Presentation on theme: "PHAR2811 Dale’s lecture 7 The Transcriptome"— Presentation transcript:
1PHAR2811 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?
2Definitions: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.
3Genome: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.
4Transcriptome: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 sequencesThere is ~5X more RNA than DNA in a cell, most of it rRNA (~80%) and tRNA (~15%)
5Transcriptome: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.
6Proteome: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
7Non-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%.
8How can the disparity between the number of sequences transcribed and translated be explained?
9Non-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.
10Non-coding RNA Ribosomal RNA (rRNA) Transfer RNA (tRNA) Small nuclear RNA (snRNA)Small nucleolar RNA (snoRNA)MicroRNA (miRNA)Short interfering RNA (siRNA)
11RNA polymerasesThere are 3 RNA polymerases in eukaryotes: RNA pol I, II & IIIRNA 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)
12RNA polymerasesAll three polymerases have >10 subunits; 500 – 700 kD BIG!!!Some of the subunits are unique to each polymeraseAll 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)
13Let’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 regionThis process is really complicated
14RNA polymerase II abbreviations TATA boxTBP: TATA binding proteinTAFs: TBP associated factorsTFII: transcription factor (RNA pol II); there are A, B. D, E, F and HCTD: C terminal Domain (of RNA pol II)
15RNA polymerase II This is the basal transcription apparatus!! TFIID TAFsTFIIERNA polymerase IITFIIFDNATATAStart siteTFIIATFIIBTFIIHTBP
16RNA polymerase II TFIID TAFs TFIIH is the only transcription factor with enzymic activity.DNATATAStart siteTBPTAFsTFIIDTFIIATFIIBRNA polymerase IITFIIFTFIIETFIIHThe CTD is phosphorylated by protein kinases; one is a subunit of TFIIH2 subunits of TFIIH unwind the DNAC-terminal Domain CTD of RNA pol II
18Gene Expression Mediator TAFs enhancer Transcriptional activator RNA pol IITAFsTBPnucleosomesTranscriptional activatorTranslational coactivators and corepressorsChromatin remodelling complexMediatorHistone modification complexActs on the basal machinery
19Other RNA polymerasesThe regulation of eukaryotic gene expression is the subject of later lecturesLet’s consider the other polymerases
20Infrastructural RNARibosomal 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.
21Processing pre-r RNAThe 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.
22Processing pre-r RNAInitially 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).
23Ribosomal 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.
24Infrastructural RNATransfer RNA is also transcribed as a long precursor containing several tRNAs joined together.Promoter lies within the coding regionRNase P releases the separate tRNAs by cleavage at the 5’ end of the tRNAs.
25RNase PRNase 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.
26Infrastructural RNAThe 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.
27Other 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???
28Other 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.
29Splicing Process where the introns are removed from the pre-mRNA Occurs in the nucleusCapping (meG at 5’ head) and polyA tailing at 3’ end carried out firstSplice sites are defined by a sequenceFormation of a “lariat” by the spliceosome (U1, U2, U4, U5 & U6 and ~10 proteins)
30Splicing Exon 1 Branch site Exon 2 AGGUAAGU YNYRAY YYYNCAGG 5’ Lariat formed when 5’ p of the intron G attaches to 2’ OH of A5’Y pyrimidineR purineN any nuc5’AG-OHAGpG
31snoRNAsnoRNA are small nucleolar RNAs between 60 and 300 nucleotides in length.RNA editing functionThey 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 conversionsaddition of pseudouridines.
32snoRNA 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.
33Alternative splicingA 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.
34Typical Human GenomeHuman 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).
35Alternative 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.
41snoRNAsnoRNAs are derived from the introns of pre-mRNA transcripts, suggesting that introns are not “junk” DNA.
42miRNA and siRNAmicroRNA (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!!!
43miRNA and siRNAThe 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.
44Pre-miRNA in the nucleus Synthesised by RNA pol IIexonintron3’5’Drosha65 – 75 nt stem loop structure ready for export to cytoplasm3’5’
45Pre-miRNA in the cytoplasm dicer3’5’RISC21 – 26 ds RNA5’3’dicersiRNATranslational inhibition of partially complementary mRNADegradation of complementary mRNA
46miRNAIt cuts off the hairpin loop and the nt pre-miRNAs are exported to the cytoplasm by exportin 5It 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.
47siRNAsiRNAs 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.
48siRNA Researchers can also introduce their own double stranded RNA. The double stranded molecules are processed by Dicer, the cytoplasmic RNase III endonuclease system.
49siRNAThe 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.
50How does miRNA and siRNA regulate gene expression? Translation repression of target sequencesmRNA destruction of target sequencesSilencing chromatin
51Translational Repression 5’UTRAAAAAAAAAAAAAAAA3’UTRProtein that binds to 5’UTRRNARecruited proteins
52mRNA destruction: sequence specific targetting siRNA and miRNA 5’UTRRNA targets sequence for destructionAAAAAAAAAAAAAAAA3’UTR
53Pharmaceutical Applications Use of modified anti-miRNA oligonucleotides (AMOs)Complementary to miRNAInhibit a particular miRNA activityExample is inhibition of miR-122Cholesterol conjugated AMO injected intraperitoneally (X2 weekly)
54Pharmaceutical Applications miR-122 is a liver specific miRNAIts target gene mRNAs are sequences involved in cholesterol regulationIncreasing the level of the target mRNAs lowers cholesterol
55Pharmaceutical Applications The AMO lowered the miR-122 which increased the target mRNA levelsThis resulted in significantly reduced plasma cholesterol levels after 4 weeks
56AMO to miR-122miR-122Inhibits translation of target mRNAs: involved in cholesterol regulation in liverIntroduce the AMO, a stabilised complementary oligonucleotide to miR-122, given intraperitoneally X2 weeklyInactivation of miR-122miR-122 target mRNAs increase lower plasma cholesterol