Control of gene expression

What is meant by ESEs/ISEs/ESSs/ISSs?

• Exonic or intronic

• Splicing

• Silencers or enhancers

What is the splicing code?

• Regulate excision of introns and joining of exons during splicing

• Facilitated by spliceosome

• Influenced by the combination of splicing and repressors, where sequence-specific RNA binding proteins can bind to them to regulate recruitment of the spliceosome

What is a constitutive exon?

• Always included in the mature mRNA

What are SR proteins?

• Activate splice sites

• RNA-binding domain and protein-interaction domain

• RNA-binding domain binds to splice site

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

• U2AF can recruit U2 for spliceosome assembly sequence

What are hnRNPs?

• Splicing repressors

  • RNA binding domain and protein-interaction domain

• Bind to the 3’ polypyrimidine tract to prevent U2AF and U2 recruitment, preventing spliceosome assembly

What are the three key factors that determine which splice site is used?

• How similar to the consensus sequence the splice sites are (‘strength’)

• Whether or not there are nearby enhancer/repressor elements, and any SR proteins / hnRNPs bound to them

  • Any RNA secondary structures that are masking splice sites

Describe influence of SXL protein in drosophila

• Controls expression of transformer gene that controls somatic sex determination and dosage compensation

• In males, no SXL present, premature termination means no transformer gene, no dosage compensation (good)

• In females, SXL prevents U2AF binding to proximal pyrimidine tract, spliceosome assembles at distal pp tract, full transformer protein translated, dosage compensation (good)

Draw diagram showing how splicing can change whether or not the SXL protein is translated

https://ibb.co/mvM5TB9

Draw a diagram showing what happens in splicing the transformer gene when the SXL protein isn’t and is present (good example of the impact of splicing repressor proteins)

https://ibb.co/TtkkCq6

What is the GU/AG rule?

• Most introns start with GU and end with AG

What are the three key cis elements of a 3’ splice site?

• Branch point consensus containing conserved adenine

• Polypyrimidine tract

• 3’ splice site

Which bases are the pyrimidines?

• Cytosine

• Thymine

• Uracil

What do trans factors do?

• Bind to cis-elements to cause transcription by recruitment of ribosome or recognition of polypyrimidine tract by U2AF

What are snRNPs?

• Small RNA-protein complexes that directly base pair with the mRNA

• Involved with spliceosome assembly

• Includes the U1-6 proteins (exl. U3?)

What type of ‘factor’ is U2AF and what does it do?

• Trans factor

• Large subunit binds to polypyrimidine tract

  • Small subunit associates with 3’ splice stie

How do we know that other proteins assemble to the U1 snRNP?

• Antibodies against U1 snRNPs block splicing

  • Other things must be recruited where those antibodies are binding

What is the chemistry process of splicing?

• Two successive transesterifications

• -OH of branch point attacks phosphate at 5’ of intron

• 5’ exon released

• Forms lariat intermediate

• -OH of 5’ exon attacks phosphate at 3’ of intron

• Ligates two exons together

• Lariat released, rapidly degraded

  • No ATP consumed, number of phosphodiester bonds is conserved

What are the four stage of spliceosome assembly?

• Early complex

• A complex

• B complex

  • RNA rearrangements into catalytic spliceosome

What is the full process of spliceosome assembly?

• U1 to 5’ splice site consensus

• U2AF to polypyrimidine tract and 3’ splice site

• U2 bps to branch point

• ATP hydrolysed

• U4-U6 and U5 recognise A complex and bind

• ATP hydrolysed

• RNA rearrangements into the catalytic spliceosome

  • U4-U6 pairing is broken

  • U1 unbinds, replaced by U6

  • U6 also binds to U2

  • Align pre-mRNA for first catalytic step

  • Generates catalytic site

How are trans-acting factors identified?

• Isolate specific factors that bind to cis-acting sequences

  • UV cross-linking

How are cis-acting elements identified?

• Identify consensus sequences that can be experimented on using mutational analysis

Describe the process of RNA purification / biotin tagging

• Use biotinylated UTP to make oligonucleotide identical to sequence querying that proteins interact with

• Incubate with protein

• Recover using streptavidin beads that bind to biotin

  • Western blot to identify presence of bound proteins, or use NMR if not a clue

Describe the process of RNA immunoprecipitation (RIP)

• Antibody-based technique to map in-vivo RNA-protein interactions

• Fix antibody against protein of interest against side of well

• Mix protein with various RNA, add to well

• Repeat washings so only protein of interest of left

• RT to extract cDNA from RNA bound in protein

• PCR or seq

Describe the process of cross-linking RNA and proteins

• Irradiate with UV

  • Forms stable covalent bonds between RNA and proteins

What are the components of mature, post-spliced mRNA?

• Coding sequence

• 5’ and 3’ untranslated region

• 5’ cap

• 3’ poly(A) tail

What is the purpose of the 5’ cap?

• Increases 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

What is the 5’ cap made of?

• N-methyl-guanosine (m7Gppp-)

How is the 5’ cap added co-transcriptionally?

• 5’ gamma phosphate removed by RTPase

• Pol II CTD activates RGTase

• RGTase adds GMP to 5’ end

• Methylated at position 7 (hence notation m7Gppp)

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

How is 5’ capping made specific?

• All and only Pol II transcripts are capped

• If Pol II promoter is changed to pol I or III there will be no capping, despite the transcript sequence being the same

  • Only di- or tri- phosphate ends will be capped, so mRNAs that have already been digested with endonucleases will not be capped

What is the tandem repeat of the CTD of RNA Pol II and how many repeats are there in yeast and mammals?

• YSPTSPS (

• 26 in yeast

  • 52 in mammals (double)

Simply, what does the CTD of RNA Pol II do?

• Activates RGTase to add GMP in the second step of capping

What evidence is there that the CTD of RNA Pol II is required for capping?

• Amanitin is an inhibitor of Pol II (allows selection for those that have taken up desired pol II)

• Cells infected with one of two version of amanitin-resistant pol II: one has standard 52 CTD repeats and other has 5

• Endogenous pol II inhibited by amanitin

• Incubated

• Capped/uncapped mRNA extracted and quantified

• In 5 repeat group, equal amounts capped and uncapped (both very low)

• In WT group, uncapped mRNA volume the same as in 5 repeat group suggesting uncapped mRNA formed by a different enzyme e.g. pol III

• In WT group, much higher capped mRNA amount

What evidence is there that capping enzymes associate with the phosphorylated version of the RNA Pol II CTD?

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

• measure capping activity of each sample using northern blotting and radiolabelled probes to the cap

• Capping activity only retained in column with phosphorylated WT CTD

What does run-on transcription mean?

• Pol II transcription does not terminate at precise regions

  • Carries on for hundreds bps downstream of what will become 3’ end

What evidence is there for run on transcription?

• Incubate nuclei with NTPs and radiolabeled UTPs in vitro

• Initiation is inhibited but RNAs that are already being transcribed are completed using the radiolabeled UTPs

• Hybridized to probes once fully synthesized

• There is radioactive signal much further downstream of the site corresponding to the future mature 3’ end

  • Signal is not constant and gradually decreases 5’ → 3’

What is the purpose of the poly(A) tail and how long is it in yeast and mammals?

• Protects from 3’ exonucleases to control rate of mRNA degradation

• Binding site for PABP (poly-a binding protein) which then binds to eI4FG

Evidence that cleavage and polyadenylation can happen independently

• Radiolabel RNA substrate

• ATP present: substrate is cleaved and polyadenylated

• ddATP present prevents elongation when incorporated, substrate is cleaved but not polyadenylated

• So polya happens after transcription and requires downstream elements and cleavage is upstream of polya

• RNA molecule that mimics pre-cleaved substrate is polya but not cleaved further so cleavage must need some downstream elements

What evidence is there that polyadenylation has two steps

• Normal substrate with AAUAAA consensus sequence has polyadenylation of 10A

• Mutated consensus sequence has no polyadenylation at all

• Mutated consensus sequence, but small poly(A) already present has polyadenylation

  • So, cleaved substrate with correct consensus is initially pola with a small poly(A) and then adenylated further in a second step

What is the consensus sequence involved in polyadenylation

AAUAAA

Why do proliferating cells have shorter 3’ UTRs

• Fewer binding sites for regulators of gene expression

Why do only 105 of ribosomes exist in the dissociated form?

• Ribosomes are 10x more abundant than dissociation factors

• When small and large subunit are associated under physiological conditions it prevents them translating

What are the prokaryotic and eukaryotic initiation factors? What do they do?

• Prok: IF3

• Euk: eIF3

• Bind to small subunit to prevent its reassociation with the large subunit so it can begin the process of translation initiation

What is the order of the sites on a ribosome (5’ → 3’)>

EPA

What is the full process of prokaryotic translation initiation?

• 30S subunit associates with IF1 and IF3

• This complex interacts with mRNA at RBS

• IF2 forms complex with GTP and initiator tRNA

• These two complexes associate into the 30S initiation complex

• Initiator tRNA is at the P site

• 50S subunit joins

• Activates intrinsic GTPase activity of IF2

• GTP hydrolysed, all initiation factors are released

• 70S initiation complex formed

  • IF2 exchanges GDP with GTP to recycle it

Some facts about the prokaryotic initiator tRNA

• All bacterial proteins are synthesised with fMet as their first amino acid as that is the aa on the charged initiator tRNA

• Formyl group rapidly lost

  • 50% proteins lose the met entirely by slower enzymatic removal

What is the Shine Delgano sequence (bases)?

• AGGAGG

What evidence is there that the Shine-Delgano sequence exists?

• Bind ribosome subunits to mRNA’s during initiation to prevent elongation

• Digest unprotected mRNA with RNase

• mRNA inside ribosome is protected

• Isolate fragments (RT) and sequence and map onto genome

  • 16s rRNA binds to AGGAGG roughly 10nt upstream of the the initiator AUG

What is the advantage of having multiple initiation sites for prokaryotes?

• Polycistronic translation

• Ribosome can be bought to each ribosome binding site individually and does not stop translating when the next SD/RBS is reached

Summarise the three steps of prokaryotic elongation in translation

• Binding of aminoacyl-tRNA to A site

• Formation of peptide bond

  • Translocation of ribosome

Fully describe the process of prokaryotic elongation in translation

• Elongation factor-Tu (EF-Tu) brings the aa-tRNA to A site, has GTPase activity

• GTP-EF-Tu is masking aminoacyl group of aa-tRNA so it cannot react with existing peptidyl-tRNA

• If codon-anticodon match is correct, conformational change in ribosome triggers GTPase activity

• GDP-EF-Tu is released, tRNA in A site moves towards P site to enable peptide bond formation

• If codon-anticodon match is incorrect, GTP-EF-Tu is released with GTP still intact

• deacylated tRNA leaves P site, exits through E site as ribosome translocates

Describe how prokaryotic ribosomes translocate using elongation factors

• GTP-EF-G only binds when EF-Tu isn’t present

• GTPase activity stimulated on binding, causes translocation three nucleotides along, 5’ → 3’

• Peptidyl-tRNA moves to P site, deacylated tRNA leaves via E site

Describe the importance of release factors for termination of prokaryotic translation

• Recognise termination codons

• Induce hydrolysis of the peptide chain from peptidyl-tRNA to release it

  • Need action of class I (RF1 and RF2) and class II

Describe how class I and class II release factors cause termination of prokaryotic translation

• Class I (RF1 and 2) recognise termination codon directly, induce hydrolysis of peptidyl-tRNA

• Class II (RF3) binds ribosome when bound to GDP

• Exchanges GDP for GTP causing conformational change that releases a class I factor

• Subsequent GTP hydrolysis releases RF3

What are the 6 key prokaryotic translation regulation mechanisms

• Incorporation of the SD sequence in to a secondary structure

• Translational coupling

• Repressor proteins

• Small non-coding RNAs (sRNAs)

• Thermosensors

• Attenuation

What is useful about the MS2 bacteriophage mRNA?

• Contains three genes (maturase, coat, replicase)

• Each gene has its own Shine-Delgano box

  • Region between each gene has potential to form secondary structure that disguises the SD box

What evidence is there that incorporating the SD box into a hairpin loop inhibits translation?

• Incorporate SD box of MS2 bacteriophage into hairpin

• Many different point mutations made in coat protein that destabilised or stabilised the hairpin, but did not alter SD sequence or protein sequence

• Translation efficiency measured in vivo

• Mutations that destabilised the hairpin had more efficient translation and vice versa

How does bacteriophage MS2 show regulation by repressor proteins?

• Replicase gene is only required to be translated in early stages of infection, but its translation is coupled to coat protein translation

• When coat protein accumulates, it is implied that enough replicase has been made

• Coat protein binds to stem loop holding SD complex of replicase gene to stabilise it and repress translation

  • Prioritizes cellular components for maturase and coat proteins that are required in greater numbers

Explain prokaryotic translational regulation by sRNAs

• Bind to mRNA

• Activate translation by competing with ds regions in secondary structures, opening the secondary structure to reveal the RBS

  • Repress translation by directly binding to or masking the RBS

Describe prokaryotic regulation of translation by thermosensors

• Secondary structures that block access to SD sequence are sensitive to temperature if proteins (e.g. heat shock proteins) that are only synthesized in response to temperature change, interfere with the stem loop

• Observed in synthesis of prfA protein in some Listeria strains

Describe the principle of prokaryotic translation regulation by attenuation

• Amino acid synthetic operons are switched off if the corresponding tRNA is abundant

• Attenuation represses translation by premature termination of transcription (coupled)

Describe the process of attenuation in the Trp operon

• Trp level is sensed by having two Trp in sequence in a short ORF

• If trp is low ribosome pauses, 2:3 region hairpin forms

• Prevents formation of RNAP-terminating hairpinn

• Allows transcription and translation of the rest of the gene

• If trp is abundant, no pause

• 3:4 RNAP-terminating hairpin forms

• RNAp cannot bind, transcription terminated

Describe prokaryotic Rho-Independent Transcription termination

• GC rich hairpin followed by run of 6 U

• Mutations that disrupt hairpin of 6U disrupt temrination

• Hairpin causes pausing

• Weak interaction between A of template DNA and 6U on RNA causes fall off of template

Describe prokaryotic rho-dependent transcription termination

• Rho protein has RNAdepenent ATPase that can act as helicase

• Rho recognises Crich regions followed by a hair pin that causes transcriptional pausing (this is a cis-acting region)

  • Rho translocates along RNA until reaches the RNAP that is being stalled at the hairpin, causes RNAP dissociation

Compare prokaryotic vs eukaryotic initiation

• Both: use start codons and a dedicated tRNA

• Both: bind the small subunit to RNA first using initiation factors

• Difference: recognition of mRNA

  • Pro: Small subunit binds SD box and initiation codon

  • Euk: small subunit binds 5’ cap and scans for first initiation codon

• Difference: GTP and ATP usage

  • Pro: Only GTP for formation of 30S initiation complex and release of initiation factors

  • Euk: Both GTP and ATP

Simply, what is the eukaryotic translation scanning model?

• 43S initiation complex binds to 5’ cap and scans 5’ → 3’ until first AUG

  • 60S subunit joins, elongation starts

What evidence is there for the eukaryotic scanning model?

• 90% of mRNAs initiate at first AUG

• Insert a new AUG between 5' cap and original AUG, initiation begins at new inserted AUG

  • Inserting hairpin loop between 5’ cap and AUG inhibits translocation, so must be translocation of 43S between the two

What evidence is there that the 5’ cap is used in eukaryotic translation initiation?

• Uncapped mRNA are translated accurately but ineffeciently

  • Addition of cap analogue reduces translation efficiency, so 43S is interacting with WT cap

Briefly, what are the three major steps of eukaryotic translation initiation?

• Formation of 43S initiation complex

• Formation of cap-binding complex at 5’ of mRNA

  • Binding to cap-binding complex and scanningD

Describe the full process of eukaryotic translation initiation:

• 1: Formation of 43S initiation complex:

  • 40S subunit associates with eIF3 and eIF2

• 2: Formation of cap-binding complex at 5’ of mRNA

  • eIF4A, eIF4E and eIF4G all associate together to form the cap-binding complex eIF4F (43S)

  • Assembly is on mRNA

• 3: Scanning

  • 43S binds to cap-bound complex

  • Scans mRNA until first AUG

  • Helicase activity of eIF4A unwinds initial secondary structure, requiring ATP

  • GTP in eIF2 is hydrolysed and released, releasing all imitation factors

eIF3

• Prevents reassociation of ribosomal subunits

eIF2

• GTP-bound

• Brings initiator tRNA to the complex

eIF4A

• Helicase

eIF4G

• Binds to polyA binding protein for transition between initiation to elongation

eIF4E

• Binds to 5’ cap

• Recruits eIF4G

What evidence is there that eukaryotic mRNAs are monocistronic

• Ribosomes scan starting at 5’ cap and eIF4A helicase activity unwinds first AUG, therefore cannot access downstream cistrons

Describe the transition between eukaryotic translational initiation to elongation

• PolyA tail is coated in PABP

• eIF4G omn 5’ cap binding complex binds to the PABP and mRNA is pseudo-circular

  • Interaction stimulates elongation

Describe internal initiation within viruses

• Picronaviruses have uncapped mRNA and long 5’ UTRs with many AUGs and stable secondary structures

• Prevents access to authentic AUG by scanning ribosome

• mRNA is not translated by scanning but instead by direct ribosome binding to internal ribosome entry site (IRES)

  • Allows cap-independent translation

What evidence is there for IRES in viruses?

• Discistronic reporter where finishing cistron A translation causes ribosome detachment

• Insert IRES between cistrons A and B

• Allows translation of cistron B

State four methods of regulating translation in eukaryotes

• eIF2-GDP phosphorylation

• eIF4E-eIF4G interactions

• Gene-specific RNA-Binding proteins

  • RNA degradation / decay

Describe how eIF2-GDP phosphorylation regulates eukaryotic translation

• Regulates initiation (otherwise would be wasteful and risky)

• eIF2-GTP hydrolysis in the 43S initiation complex releases all initiation factors, beginning elongation

• Recycling back to eIF2-GTP requires guanine exchange factor eIF2B

• If eIF2-GDP is phosphorylated quickly, it will bind to eIF2B and prevent it recycling GTP of both that complex and other complexes

• Also prevents eIF11 binding to 5’ cap, preventing initiation

  • Very little eIF2B present, so very rapid prevention of GTP recycling

How is eIF2-GDP phosphorylation part of the antiviral response?

• Protein kinase R phosphorylates eIF2-GDP

• Activated by presence of dsRNA e.g. with rotaviruses

  • Prevents translation of viral RNA

Describe how eIF4E-eIF4G interactions regulate eukaryotic translation

• Once bound to 5’ cap, eIF4E recruits eIF4G

• Can be modulated by 4E-BP which binds to eIF4E before eIF4G can bind

  • Phosphorylation of 4E-BP releases it

Changes in cellular mRNA levels can be achieved by….

modulating transcription rates, decay rates, or simultaneously both

How can mRNA degradation be measured experimentally?

• Block transcription of the gene by inhibiting RNA pol II or cloning gene of interest under a regulatable promotor

• Changes in mRNA levels are then reflected only by degradation as no synthesis

Describe non-specific mRNA decay and the two pathways that achieve this

• Shortening of polyA tail and then:

• Decapping or

• 3’ → 5’

Describe the non-specific decapping pathway of mRNA degradation

• polyA tail is shortened

• Lsm protein binds to shortened polyA

• Promotes decapping

• Can now be degraded 5’ → 3’ by XRN1 exonuclease

Describe the 3’ → 5’ pathway of mRNA degradation

• Exosome degradation

Describe transcript specific mRNA decay

• Involves both cis elements and trans factors

• Trans factors: RNA binding protein recruits exosome to destabilizing cis element

• Cis element involved: AU-rich elements (ARE)

• Recognized by 3’ → 5’ action of exosome

Describe the biogenesis of miRNAs in animals

• Genetically encoded imperfect dsRNA duplexes processed by drosha in nucleus into pre-microRNA

• Exported to cytoplasm

• Cut by dicer

• Mature microRNA is 21nt ssRNA

Describe the biogenesis of siRNAs in animals

• Less than 30bp dsRNA will not cause an interferon response

• Or can insert small hairpins

• Cut by dicer into siRNAs

How to identify binding sites of RNA binding proteins

• CLIP-Seq

• Proteins and RNA are cross-linked in vivo using UV

• Extract

• Treat with RNase so only RNA fragments protected by proteins remain

• Add antibodies specific to RNABP to capture

• Precipitated, RT RNA into DNA, sequence DNA

• Map sequences onto genome

Where do miRNAs bind to mRNA and do they function in prokaryotes or eukarytoes?

• 3’ UTRs near cap (causes decapping)

  • Eukaryotes only

How are cells able to identify an siRNA as an siRNA?

• 2nt 3’ overhangs

  21nt

• 5’ phosphate, 3’ hydroxyl

How do siRNA cause mRNA degredation

• Loaded on RISC, associated with Ago2

• Binds anywhere in the mRNA

• Perfect complementarity

• Causes formation of A-form helix which allows the duplex to align with the catalytic residues inside Ago2

• Endonucleolytic cleavage

Why do microRNAs not cause endonucleolytic cleavage?

• Bind with imperfect complementarity

• Not form A-form helix

• Does not align with catalytic residues inside Ago2

How do microRNAs cause mRNA degredation?

• Bind in 3’ UTR

• First 2-8 nt ‘seed’ bind perfectly, the rest is imperfect

• Need multiple miRNA binding for mRNA degradation (causes recruitment of 3’ → 5’ exonucleases)

What is different about microRNA usage in plants?

• Binds with perfect complementarity, which then allows Ago2 function