IT10

What processing reactions is pre-mRNA subject to in eukaryotes?

• splicing

• 5’ end capping

• 3’ end formation

what is the role of the processing reactions we do to pre-mRNA?

• facilitates nuclear export

why do eukaryotes require pre-mRNA processing but prokaryotes dont?

• they lack the separation of cells into different organelles

  • nucleus - transcription

  • cytoplasm - translation

• this separation in eukaryotes creates a requirement for transport out of the nucleus and into the cytoplasm

which enzymes are used in the 5’ end capping reaction?

• RNA triphosphatase

• Guanylyltransferase

• methyltransferase

describe the process of the capping reaction?

• Triphosphatase first reacts

  • produces a diphosphate

• Then the guanylyl transferase adds the guanosine

  • GTP is associated by a 5’ 5’ linkage

  • triphosphate linkage formed

• Then a methyl transferase reacts

• This modification produces a guanosyl nucleotide at the 5’ end

what protein binds the 5’ cap?

• cap binding complex

what does the CPC recognise?

guanosyl nucleotide at the 5’ end of mRNA

why do only eukaryotes require splicing?

• in prokaryotes, the ORF is in a continuous fashion

• in eukarytoes, the ORF is disrupted

  • the coding region of the DNA is segmented by intervening intron seqeunces

  • these split the coding exons

• To create an mRNA with a continuous ORF for ribosomal translation, exons need to be fused, and introns are removed

  • this is splicing

  • this is done co-transcriptionally

what do the snRNAs in the spliceosome accommodate?

• associate with particular pre-mRNA splice sites via hybridisation of sense to complementary sequences

• this is used for splicing to occur

what are cleave and polyadenylation sequences recognised by?

these are recognised by cleavage and poly adenylation factors

what do cleavage and polyadenylation factors do?

• these cleave the pre-mRNA, producing a mature mRNA and a unprotected 5’ end

  • the pre-mRNA can be modified by the polyadenylation reaction

    • this occurs with every mRNA - all have a polyA tail

    • this makes it functional

  • this also produces an unprotected 5’ end - exoribonucleases use a torpedo model to degrade this mRNA, causing dissociation of RNA polymerase-II from the DNA

What binds exon junctions?

• exon junction complexes

describe how viruses, such as influenza van use cap snatching?

• it associated with a host polymerase at the S5p CTD which is starting to transcribe an mRNA, cuts the cap off and associates it with its own mRNA

• so it uses the hosts cap from a host mRNA

• without this cap, the host mRNA is destabilised and degraded

  • this can supress an immune response from the cell

what problem does cap snatching in influenza overcome?

• the viral polymerase lavcks a CTD, and so cannot be capped

• the use of the host cap allows the production of a stable mRNA for translation

advantage of cap snatching for virus?

the use of the host cap prevents the host mRNA from being stabilised and so it is degraded - this can be used to suppress an immune response by the host

describe how the herpes simplex virus can make use of pauses in transcription to prevent an immune response

• pauses in transcription are typically overcome by pTEFb - which phosphorylates DSIF and NELF and the serII of the CTD of RNAP-II

• viruses such as the herpes simplex virus can express ICP22, this associated with pTEFb, preventing its phosphorylation activity - this prevents the host polymerase from escaping the initial arrest, and so can prevent host immune genes from being transcribed

how can mutations in processing signals cause disease?

• premRNA processing occurs at key cis elements - these have distinct sequences that are recognised by trans factors

• mutations in these can have large consequences for mRNA maturation, as it can prevent recognition by specific transactors

how can polyA mutations alter gene expression>

• Mutations affecting poly(A) signals can reduce or increase cleavage and polyadenylation. Total output of mRNA can be affected.

what are the two potential types of mutations affecting splicing?

• cis-element mutations

  • nt changes affect sequences that affect the association of RNA binding proteins

  • this affects pre-mRNA processing

• trans element mutations

  • mutations in the genes of proteins that associate with the cis elements

describe how mutations in splice signals can cause frontotemporal dementia?

• related to alzeihmers disease

• distortion of the Microtubule associated proteins

  • different isoforms - alternative splicing

  • ratio of isoforms can be distorted, resulting in the formation of fibres which cause cell death

describe how mutations in splice signals can cause Duchenne muscular dystrophy ?

• mutations can render the DM protein less funcitonal

• muscle degeneration caused by mutations in the dystrophy gene

• in some cases, these disrupt splicing enhancers or create splicing silencers

describe how fault alternative splicing of MAPT can occur?

• In the Tau gene, there are exons 9, 10 and 11

• The tau gene produces 2 isoforms

  • tau3 isoform - lacks exon 10

  • tau4 - isoform - includes exon10

• Splicing is directed by elements in exon 10

  • splicing enhancer at the 5’ end

  • splicing silencer - middle

  • splicing silencer - 3’ end

• If the seqeunces are present, there will be a specific ratio of tau3 and 4 mRNAs, and the proteins produced

  • mutations in the splicing enhancer reduce the amount of tau3 produced

    • tau4 is elevated

    • higher number of E10 inclusion

  • if the silencers are mutated, tau4 isoform is elevated, less likley that e10 inclusion is supressed

  → both can produce a distortion of tau3 and 4, leading to disease

how can alternative splicing cause DMD?

• In DMD, exon 31 mutations can introduce an exon splicing silencer

  • this associated with hnRNP A1, suppressing the recognition of the 3’ splice site

  • this leads to exclusion of exon31

  • produces a less functional protein, resulting in destabilisation of muscle cells, and causing DMD

→ most DMD patients are associated with deletions, rather than alternative splicing

describe how mutations in cis elements can cause cancer in the KIT oncogene

• KIT encodes an RTK

• mutations at the end of the gene distort splicing due to the deletion of a 3’ splice site

• this produce an mRNA encoding a functional kinase component, but lacks a control region

  • this produces a constitutively active kinase

  • can cause gastrointestinal tumour

describe how mutations in cis elements can cause cancer in the KLF6 tumour suppressor

• tumour supressor transcription factor inactive in prostate cancer

• point mutation in intron of a gene produces an ISE - this activates cryptic 5’ splice sites

• resulting alternative proteins lack DNA binding domains - may have a dominant negative effect

describe how trans factor mutations can cause retinitis pigmentosa

• mutations in factors or proteins associated with maturation or the association of snRNAs (component of the spliceosome)

  • mutations in these proteins can have dramatic consequences

• retinal disease

• common cause of blindness

describe how trans factor mutations can cause prader-willi syndrome

• mutations indifferent rgions of the genome

• deletion of paternal SNURF-SNRPN allele causes disease by loosing many snoRNAs

• causes wide range of symptoms - eg. restricted growth and musce weakness, learning difficulties

in-depth detail for how trans factor mutations cause prader-willi syndrom

• There is a cryptic 5’ splice site that is notionally suppressed by the adociatio with a snoRNA - snord115, which regulate splicing of the serotonin receptor premRNA via base pairing, preventing U1 snRNA association

  • this promotes recognition of the correct splice site

• In PWS< deletion I chromosome 15, which encodes SNORD115 leads to absence of this suppressive snoRNA

• this means the cryptic splice site is not masked, and so it can be recognised, leading to part of exon5 being excised, altering the signalling capacity of the serotonin receptor

Give examples of how alterations in splicing can cause hallmarks of cancer?

• VEGFA can induce angiogenesis

• BCL2L1 can resist cell death

• CD44 can cause tumour promoting inflammation

→ distortion of splicing events can cause inclusion of cryptic exons or exclusion change the premRNA, creating a protein that lead to cancer hallmarks

how can mutations in transfactors and splice regulators cause mis-splicing in cancers?

• can be in U2 snRNP associated splice factors - this associates with the branchpoint

• can be in sf3b1 - this alters the recognition of 3’ splice sites, causing alternative splicing

• can be in u1 sn~RNP - this recognises the 5’ splice site via base pairing

what blood disorders can be caused by mutations in polyA signals?

thallassemia and thrombophilia

typical polyA signal?

AATAAA

how is thallasemia caused by loss of funciton mutations in cis elements?

• In the globin genes - Alpha and Beta, mutations to hitting the polyA sequence disotrt the recognition of the end of the globin premRNA

• this produces less funcitonal mRNA due to supression of polyadenylation

• this alters the amount of b and alpha globin chains

• this destabilises blood cells, resulting in increased haemolysis

how can diseases such as IPEX syndrome and fabry disease be caused by LOF point mutations in cis elements?

• mutation of the polyA signal in genes such as foxp3, involved in Tcell maturation can prevent recognition of the polYA signal

  • this can lead to over stimulation of T cells, forming IPEX sydrome

• mutations in alpha-galA polyA sequence via a deletion can prevent polyA site recognition, causing issues with sugar metabolism

  • this leads to fabry disease

what can GOF point mutations in cis elements if mRNA do?

can enhance splice site recognition, causing more mRNA to be made

how does GOF point mutations cause thrombosis

• the prothrombin protein is responsible for blood coagulation, this contains premRNA processing signals that direct cleavage and polyadneylation - these signals are weak, which can reduce mRNA production

• Mutations in this can enhance splice site recognition, enhancing the mRNA made and so enhancing the proteim

  • eg mutations changing the C or G → A

    • this can make individuals more prone to clots

describe how mutations in polyA binding proteins can cause disease with example

The disease is caused by an alteration in the ORF of the poly(A) binding protein. The alteration typically consists of the insertion of several additional GCG repeats expanding the polyalanine stretch in PABP significantly. This results in an increased aggregation of PABP in the nucleus.

This can cause ocular muscular dystrophy

describe how alternative poly adenylation can cause cancer

• Many changes associated with disease cause mRNA to contain different features

  • this can be 3’UTRs that are associated with particular phenotypes

  • most genes have different cleavage and polyadenylation sites

    • this produces mRNAs that differ in their 3’ sequences

    • may have some sequences that are removed due to the multiple polyA sites

      • proximal or distal polyA sites might be used

      • there can also be intronic polyA sites

• In cancer, there seems to be a preferred use of proximal sites - this produces mRNAs with shorter 3’ UTRs

  • UTRs are typically important in regulation of mRNA expression by altering transaltion

    • this can be through miRNA binding to the 3’UTR

  • therefore, shorter UTRs can mean less regulation, leading to to higher expression

What problem does viral polymerase have at the 3’ end of genes?

• cannot do cleavage and polyadenylation due to the lack of a CTD to associate with the correct factors

How does vital polymerase produce a polyA tail?

• The viral transcriptase transcribes a polyA tail by going over a uracil stretch

• This has slippage - goes back and forth over this, allowing the production of the polyA without the requirement of polyA polymerase

  • this provides an opportunity to alter the expression of the host genes

how does viral polymerase inhibit polyA tail formation and cleavage in host mRNAs

• one of its genes, NS1 is translated by the mRNAs

• this becomes nuclear localised

  • this binds and sequesters the polyA complex - this contains CPSF and the other factors

  • this inactivates it

  • this prevents polyadenylation of host mRNAs

• this also sequesters polyA binding protein 2

• can inactivate the U6 snRNP, altering host gene splicing

what are the types of potential ~RNA therapeutics?

• use of small RNAs

• use of mRNAs

what small RNAs can be used therapeutically?

• antisense oligonucleotides

• aptamers

• siRNAs

how can aptamers be used in therapeutics

RNA molecules with structures that can inhibit enzymes/receptors

how can siRNAs be used in therapeutics

• small interfereing RNAs

• supress certain genes / translation of mRNAs / cause degradation of mRNAs

describe the use of patisiran as an RNA therapeutic?

• Associated with combatting the formation of transthyretin fibrils

  • these can aggregate in the heart and kidneys in the liver of those with the aggregating isoform

  • these are transported in the blood, causing amyloidosis

    • results in death

  • mutation in these patients creates an isoform that forms fibrils

  • inject an siRNA into the liver - this prevents the translation of this mRNA isoform, causing cleavage and degradation

    • this supresses fibril formation

why do mRNA function need to be understood for mRNA vaccine design?

• to create these drugs, mRNAs need to be understood

  • functianl mRNAs contain caps, UTRs, ORF and a polyA tail

  • making these at scale is difficult

  • need the cap to ensure stability and initiation

  • 5’UTR can aid stabilisation of the mRNA

• mRNA drugs - within 3 weeks, they are degraded

  • this is becuase everything in the mRNA is already present in the cell

  • however, may not get enough - need a balance between things that stabilise and destabilise

why was the immunogenicity in mRNA vaccines initially a problem?

• But mRNAs may be recognised as viral RNAs, causing the innate immune response

  • this is part of the antiviral immune response

why are liposomes used to get mRNAs into cells?

• But mRNAs are highly charged - difficult to get into the cell

  • need to be captured into liposomes

• Liposomes are used as carriers to get small RNAs into the cell

how can mRNAs for therapeutics be made?

in vitro transcription

what base can be incorporated to improve mRNA vaccine efficacy?

psuedouridine

describe how mRNA vaccines work

• mRNAs are packaged into liposomes, transported into cells where they are released

• They are taken up by dendritic cells - this translates the spike protien, which are presented to B-cells, allowing production of the antibodies

  • this allows neutralisation of the virus

example of a membraneless organelle in the nucleus?

• the nucleolus

  • very distinct - pol1, ribosomal RNAs are made, snoRNAs are present, ribosomes made

  • distinct regions

what are MLOs?

membrane less organelles

regions of the cell that behave like organelle, containing specific chemistry and environment, but lack membranes

describe the structure of nuclear pore complexes?

• baskets in the nucleus with nuclear pore rings - RNA docks here through the use of RNA binding proteins

• mRNAs are not naked!! - RNAs are coated in proteins that have function

• the RNA binding proteins are associated with the basket, and thdre is a liquid liquid phase region

  • this contains proteins, fibrils that create a liquid

  • RNA has to go through this

  • RNA binding proteins and export factors make RNAs compatible to go through this

• there are also protrusions, which act as links to the cytoskeleton, allowing targeting of mRNAs

what proteins are used to import and export small RNAs?

• karyopherins

describe the process of export of small RNAs out of the nucleus?

• ran-GTPs associate with exportins

• captures the cargo

• exported in a targeted manner via the nuclear export signal

• in the cytoplasm RAN-GAP phosphatase takes a phosphate off of the RAN-GTP, causing dissociation

  • this release cargo and exportin

describe the import of different smallRNAs and proteins into the nucleus?

• Importins are also important

  • associate with cargo with an NLS

  • this is imported

  • RAN-GTP associates, causoing dissociation and allows recrucling of the importin

why may some RNAs need to move back into the nucleus?

snRNAs - U1, U2 are made and processed into the nucleus, released into the cytoplasm for loading with the sn binding proteins and then reimported

what types of RNAs use the karyopherin mediated mechanism of export/import?

• tRNA, microRNAs and snRNAs also use the same process

• Have specific exportins and use the same mechanism

how do rRNAs and ribosomes leave the nucleus?

ribosomes are budded out (do not use nuclear pore complexes!!!)

do mRNAs use a ran-GTP mediated mechanism for nuclear import/export?

no

how can mRNAs be moved in and out of the nucleus?

• mRNAs go through a series of associations with RNA binding proteins, such as the cap binding proteins, EJCs, TREX proteins to create an export competent mRNA

  • these bound factors act as adaptors for general export factors - eg. NXF1

• NXF1 can then guide mRNA through the chromatin - this allows association of the nuclear pore complex with TREX

• In the cytoplasm, the cap protein will be exchanged for translation initiation factors, and many EJCs will be removed. the polyA binding protein is also exchanged for a cytoplasmic PABP

example of general mRNA export factor?

NXF1

what are TREX proteins?

transcription associated export factors

how can gene gating be used in yeast?

• Gene gating can be used in yeast to move the gene towards a nuclear pore, making it easier to export

  • PPIs might explain this

    • When the gene is switched on, multiple protein protein interactions mediated by SAGA generate a network that pulls the gene towards the nuclear pore complex. This ensures that the pre-mRNA is synthesized close to the pore, ready for export.

what parts of mRNA can control localisation?

3’ UTR seqeunces

experimental evidence for how RNA binding proteins are important for RNA localisation?

• Example: oskar in oocytes

  • has 4 exons

  • it is spliced, this deposits EJCs upstream of each splicing junction

  • the EJCs between exon 1 and 2 is important for firecting the RNA in the oocyte

  • can make some with exon junction complexes/lacking it - eg. can put in a plasmid with a gene with fused exons

  • normal RNAs go to the posterior pole

    • the ones without this do not localise

  → proteins deposited on RNA can have an implication on where the RNA ends up

examples of cells in which mRNAP localisation matters?

• neurones

• migrating fibroblasts

• cell adhesion in epithelial cells

how does mRNA localisation matter in neurones?

• very long!!

• move mRNAs to specific regions for translation

• polarity is made via the transport of mRNAs that encode the specific proteins

how does mRNA localisation matter in migrating fibroblasts?

• focal adhesions

• b-acting mRNAs expression is a very targeted mechanism

• need to be moved to the pole of the cell to make the cytoskeleton and FA contacts

how does mRNA localisation matter in tight junctions?

beta-cadherins are transported and translated to specific junctions

describe the localisation of mRNA in neuronal cells

• 3’UTR sequences are used to regulate localisation, as they contain sequence elements that are recognition sites for RNA binding proteins

  • this allows specific proteins to associate with particular mRNAs

• Some of these proteins/seqeunces might prevent ribosome association

  • miRNAs can prevent association via hybridisation

  • liquid liquid phase spearation

  • 
    

how can signals in neurones be used to regulate mRNA localisation?

• those causing phosphorylation can cause RNA binding proteins to dissociate from mRNA

describe how localisation of mRNAs can be regulated by alternative cleavage and polyadenylation?

• alternative splicing and alternative cleavage and polyadenylation can be used

• those with specific 3’UTRs can accomodate different proteins

  • this allows different distribution

• this alternative splicing allows you to create a downstream fate of the mRNA

• may make a long or sort mRNA, depending on which APA site you use

• long UTR - more proteins likely to bind

  • these can prevent migration, or aid migration

• can also have different terminal exons

  • these produce a different 3’UTR

  • this can alter the localisation

describe how RNA binding proteins can cause LLPS?

Many RNA binding proteins have intrinsically disordered regions that provides multivalency and many membrane less organelles are RNAP granules.

what chemistry can drive LLPS? how can this produce RNAP granules?

• Cation - pi

• pi - pi

• electrostatics

• transient cross B-sheet contacts

• RNA-RNA scaffolds

• Poly ions

• Disordered domains and folded multivalent domain interactions

• Uses specific chemistry

  • this creates a chemistry attractive to some molecules - such as RNAs and proteins

  • important to understand how they are made

examples of how MLOs are used to organise the nucleoplasm and cytoplasm?

• Nucleolus - rRNA transcription and processing

• Cajal bodies mediate maturation of snRNAs, snoRNAs and telomerase RNA are also present

• Nuclear pore complex mediate export import into the nucleus

• Stress granules are formed undet stress

• Paraspeckles are formed under stress by IncRNA Neat1

how are paraspeckles formed? what is the function of this?

• formed in cell stress

• created to sequester splicing components in stress

• involves ncRNA - NEAT1

  • normally made in non-stress conditions

  • shortRNA molecule

  • under stress, creates a very long one

  • this causes the association of proteins, RNAs, which is attractive to splice factors, inducing sequestering of the proteins

describe the use of p-bodies and stress granules in the cytoplasm?

• stress granules sequester mRNAs in stress, and are then released

  • very dynamic!!

• P-bodies are more permanent fixtures, sequestering poorly transalted mRNAs

  • keeps RNA safe from degradation

  • regulate the pool of mRNAs available to the translation machinery

• enriched in mRNAs free of trnslation factors and not ribosome engaged

• funciton is not clear - includes storage of mRNA and regulation of transaltion

  • may serve as proteome biffers

how can MLOs be formed and disassembled rapidly with cues?

• Processes are very dynamic!! - G33P protein is essential to form stress granules

  • this clusters and forms distinct RNA granules

  • this can be in response to the presence of drugs etc

  • select mRNAs will associated - these are not translated

  • eg. cancer cells cant make stress granules - less able to deal with certain drug treatments

why are MLOs highly dynamic?

• lack of membrane allows change of constituents

how can MLOs specifically sequester specific mRNAs?

• Membraneless organelles are made with different proteins

  • these produce MLOs that selectively takes up specific RNAs

how can mRNAs be transported via RNP granules?

• Eg. G3BP1 - many proteins are associated and can phase separate

  • phase separation creates a ‘transport bubble’

  • this transports with motor proteins, which can move the bubble along the cytoskeleton to the required destination

• These MLOs prevent mRNA acess to ribosomes - this ensures efficient transport and only translated in specific positions

how can RNA transport granules ‘hitchhike’?

• Some transport granules may also hitch onto membrane engulfed organelles

  • transporting of membrane engulfed organelles is very fast as these vesicles provide transport fuel via vesicular glycolysis

how can RNA be moved between cells?

extracelular vesicles

how are extracellular vesicles for RNA transfer between cells made

• these exosomes can be made in different ways

  • endosomes, packaged with molecules

  • can fuse with the membeane and release into the extracellular space

    • can then move around and influence recipient cell via recpetor-ligand interaction or fuse with the cell and release the cargo

  → this can allow transport of cellular components between cells

how can angiogenesis be promoted using miRNAs?

• influences epithelia - angiogenesis promoting environment and branching of blood vessels towards the tumour

• produces a particular subset of miRNA in exosomes

• this inhibits processes associated with supressing angiogenesis, allowing secretion of proteins that stimulate angiogenesis

how can tunnelling nanotubes be used to transfer material between cells?

• mRNAs, protein and organelles can also be transported via tunnelling nanotubes.

• Tunnelling nanotubes enable cell to cell communication and material exchange over “large” distances.

• Tunnelling nanotubes are have been implicated in a number of processes including heart development and tumor micro-environments.

what is the templates for translation in prokaryotes?

• Bacterial genomes are operonic

  • multiple ORFs (genes) are arranged in a continuous fashion

  • contains one promoter controlling the transcription of multiple genes

  → the fundamental difference is that one mRNA is produced to encode multiple genes

  • this produces a polycistronic mRNA

    • because it contains multiple cistrons or ORFs

  • in eukaryotes, this is not the same

what is the templates for translation in eukaryotes?

• one monocistronic mRNA

• This means one gene can produce multiple mRNAs through alternative splicing

  • One mRNA encodes one polypeptide

why do prokaryotic mRNAs not need to be processed?

• polycistronic mRNAs contain multiple ORFs, each with their own shine dalgarno sequence

what is the shine dalgarno sequence?

• a conserved GGAGG sequence 5-9nts upstream of the AUG start codon allowing ribosomes to initiate at internal mRNA sites via a co-transcriptional processwht

why is there tight spacing between the SDS and the start AUG in bacteria?

this is because the SDS is important for the 30S subunit recruitment to mRNA

how does the small ribosomal subunit engage with mRNA in prokaryotes?

The small ribosomal subunit engages with the RNA through an RNA-RNA mediated interaction

• ribosomes consist of proteins and RNA

• the 16S rRNA in the 30S subunit contains a segment that is complementary to the sequences of the SDS

• this allows the ribosomal small subunit to engage with the RNA via RNA-RNA hybridisation interaction

what does the engagement of the small ribosomal subunit do?

• this positions the peptidyl transferase site of the ribosome directly at the AUG start codon

  • AUG is in the P-site

• The next triplet is engaged in the A site, which is blocked by initiation factors such as IF3 and IF1

what factors block the A site during binding of the small ribosomal subunit to prokarytic mRNAs?

• IF3 and IF1 block the A site engagement with the second codon

What associates once the 30S subunit has engaged in bacteria?

• the ternary complex associates

what is the ternary complex in bacterial translation initiation?

contains IF2, fmet charged tRNA and GTP

• this associates with the AUG

what happens once the ternary complex has associated with the 30S subunit in translation initiation in bacteria?

• initiation factors such as IF3 and IF1 can then dissociate

  • this opens up the A site, allowing the next amino acid charged tRNA to be brought via triplet formation

  • get peptide bond formation

  • the ribosome can then continue elongation