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?

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

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

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

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

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 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

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 Spliceosome, made up of 5 snRNPs and ~150 proteins exons

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

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 Synthesised by RNA pol II exon intron 3’ 5’ Drosha 65 – 75 nt stem loop structure ready for export to cytoplasm 3’ 5’

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

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 5’UTR AAAAAAAAAAAAAAAA 3’UTR Protein that binds to 5’UTR RNA Recruited proteins

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

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-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