patrick (L14-15)

Question 1

3 mRNA processing steps that are eukaryotic specialities

Answer 1

5’ capping
rna splicing
3’ polysadenylation

IMPORTANTCE
mRNA stability
translation
protein function

Question 2

overview of pre-mrna processing

Answer 2

transcription, 5’ capping
cleavage at poly A site (by endonuclease)
polyadenylation (by polyA polymerase PAP and ATP)
RNA splicing (to remove introns)

Question 3

rna polymerase II structure

Answer 3

C terminal domain - ctd
Rna pol tail is a long part of the rna pol, has diff loops
Serine at position 5 where phosphorylation happens.

Question 4

transcription includes modifications of?

Answer 4

includes modification of 5’ and 3’ end of mrna
CTD phosphorylation of rna pol II is crucial for mrna processing proteins

processing factors for capping / poly adenylation / splicing etc are attached to the Pol II CTD tail and hop to specific pre mRNA sites during its transcription

Tail is a small platform to recruit proteins for processing and bring them closer to the mrna that is means to be processed
Phosphorylated at different positions
This goes with other processing enzymes and proteins

Question 5

uses of 5’ capping modification

Answer 5

• modifications distinguished mrna from other rna molecules
• 5’ cap helps mrna to be properly processed and exported to cytosol
• stabilises mrna
• required for efficient translation

(( Stabilises mrna. (rna is very unstable molecule, the cap at 5’ end protects it because the enzyme starts to digest it from the ends, not the middle)
Cap important for later export because this all happens in the nucleus. N is important for efficient translation later on. Ribosome recognises ))

Question 6

steps of 5’ capping

Answer 6

1. Phosphatase removes a phosphate group (Pi) from the 5’ end
2. The guanosine is methylated by guanyl transferase (PPi is removed by GTP)
3. a second methylation of the following base downstream of methyl guanosine (now both the base and the ribose are methylated)

Question 7

importance of step by step modifications

Answer 7

allows cells to monitor the intactness and completeness and correctness of transcripts

Question 8

crucial proteins needed for 3’ polyadenylation

Answer 8

polyA addition
crucial proteins:
- CPSF (cleavage and polyadenylation specificity factor)
- CstF (cleavage stimulation factor F)
- PAP (polyA polymerase)
- PABP (polyA binding protein)

Question 9

mechanism of 3’ polyadenylation

step 1 - forming polyA tail

Answer 9

The C terminal domain, we have a polyadenylation factor sitting there
Waiting for AAUAAA to pop up, then they chomp up on this mrna
Cleavage factors cleave and remove 3’ sequence that is no longer required
PolyA polymerase binds to the sequence and starts adding As to the end of the sequence
These A’s are added to the 3’ tail and covered by poly a binding proteins
PolyA continues until there enough of them bad bois

Question 10

mechanism of 3’ polyadenylation

step 2 - building the platform

Answer 10

The AAUAAA is where the cleavage and polyadenylation specificity binds to
The GU sequence has to be cleaved off.
Recognised by cleavage factor f or something
Cleavage factor 1 and 2 that recognise the CA region and bend the whole thing and yeah build a kind of platform for the next step/slide

Question 11

mechanism of 3’ polyadenylation

step 3 - PAP binding

Answer 11

PolyA polymerase binds to this.
Cleavage factors help to define where PAP should sit
Cleavage properties of PAP cut off what isnt required (which is then degraded)

Question 12

mechanism of 3’ polyadenylation

step 4 - actual polyadenylation

Answer 12

Polyadenylation is rather slow and is energy dependent (uses ATP and removes PPi)

CStF, CFI and CFII are no longer required so are removed from site of action

Question 13

mechanism of 3’ polyadenylation

step 5 - role of PABII

Answer 13

Once enough polyAs are added, the polyA binding proteins comes in and binds to the polyA structure
Can cover 12 polyAs

rapid polyadenylation!
PABII helps polymerase A to be faster in adding more polyAs
Stabilise polA activity
PAP unbinds and the polyA tail is complete

Question 14

role of spliceosome

Answer 14

gets rid of introns
it has sequence specificity so knows where to bind.
End tail is cleaved and poly a tail is added

Question 15

eukaryotic mrna processing

Answer 15

removes introns
exons are ligated to produce the protein
pre-mRNAs (genes) have different sizes

Question 16

principle of splicing

it is a two step transesterification

Answer 16

we need components that recognise

• 5’ end splice site
• 3’ end splice site
• branch point (A) in the intron sequence

remove introns and ligate the exons - lariat structure is made
STEPS:
1. Hydroxyl group binds at A - Forms an incomplete or still attached lariat to the 3’ exon
2. Loop removed from 3’ exon
3. Two exons ligated by esterification reaction

Question 17

splicing specificity and structure of the complex

Answer 17

splicing must be precise to the exact nucleotide
small nuclear RNAs are complexed with at least 7 protein subunits (small nuclear ribonulceoprotein particles snRNPs)
spliceosome is very complex = 5 snRNPs + 200 other proteins
only when splicing is complete does the mature mRNA exit the nucleus

splice sites in premRNA are specified by nucleotide sequences
At branching point A there is a specific sequence that guides the splicing machinery to find where we have the intron exon junctions

Question 18

role of U1 / U2AF /U2 / U4 / U6-U5 in splicing

Answer 18

LOOK AT DIAGRAM IN L14 S24

Question 19

how do snRNPs give specificity in splicing?

Answer 19

U1 snrnp has an rna sequence that matches the sequence found at the intron exon junction at the 5’ site

So this rna gives us 100% specificity to find the specific site where the splicing occurs

U6 and u2 how they interact with each other and stabilised. Can bind the mrna and cleave and remove the intron.

Question 20

how do snRNPs accelerate splicing by RNA-RNA basepairing

Answer 20

LOOK AT DIAGRAM IN L14 S 26

We need srna
Mutate the pre-mrna
The exon/intron junction is not recognised because there is a base pair missing - U1 snrnp wont recognise mrna any more so no more cleavage.

Question 21

experimental evidence for splicing/splicing products

Answer 21

DONT UNDERSTAND IT. DONT KNOW IF I EVEN SHOULD.

ANYWAY L14 S27

Question 22

exon definition hypothesis ????

Answer 22

cell do not only rely on snRNPs for splicing
cells do not only rely on snRNPs for splicing

We have sequences at intron exon junctions that already indicate that the cell is not relying on simple distances or sizes of exons and introns
It’s a huge variation with introns / exon sizes are a lot more uniformed
Exon conservation in size among many organisms. Splicing uss this info to give more accuracy and protect the exons

Question 23

2 guidances for snRNPs to find the exon intron junctions

Answer 23

1. ESE - exonic splicing enhancers. Support splicing.
   This is where SR proteins bind to - they are rich in serine (S) and arginine (R)
   They cover or shield the exon - helps snrnps find the exon junctions. Wont happen somewhere else by accident
2. HnRNPs - they bind to introns and wrap them up
   Makes it more easily found
   Movement of the branch point A to 5’ intron exon junction is mediated because introns are very long sometimes
   SR proteins are positively charged to bind to negative mrna

Question 24

why do we splice??

Answer 24

splicing is not only required to convert premrna in accurate mrna (generation of templates used for translation)

splicing serves as mechanism for cells to generate diversity by creating alternative mRNA (by alternative splicing)

In terms of evolution - better protein transcript, we have 3 exons and 2 introns, and the 3 exons usually produce a template

Question 25

why do we have capping??

Answer 25

Mrna is not a sequence that can leave the nucleus without any help Interaction with different proteins that stay on mrna till it is translated. Cap binding complex is helping the mrna to be exported and reach the nucleus while being protected

Question 26

definition and usefulness of alternative splicing

Answer 26

LOOK AT DIAGRAM IN L14 S32 We have 3 exons to introns The 3 exons could produce a template If there is a mutation at an exon - no longer recognised and get a new sequence. There are other principles of how a sequence can be mutagenised, would result in splicing machinery recognising new parts of the mrna or to not recognise the usual parts of the mrna This could produce new mRNAs Should not happen - protein may become malfunctional Seems to be that the development of us (complex orgasism) benefit from such mutagenesis - from one ancestor gene you get a lot of other mrnas. One gene can suddenly code for a lot of different mrnas with lots of different functions Some might even encode some beneficial functions, or some harmful functions

Question 27

dystrophin

Answer 27

A lot of flexibility in our organisms to allow this to happen Dystrophin is a huge large mrna - there are different dystrophin variants existing, which are sometimes expressed in different cell types. So you can generate variation by alternative splicing

Question 28

muscle cells

Answer 28

Component of muscle cells - myosin mrna transcripts Different sorts of mrna you would find in different tissues. Myosin is spliced in diff ways depending on the cell type. Gives diff myosins with diff functions

Question 29

DSCAM

Answer 29

``` Down Syndrome Cell Adhesion Molecule huge complex made up of: - 24 exons - 10 immunoglobulin domains - 6 fibronectin domains ``` Alternative splicing sites can change transcripts significantly Lots of variants in each of the exons giving a huge amount of variants changing transcription completely. Very powerful splicing tools to generate different functions

Question 30

define RNA editing

Answer 30

[FIRST MECHANISM OF POST TRANSCRIPTIONAL GENE REGULATION] it alters the sequences of some pre mRNAs - rna editing occurs in mammals a very unexpected example is the process of rna editing, which alters the sequences of mRNAs once they are transcribed (Different to rna capping and polyadenylation of splicing Rna editing happens in all eukaryotes)

Question 31

RNA editing mechanisms

Answer 31

base insertions (usually uracil) cytosine deamination to uracil adenine deamination to inosine

Question 32

1. base insertion

Answer 32

LOOK AT DIAGRAM ON L15 S5 It can result in completely different sequences Rna transcript has some regions where we find proper rna editing a uracil to be inserted.. If you have an unedited rna and an edited rna, GUIDE RNAs directs the insertion of uracil Guide rnas bind to the rna stretches and direct the system to insert the uracil → guide RNA1 and 2

Question 33

2. cytosine deamination to uracil

Answer 33

it can alter protein sequences --> alteration of protein function native sequence becomes editted by a base losing its amino group (by deamination) Causes base change and coding sequence change and new protein with new protein function

Question 34

the editing of apolipoprotein B - the editosome

Answer 34

cytosine deamination to uracil 1. a cytidine deaminase activity is involved (APOBEC, ApoB mRNA editing enzyme catalytics subunit) 2. another protein, ACF (APOBEC, complementation factor) is alos needed 3. both recognise the sequences flanking the C to be edited

Question 35

function of apolipoprotein

Answer 35

Apolipoprotein is very important in binding to (bad) cholesterol (low density lipoprotein which can be transported around the body) Apoprotein is modified by APOBEC1 with the help of ACF (editosome, green is anchor to sport where it needs to edit the mrna)

Question 36

difference between deamination of ApoB100 protein in liver and in intestine

Answer 36

In liver cell we have the full length protein produced from the mrna This is important to circulate and bind to the LDL (low density lipoprotein) In the intestine, the CAA codon was changed in the ApoB100 protein, change to a UAA codon This results in an ApoB48 protein which cannot bind to the LDL receptor so cannot transport LDL Editing gives specificity in diff cell types and a regulatory mechanism adds plasticity in tissues and variation in functionality

Question 37

3. adenine deamination to inosine

Answer 37

it can alter protein sequences --> alteration of protein function an amino acid is removed from the adenosine which resulted in an inosine which results in a base change which causes a different amino acid sequence

Question 38

what is ADAR

Answer 38

Adenosine Deaminase Acting on Rna A to I editing affects more than a 1000 genes in humans examples of mammalian A to I editing in L15 S12

Question 39

types of consequences from deamination

Answer 39

- Changes splicing - Self editing adar, autoregulate adar activity - Diff receptors that are modified at the deamination - Neural cell adhesion molecule / phosphodiesterase, intron editing, affects splicing - 3’ UTR editing (untranslated region) - they are always flanking (?) the mrna at the 3’ and 5’ end, and can stabilise mrna too

Question 40

RNA editing of glutamate receptor subunit GluR-B

Answer 40

- A to I editing of the pre mrna that codes for a transmitter gated ion channel in the brain - causes a glutamine (Q) to arginine (R) change that alters the Ca2+ permeability of the channel - mutant mice lacking the ADAR gene are prone to epileptic seizures and die in infancy - direct prodcution of the edited form of the gated ion channel rescues mice lacking the ADAR (normal development) Deamination of adenine can change bases and turn G into A GluRB has 4 transmembrane domains sits in the plasma membrane If you have glutamine, this GluRB codes for a channel used in Ca influx. If a G is blocked then no Ca comes in (malfunction of this transporter) You need to have the Q version of this receptor but in brain tissue, you should not have this ACTIVE Q version

Question 41

proteins from edited mrna are involved in CNS function | EXAMPLES

Answer 41

glutamate receptors serotonin recetors dna repair enzymes ADAR mutant flies suffer from neurodegeneration

Question 42

possible explanataions for evolvement of rna editing

Answer 42

- system to revise mistakes in transcription - enhancement of genome/transcriptome plasticity - once evolved as defense system to inactivate retroviral mRNA/retrotransposons

Question 43

ribozymes

Answer 43

[SECOND MECHANISM OF POST TRANSCRIPTIONAL GENE REGULATION] Ribonucleic acid enzymes: catalytic RNAs enzymes that can affect and change mRNAs - RNA molecules that possess catalytic / enzyme like activities are called ribozymes - has revolutionised our view of biological catalysis - This rnas can bind to mrna and cleave it to destroy rna

Question 44

intron rna has catalytic activity - ribozymes

Answer 44

LOOK AT DIAGRAMS IN L15 S19 Splicing is mediated by spliceosome (complex process) They can splice in the autocatalytic process without a spliceosome Introns have self splicing activity

Question 45

RNAse P has nuclease activity - ribozyme

Answer 45

- RNAse P known to contain an RNA component along with the protein component - it is now known that the RNA of RNAse P is intimately involved in the catalytic mechanism of the nuclease - at high Mg ion conc, the pure RNA component is enzymatically active in cleaving E coli tRNAs Ribonuclease is involved in the processing of trna and mrna often not produced by polymerase 3 Rna P consists of 101 proteins and H1RNA H1RNA recognises precursor trna Cleaves it and activates the trna so it can deliver the aa to the ribosomes the peptidyl transferase function of the ribosome is encoded (and mediated) by an RNA molecule (ribozyme)

Question 46

types of ribozymes?

Answer 46

- hammerhead ribozyme (plant virus) - hairpin ribozyme (plant virus) - hepatitis delta ribozyme (human virus) - neurospora VS ribozyme (mitochondrial mt RNA) - group I and group II intron ribozyme (rRNA and mt RNA) - RNAse P (trna maturation) - ribosome (translation) - spliceosome (splicing) Found in eukaryotes and in viruses Viruses have around 3 protein coding genes only But they can manipulate their hosts with only these 3

Question 47

ribosomes

Answer 47

ribosomes consists of rRNA and ribosomal proteins RNA core of ribosomes are essential for protein synthesis ribsomal proteins stabilise RNA core rRNAs of ribosome are ribozymes

Question 48

hammerhead ribozyme

Answer 48

plant virus discovered in smal rna satellites of small viruses in 1986 LOOK AT DIAGRAM IN L15 S23 S structure is the substrate The rest is the ribozyme Ribozyme binds to substrate region and cleaves it Recognises substrates by sequence complementarity Can be used for therapy?

Question 49

ribozyme therapy

Answer 49

in cancer tissue we have specific sequences pleiotrophin cannot generate a synthetic ribozyme that would recognise the pleiotrophin RNA they hope to use the ribozyme to cut up the mrna of this cancer tissue the process is to take immunity cells like cd4 and cd34 and add the synthetic ribozymes into the cells and back into the cancerous human STEM CELL THERAPY

Question 50

processing of rRNA

Answer 50

the processing of some of these RNA is done by polymerase III nucleolus is a sub location of the nucleus LOOK AT DIAGRAM IN L15 S25

Question 51

chemical modification of bases - ribosome synthesis

Answer 51

post trancriptional modification of rRNAs by - snoRNPs (small nucleolar ribonucleoproteins) - snoRNAs - guide RNAs used as template to conduct modifications once they enter the nucleolus they undergo some modifications in order to process this ribosomal RNA accurately is to process the pre ribosomal RNA what is crucial in this process is a modification of some base points snoRNPs are active and found in the nucleus

Question 52

regulation of nuclear export of mRNAs

Answer 52

[THIRD MECHANISM OF POST TRANSCRIPTIONAL GENE REGULATION] when mrna is ready for translation it needs to leave the nucleus first by export, to get to the cytosol mrna is heavily covered in proteins and it's cap binding complex at the 5 prime end of the strand allows the mRNA to go through the nuclear pore and reach the cytosol this is an energy dependent and active process mRNA is restricted by the movement of the molecules from the cytosol and nucleus the small sized molecules can just diffuse while big sized molecules need active transport which means it requires energy

Question 53

miRNA

Answer 53

[FOURTH MECHANISM OF POST TRANSCRIPTIONAL GENE REGULATION] if the mrna is not exported into the cytosol then it will be the graded in the nucleus - often expressed in the junk or intragenically - microRNA is very important, it can stifle production of a protein by interacting with the true mrna (thereby preventing its translation) - more than 100s of miRNAs can be expressed from the genomes of animals and plants

Question 54

more info about miRNAs (function and where they can be found)

Answer 54

very small 22-25 nucleotides long found in the junk part of the DNA took time to identify their existence and function THEY ARE FUNDAMENTAL IN TRANSCRIPTION AND REGULATION OF TRANSLATION almost all processes found in our body and eukaryotes are heavily dependent on this micro RNA plants can produce these small RNAs and shuffle them into pathogens these small mrna are not recognised by the pathogens and so they accidentally use them to kill themselves this happens in our body too to get rid of pathogens, as DEFENCE pathogens also produce small RNA and secrete them into the host organism to manipulate host signalling processes

Question 55

what are microRNAs? (again)

Answer 55

small noncoding RNA molecules that can regulate eukaryotic gene expression: DEGARDATION AND TRANSLATABILITY micro RNA are very short and are single stranded this means that they have a matching/complementary mrna in a very specific way using the RNA induced silencing complex they can bind to the specific sequence on the mrna and block its translation RISC - RNA induced silencing complex

Question 56

miRNA formation

Answer 56

LOOK AT DIAGRAM IN L15 S35

Question 57

miRNA function in gene knock out or knock down

Answer 57

LOOK AT DIAGRAM L15 S36 cropping step gets rid of the legs of the the mrna dicing gets rid of its head the double strand separate into two single strands one of them will be degraded and digested by binding to the RIS complex and splicing or it can use other helper proteins with the RIS complex to prevent translation bind to mrna and block ribosomal activity

Question 58

miRNAs are specific in what ways?

Answer 58

TISSUE SPECIFIC DEVELOPMENTAL SPECIFIC 1. titrate the levels of key regulatory proteins (important in early development, cell proliferation, cell death and neurodevelopment) 2. dicer is deliberately disrupted in fertilised eggs (block the generation of all miRNAs at this developmental stage) 3. if dicer is deliberately inactivated in development in specific tissues (like in limb musculature), severe growth defects will be caused since miRNAS can fine tune protein levels THIS FUNCTION OF MIRNAS REGULATING GENE EXPRESSION COULD BE AS IMPORTANT AS TRANSCRIPTION FACTORS