Eukaryotic Transcription and Splicing

Describe the CTD of RNAPII and what subunit it is on

• B1

• Unique CTD tandem repeats of YSPTSPS, all of which can be phosphorylated so site of post-translational modifications

• Flexible (not seen in x-ray structure(

• 52 repeats in mammals, 26 in yeast

How can the hyper- and hypo- phosphorylated forms of RNAPB1 be distinguished experimentally?

• SDS-PAGE

• Phosphorylation increases mass so decreases movement

What are the hyper- and hypo- phosphorylated forms of RNAPB1 associated with?

• Hypo = initiation

• Hyper = elongation and termination

Describe the two main RNAPII promoters that CAGE mapping has revealed

• Sharp promoters: single TSS, TATA box and/or initiator elements

• Broad promoters: multiple TSS over 150nt stretch, lack defined elements

What is required for accurate in vitro initiation by RNAP?

• Evidence from in vitro transcription assays using mutated DNA templates

• Either TATA box or initiation element are required, mutation of both loses accuracy

What are the similarities between the euk core promoter cis-element properties an the prok. -35 and -10 box

• Single copy

• Fixes position relative to TSS, conserved

• Asymmetric

• Orientation specific

What cis-acting elements are used for transcription?

• Core promoter cis-elements alone are sufficient for basal level of in vitro transcription

• Upstream cis-elements e.g. enhancers are binding sites for TFs and required for in vivo transcription, very strong

How can enhancers be identified experimentally? Explain and add a diagram

• Enhancer trap assay

• Potential enhancer fragment cloned downstream of reporter gene e.g. GFP in both orientations (action is directional so need both if unsure)

• True enhancer is able to upregulate transcription even when downstream of the promoter

• Result visualized by reporter gene

https://ibb.co/ZscRdyX

In the context of transcription, what is the main trans-acting factor and what is it required for?

• General transcription factors

• Accurate TATA-dependent initiation

• Functionally similar to euk RNAP sigma factors

General transcription factors for initiation are functionally similar to…

• Prokaryotic RNAP sigma subunits

What evidence is there that general transcription factors are required for accurate TATA-dependent initiation? Include a diagram

• Northern blotting

• RNAPII alone with the template results in a variety of transcripts produced at low levels (inconsistent ladder)

• RNAPII with a GTF extract and template results in a single, larger band, so transcription is initiated at the same place every time

• https://ibb.co/KxPrsGTz

What is the TATA box?

• Binding site for TFs required for RNAP assembly at transcription start site of eukaryotic promotor regions

What is the advantage of using general transcription factors (GTFs) to mediate accurate transcription initiation?

• Different GTFs can be present in different tissues or under different environments

• Allow for tissue and environmental regulation of what genes are transcribed and when

Describe the complete process of euk initiation complex formation

• TFIID binds TATA box

• TFIIA stabilises 80degree bend formed

• TFIIB binds, establishing polarity

• TFIIF binds RNAP II

• This complex interacts with the complex assembled on the RNA

• Positions RNAP so it is facing downstream of the complex

• TFIIE binds TFIIH, recruited downstream of RNAP

• Enhancer-bound proteins interact with CTD of RNAP

• TFIIH CDK7 activity phosphorylates RNAP CTD and enhancer-bound proteins after ~10bp are synthesized

• Release of transcription factors and RNAP stably enters elongation

What is the rate limiting step of transcription?

• Formation of pre-initiation complex

Why is RNAP II the only one really discussed?

• Most studied polymerase

• Responsible for most of mRNA synthesis

What happens when TFIID binds the TATA box? What is needed to assist this?

• Binding TATA box locally distorts DNA in the minor groove due to minimal stacking, causing 80degree bend

• This needs to be stabilized by TFIIA (not a strict GTF)

How is the initiation complex made assymetric? Why?

• TFIIB positioning

• ‘B reader’ domain faces upstream

• Determines polarity as determines direction of RNAP binnding

Why is RNAP binding dependent on TFIIF?

• RNAP has no affinity for DNA alone

• Needs TFIIF to interact with TFIIB to be recruited to DNA

What are the three enzymatic properties of TFIIH?

• DNA helicase

• DNA translocase

• CDK7 (cyclin-dependent kinase)

What is the code for the RNAP CTD tandem repeats? What is significant about these amino acids?

• YSPTSPS

• High density hydrophobic and polar

• All can be phosphorylated by TFIIH

In shortened notation, what is the complete process of the assembly of RNAP in transcription initiation?

• TFIID:TATA

• TFIIA assembles

• TFIIF:RNAP

• Assembles

• TFIIE:TFIIH

• Assembles

• Enhancer-bound proteins interact with RNAP CTD

• TFIIH phosphorylates CTD and proteins after 10bp synthesized

• Release of TFs

• RNAP stably enters elongation

In general, how are cis-acting elements and trans-acting factors identified?

• Cis by mutational analysis of believed consensus sequences

• Trans by isolating proteins that bind to cis-acting sequences

State four methods of studying trans-acting factors of transcription

• RNA purification / biotin tagging

• RNA immunoprecipitation

• Genome-wide analyses

• RNA footprinting

Describe the process of biotin tagging for analysis of trans factors

• Make oligonucleotide identical to sequence that is being analyzed for interaction with protein of interest, but use biotinylated UTP

• Incubate with protein

• Recover RNA-protein complexes using streptavidin beads

• Western blot or NMR to identify presence of bound proteins (e.g. mass shift)

If a protein has been tagged with biotin, what can be used to purify it?

• Streptavidin beads

Describe RIP

• Mix protein in question with various RNA fragments

• Fix antibody against protein to side of the well, repeat washing so only bound protein is left

• Add RT to extract cDNA

• Detect using PCR or sequencing

• Can use progressively smaller fragments or mutational analysis for more specific binding regions

Describe the process off RNA footprinting

• UV crosses RNA and proteins covalently

• Digest mildly with -OH radicals so each strand only hit once

• SDS page against RNA also mildly digested, no protein

• Will form a ladder of n, n-1, n-2 etc

• Protein + DNA column will have a gap (footprint) where the protein has protected the RNA

• Map back onto the fragment

What are the advantages to using genome wide approaches to genetic analyses?

• Insight into how expression is coordinated for groups of genes

• Foundation of finding where to start in research, narrows down to several 200bp regions rather than trying to evaluate all 3 billion bps in one go

• Systematically identifies targets of RNA binding proteins, can be automated

Mature mRNA’s contain…

• Coding sequence

• 5’ and 3’ UTR

• 5’ cap

• polyA

• Exons only

What is the 5’ cap sequence?

• N-methyl-guanosine (m7Gppp-)

What is the purpose of the 5’ cap?

• Increase splicing efficiency of introns close to 5’ end

• Required for export to cytoplasm

• Binding site for eIF4G in efficient translation initiation

• Protection from 5’ exonucleases

Cap formation is… why?

• Co-transcriptional

• RGTase activity is dependent on activation by phosphorylated RNAP II CTD

Describe the full process of cap-formation

• 5’ gamma phosphate from pre-mRNA is removed by RTPase

• GMP added by phosphorylated RNAP II CTD-activated RGTase

• Methylated at position 7

• Final step in yeast, in mammals some nucleotides are then individually modified

How is capping made specific? What evidence is there for this?

• All and only RNAP II transcripts are capped

• Change RNAP II promoter to I/III promoter, no capping despite transcript sequence remaining the same

• Only di- or tri-phosphate ends are capped

• mRNAs already digested with endonucleases are not capped

What evidence is there that the RNAP II CTD is required for capping?

• Cells infected amanitin-resistant RNAP II: either a 52 repeat CTD (wt) or 5 repeats (mutant)

• Amanitin added to inhibit endogenous RNAP II

• Cell incubated, mRNA extracted and quantified

• Fraction of capped mRNA much lower in mutant group than in wt, suggesting length of CTD is crucial for capping

• Equal amouunt of uncapped mRNA is equal, suggesting uncapped mRNA is formed by a different enzyme e.g. RNAP III

What evidence is there that the RNAP II CTD needs to be phosphorylated to activate capping enzymes?

• Pass nuclear extract through one of three affinity columns: WT CTD, mutant CTD, WT phosphorylated CTD

• Measure capping activity of sample retained in each column using northern blotting

• Observe capping activity only retained in column with phosphorylated WT CTD

Is the polyA tail encoded?

• No

What is the length of the polyA tail in mammals vs yeast?

• ~240nt in mammals, 120nt in yeast

What is the relationship between the polyA tail and mRNA decay

• PolyA length deceases during export to cytoplasm and as the mRNA “ages” in the cytoplasm

• Rate of shortening is transcript-specific

• When too short, unprotected from 3’ exonucleases which can then degrade

What is run-on transcription?

• RNAP II can carry on for hundreds to thousands of bps downstream of what will become the mature 3’ end as it does not normally terminate transcription at precise positions

What evidence is there for run-on transcription?

• Incubate nuclei with NTPs and radiolabelled UTPs in vitro

• Transcription initiation is inhibited but RNAs already being transcribed are completed using the labelled UTP

• Fully synthesized RNA is cleaved into 100bp fragments

• Hybridized to probes to determine where transcription has carried on until

• Signal is not constant, gradually decreases 5’ → 3’ showing decrease in likelihood of polymerase continuing the further from the mature 3’ end, but no specific terminations site

What re the two cis sequences involved in cleavage and polyadenylation?

• AAUAAA 12-30nt upstream of cleavage site

• GU-rich sequences up to 330nt downstream of cleavage site

What evidence is there that 3’ cleavage and polyadenylation occur independently?

• Radiolabel RNA substrate

• ATP present: cleavage and polyA

• ddATP prevents elongation when incorporated, cleavage but no polyA, so polyA is post-transcription and requires downstream elements

• RNA molecule that mimics a cleaved substrate is polyA but not cleaved further, so cleavage requires downstream elements that it removes

What evidence is there that polyadenylation has two steps?

• Normal substrate with AAUAAA consensus: polyA of 10A

• Mutated AAGAA: no polyA at all

• Mutated AAGAA but with small polyA present: polyA occurs

Summarise the process of polyadenylation

• Cleavage stimulation factor binds GU-rich cis-element

• Cleavage polyadenylation specificity factor binds AAUAA cis elements

• Cleavage upstream of AAUAA

• PolyA

In summary, what is splicing controlled by?

• Gene-specific cis-acting enhancers and silencers

What is the splicing code?

• Interactions between different combinations of enhancers and silencers (cis) with activators and repressors (trans) which dictates the assembly of the spliceosome

What is a constitutive exon?

• Always included in the mature mRNA

State the splicing activators and repressors (trans)

• Activators: SR proteins

• Repressor: hnRNPs

Are activators and repressors cis or trans?

• Trans

Are enhancers and silencers cis or trans?

• Cis

Describe how SR proteins regulate splicing

• Trans-acting activator

• Activate use of splice sites

• Contain RNA-binding domain and protein-interaction domain

• Protein interaction domain recruits U2AF to polypyrimidine sequence close to 3’ splice site

How do trans-factors regulate splicing on a genome-wide scale?

• Different activators and repressors are only present in certain cells and at certain times of their lifecycles, or expressed in response to certain environments

What three factors determine which splice sites are used?

• Consensus sequence match (‘strength’)

• Nearby enhancer/silencer cis sequences, and any activators/repressors bound to them

• Any RNA secondary structures that have masked splice sites

How is the SXL protein in Drosophila a good example of the main principles of splicing? Draw two diagrams

• Whether SXL is synthesized is dependent on which exons are included, which varies between males and females e.g. due to formation of RNA secondary structure or presence of repressor proteins in males

• https://ibb.co/FLgt3hgg

• This protein then acts as a trans-factor repressor, competing with U2AF for the polypyrimidine sequence so that the full transformer protein is made in females for dosage compensation

• https://ibb.co/fVfPJZLn

Relationship between exons and what they code

• Not all encode proteins

• Some encode 5’ and 3’ UTR

Most introns start with: and end with:

• Start GU, end AG

What are the pyrimidines?

• C U T

Describe how snRNPs regulate splicing

• Trans acting activator

• Directly base pair with template cis-elements, help spliceosome assembly

• U1 bps with 5’ splice site consensus cis element

• U2 bps with branch point cis element

• U2AF large subunit binds polypyrimidine tract cis element, small associates with 3’ splice site cis element

Describe how hnRNPs regulate splicing

• Trans acting repressors

• Contain RNA binding domain and protein interaction domain

• Repress splice sites by competing with U2AF for binding to polyprimidine tract cis element to prevent spliceosome assembly

Describe the effects of mutations in cis elements of splicing

• Can completely inactivate splice sites, so nearby cryptic splice site with greater match to consensus than mutation has to be used

• Some 5’ splice site consensus mutations can be reversed by complementary mutations in U1 trans factors

What evidence is there that other proteins associate with U1 during spliceosome assembly?

• Anti-U1 antibodies added to the reaction blocks splicing

Simply, how was the spliceosome discovered experimentally?

• Analysis of in vitro splicing on native electrophoretic gels

Spliceosome assembly requires…

• ATP

Describe the ordered formation of the spliceosome

• U1 bps 5’ splice site consensus

• U2AF large subunit binds polypyrimidine tract

• U2AF small subunit binds 3’ splice site consensus

• U2 bps branch point consensus, requires ATP hydrolysis

• U4/6 and U5 bind, requires ATP hydrolysis

• U4-U6 pairing broken

• U6 replaces U1 and binds U2 snRNA

• These all align pre-mRNA for first catalytic step and generates catalytic site

Key difference between euk and prok RNAP

• Prok RNAP CTD used for activator binding e.g. CAP

• Euk RNAP CTD is made of the tandem repeats that allow hypo/hyperphopshorylated states