

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.

Alternative splicing

introns

exons

Alternative splicing

introns

exons

Spliceosome, made up of 5 snRNPs and ~150 proteins

Alternative splicing

introns

exons

Spliceosome, made up of 5 snRNPs and ~150 proteins

OR

introns

exons

OR

introns

exons

snoRNA

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

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!!!

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.

Pre-miRNA in the nucleus

exon

intron

5’ 3’

5’ 3’

65 – 75 nt stem loop structure ready for export to cytoplasm

Synthesised by RNA pol II

Drosha

Pre-miRNA in the cytoplasm

21 – 26 ds RNA

5’3’

3’5’

dicer

3’ 5’RISC

Translational inhibition of partially complementary mRNA

Degradation of complementary mRNA

siRNA

dicer

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.

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.

siRNA

• Researchers can also introduce their own double stranded RNA.

• The double stranded molecules are processed by Dicer, the cytoplasmic RNase III endonuclease system.

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.

How does miRNA and siRNA regulate gene expression?

• Translation repression of target sequences

• mRNA destruction of target sequences

• Silencing chromatin

Translational Repression

AAAAAAAAAAAAAAAA

3’UTR

5’UTR

RNARecruited proteins

Protein that binds to 5’UTR

mRNA destruction: sequence specific targetting siRNA and

miRNA

AAAAAAAAAAAAAAAA

3’UTR

5’UTR

RNA targets sequence for destruction

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)

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

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

AMO to miR-122miR-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