13_10 DNA Transcription and Splicing.pdf

UCL Cancer Institute Faculty of Medical Sciences DNA transcription and splicing Dr Sarah Koushyar [email protected] MSc Cancer 2 Part 1 Basics of transcription and bursting Learning Outcomes Following this topic, students should be able to: 1. Understand and recap the steps involved from...

UCL Cancer Institute Faculty of Medical Sciences DNA transcription and splicing Dr Sarah Koushyar [email protected] MSc Cancer 2 Part 1 Basics of transcription and bursting Learning Outcomes Following this topic, students should be able to: 1. Understand and recap the steps involved from DNA to mature mRNA. 2. Define key transcription terms. 3. Understand the dynamics of transcription in a single cell. 3 The central dogma 4 From the DNA to a protein Both strands of DNA can be used for transcription depending on where the gene body is. DNA replication *reverse transcription nucleus cytosol Viruses can reverse transcribe 5 Measuring transcription output gel as apposed to 2) Northern blot agarose acrylamide gel in WB 1) RT-PCR Reverse transcription PCR Extract DNA using trysol 3) RNA-seq What do all these techniques have in common? are their limitations? What are their * population based rather than single cell. Average What read out of transcription alignment * limitations? Population based rather than single cell. Average read out of transcription. 6 Transcription as discontinuous bursts Single cell RNA seq Single molecule FISH C-fos* GFP labelled C-fos *low sequencing depth due to low RNA amount per cell https://www.cell.com/molecular-cell/pdfExtended/S1097-2765(18)30308-3 Because the original starting material is low doi: 10.1016/j.celrep.2014.05.053. Epub 2014 Jun 26. How is transcription regulated? 1. 2. 3. 4. Cis-acting regulatory elements (CREs). Non-coding DNA regions. Regulate transcription on the same strand of DNA. Trans-acting regulatory elements (TREs). coding DNA regions. Regulate transcription through their gene products. Promoters (proximal) Enhancers (proximal/distal)-activators bind Silencers (proximal/distal)-repressors bind Insulators 1. 2. they are typically methylated to silence gene transcription Enhancers/silencers that are distant are brought close to the coding region through the folding of the DNA Transcription factors Chromatin modifiers 7 • Gene regulatory proteins (activators/repressors)-bind to CREs • General TFs. Bind to the promoter/RNA pol. Summary part 1 Central dogma – flow of genetic information from DNA ->RNA -> protein. Various methods can measure the amount of mRNA. Most techniques do not consider single cell heterogeneity. Transcription occurs in sporadic bursts within individual cells. These bursts are influenced by CREs and TREs. 8 9 Part 2 Initiation, elongation and termination of DNA transcription Learning Outcomes Following this topic, students should be able to: • Understand and define the key stages of DNA transcription. • Describe nascent RNA modification during transcription. • Understand the complexity involved during transcription. 10 11 Central dogma not every promoter will have these promoter sequences in the non-coding region First step. DNA pre-mRNA Transcription initiation core components 12 1. DNA helicase. Needed to create the transcription bubble. 2. Promoter region. CRE. Site of transcription. INR-initiator. DPE – downstream promoter element. 3. RNA polymerases: I = 18S rRNA, 28S rRNA. II = mRNA, miRNA, snRNA, siRNA. RNA Pol II has Cystine rich C terminal domain III = tRNA, 5S rRNA. 4. General transcription factors. TREs. Phosphorylation of S2 and S5 needed throughout transcription Transcription initiation 1. TBP (subunit of TFIID) aided by TFIIA binds to the TATA box allowing TFIID to bind. This distorts the TATA box, marking the promoter as active. 2. TFIIB binds to the active promoter via the BRE. 3. TFIIB recruits RNA pol II to the INR site. TFIIF stabilises RNA pol onto the DNA. TFIIH helicase unwinds the DNA. Abortive transcription in this complex. Only short pieces of RNA are made. Has kinase activity 4. CTD of RNA II is phosphorylated by TFIIH (aided by TFIIE by creating a docking site for TFIIH). This stabilises the initiation complex, ready for RNA elongation. TFIID has Acetyl transferase activity: adds an acetyl group - opens up the DNA as the group is + charged (repulsion). So it is able to distort the DNA 13 The essential components are not enough The enhancer would have been a distant enhancer - but the DNA loop brings them closer (in 3D space) • • • • Gene transcription is rapid and needs to be modulated by external cues. In vivo – more complicated. Regulation of each gene is tightly controlled. CREs and TREs are important in controlling each individual genes needed for cell identity. DNA is looped by cohesin to bring enhancers to closer contact with the promoter (helped by mediators). 14 Higher level of genome organisation • • • Higher order genome organisation into chromatin structure can restrict gene transcription. Chromatin structure is altered by histone modifications and chromatin remodeling complexes. Euchromatin vs heterochromatin. 15 16 Transcription elongation elongation can steer RNA pol to promote elongation or stall the enemy in cases of cell stress • • • • RNA pol II has left the promoter region and is producing the pre-mRNA transcript in a 5-3’ direction. Phosphorylated residues on the CRD of RNA pol II by TFIIH and P-TEFb recruit modifying enzymes as the mRNA is being transcribed. Elongation factors bind to RNA pol II to aid the elongation of mRNA by driving the direction of RNA pol II. These elongation factors include PAF1 and P-TEFb. PAF1 recruits MLL to trimethylate a histone. NURF is then recruited to remodelling the histones so RNA pol can pass through. Elongation factors Methylation in this case - can enhance gene transcription (PAF1) 5’ Phosphodiester bond Ɣ β ⍺ N N NDP NTP Diphosphate RNA N Triphosphatase N GTP Pii N Guanylyltransferase N N at the 5’ end of the pre-mRNA - it is capped with guanine that is methylated (the gamma P is removed before this, to make space for the P of the Guanine The guanine is flipped so that it is not confused for a normal G and not Methyl transferase Pii N N Transcription termination 1. RNA pol II reaches the 3’ UTR. 2. Within the 3’UTR, there is a polyadenylation sequence and AAUAAA is transcribed at the 3’ end of the mRNA. 3. CPSF (recruitment aided by the CRD domain of RNA pol II) recognises this signal. 4. Recruits a RNA cleavage complex, (CstF, CFI, CFII). 5. Exonuclease cleaves approx. 30nt downstream from the polyadenylation sequence. 6. Phosphatases remove the phosphates from the ser residues in the CRD. RNA pol II is released. 17 Polyadenylation 7. Once mRNA is cleaved, RNA cleavage complex recruit polyA polymerase (PAP). 8. PAP adds As to the 3’ end. 9. polyA binding proteins (PAB) bind along the A tail to stabilise PAP. 10. When enough As have been added, PAB and PAP fall off the mRNA. 11. This leaves the completed pre-mRNA molecule. Still has introns 18 Summary part 2 1. Three steps to transcription, initiation, elongation and termination. 2. General TFs, RNA pol II and a core promoter region is needed for transcription. 3. CREs, TREs and organisation of DNA into higher structures fine tune gene expression. 4. pre-mRNA is modified both at the 5’ and 3’ end. 19 20 Part 3 mRNA splicing and cancer Learning Outcomes Following this topic, students should be able to: • Recall modifications to the pre-mRNA molecule and understand why these are important. • Understand the mechanism of mRNA splicing. • Associate how mRNA splicing leads to mRNA transcript diversity. • Explain the link between transcription deregulation in cancer. 20,000 genes but over 2 million proteins - how does that happen? 21 22 mRNA maturation • Removes intronic regions. • Enables a variety of protein isoforms to be made from a single gene. • Aids nuclear export. • Prevents 5’ exonuclease degradation. • Recognition signal for tRNA. • Stabilises the pre-mRNA molecule. • protects against cytosolic enzyme degradation. 23 Splicing Splicing is facilitated by the splicesome. Relatively large complex Contain 5 small nuclear RNA (snRNA) and various ribonuclear proteins to form small nuclear riboprotein proteins U1, U2, U4, U5, U6. Precatalytic splicesome When they come together in a particular order they produce the active splice some Catalytic active splicesome there is no U3 24 Splicing steps Introns contain conserved sequences to guide the splicing process: 1. 5’GU sequence – the 5’ splice site. 2. A branch site – located near a pyrimidine rich area has an adenine in it - important 3. 3’ AG sequence – the 3’ splice site. U2 binds through the recruitment of BBP and U2AF Splicing steps 25 Introns contain conserved sequences to guide the splicing process: 1. 5’GU sequence – the 5’ splice site. 2. A branch site – located near a pyrimidine rich area 3. 3’ AG sequence – the 3’ splice site. Between the OH group on the adenine and the 5’ G • first cut Alternative splicing • Approx 20000 protein coding genes in our genome. • One gene = one pre-mRNA = one protein. • But there are over 2,000,000 proteins in our body. • This is due to alternative splicing. • Alternative splicing is regulated by TREs acting on CREs on the pre-mRNA. 26 Alternative splicing • Approx 20000 protein coding genes in our genome. • One gene = one pre-mRNA = one protein. • But there are over 2,000,000 proteins in our body. • This is due to alternative splicing. • Alternative splicing is regulated by TREs acting on CREs on the pre-mRNA. 27 Transcription and cancer Many of the hallmarks are the product of a deregulated transcriptional profile What can cause transcription deregulation? 1. Mutations in signalling pathways that regulate transcriptional control. 2. Alterations in CREs/TREs, histone regulators, TFs. ‘By directly targeting the expression of transcriptional regulators in cancer, more comprehensive tumour control could, in principle, be achieved’ https://www-nature-com.libproxy.ucl.ac.uk/articles/nrc4018#MOESM17 28 Transcription and cancer Modes of targeting transcriptional regulators https://www-nature-com.libproxy.ucl.ac.uk/articles/nrc4018#MOESM17 29 30 Splicing and cancer Alterations to alternative splicing can be caused by: 1. Mutations in splicing factor genes – putative cancer drivers. 2. mutations in splice sites. 3. exon skipping and alterative 3' splice sites used in cancer cells. https://doi.org/10.1080/21541264.2016.1268245 Impacts and mechanisms of alternative mRNA splicing in cancer metabolism, immune response, and therapeutics (cell.com) 31 Splicing and cancer therapy 1. Targeting de-regulated splicing via antisense oligonucleotides (AONs). 2. Small molecule compounds modulating the activity of splicing factors. AONs function similarly to snRNAs *not yet approved by the FDA Frontiers | Therapeutic Targeting of Alternative Splicing: A New Frontier in Cancer Treatment (frontiersin.org) Therapeutic targeting of splicing in cancer | Nature Medicine Summary part 3 Splicing is necessary to produce an in-frame coding mRNA. Alternative splicing enhances protein diversity. De-regulation of transcription and splicing are linked to the cancer hallmarks. 32 Optional reading 1. Transcriptional Burst Initiation and Polymerase Pause Release Are Key Control Points of Transcriptional Regulation: https://pubmed.ncbi.nlm.nih.gov/30554946/ 2. Transcriptional Regulation: Molecules, Involved Mechanisms, and Misregulation: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471904/ 3. Review Series focusing on ‘Modes of Transcriptional Regulation’: https://www.nature.com/collections/pfdsgqgzck 4. The role of alternative splicing in cancer: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423477/ 33