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1. MOLECULAR CELL BIO > patrick (L14-15) > Flashcards

patrick (L14-15) Flashcards

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1
Q

3 mRNA processing steps that are eukaryotic specialities

A

5’ capping
rna splicing
3’ polysadenylation

IMPORTANTCE
mRNA stability
translation
protein function

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2
Q

overview of pre-mrna processing

A

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

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3
Q

rna polymerase II structure

A

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.

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4
Q

transcription includes modifications of?

A

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

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5
Q

uses of 5’ capping modification

A
  • 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 ))

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6
Q

steps of 5’ capping

A
  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)
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7
Q

importance of step by step modifications

A

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

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8
Q

crucial proteins needed for 3’ polyadenylation

A 
polyA addition
crucial proteins:
- CPSF (cleavage and polyadenylation specificity factor)
- CstF (cleavage stimulation factor F)
- PAP (polyA polymerase)
- PABP (polyA binding protein)
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9
Q

mechanism of 3’ polyadenylation

step 1 - forming polyA tail

A

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

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10
Q

mechanism of 3’ polyadenylation

step 2 - building the platform

A

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

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11
Q

mechanism of 3’ polyadenylation

step 3 - PAP binding

A

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)

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12
Q

mechanism of 3’ polyadenylation

step 4 - actual polyadenylation

A

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

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13
Q

mechanism of 3’ polyadenylation

step 5 - role of PABII

A

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

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14
Q

role of spliceosome

A

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

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15
Q

eukaryotic mrna processing

A

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

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16
Q

principle of splicing

it is a two step transesterification

A

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

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17
Q

splicing specificity and structure of the complex

A

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

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18
Q

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

A

LOOK AT DIAGRAM IN L14 S24

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19
Q

how do snRNPs give specificity in splicing?

A

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.

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20
Q

how do snRNPs accelerate splicing by RNA-RNA basepairing

A

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.

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21
Q

experimental evidence for splicing/splicing products

A

DONT UNDERSTAND IT. DONT KNOW IF I EVEN SHOULD.

ANYWAY L14 S27

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22
Q

exon definition hypothesis ????

A

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

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23
Q

2 guidances for snRNPs to find the exon intron junctions

A
  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
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24
Q

why do we splice??

A

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

25
Q

why do we have capping??

A

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

26
Q

definition and usefulness of alternative splicing

A

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

27
Q

dystrophin

A

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

28
Q

muscle cells

A

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

29
Q

DSCAM

A 
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

30
Q

define RNA editing

A

[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)

31
Q

RNA editing mechanisms

A

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

32
Q
  1. base insertion
A

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

33
Q
  1. cytosine deamination to uracil
A

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

34
Q

the editing of apolipoprotein B - the editosome

A

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
35
Q

function of apolipoprotein

A

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)

36
Q

difference between deamination of ApoB100 protein in liver and in intestine

A

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

37
Q
  1. adenine deamination to inosine
A

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

38
Q

what is ADAR

A

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

39
Q

types of consequences from deamination

A
  • 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
40
Q

RNA editing of glutamate receptor subunit GluR-B

A
  • 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

41
Q

proteins from edited mrna are involved in CNS function

EXAMPLES

A

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

42
Q

possible explanataions for evolvement of rna editing

A
  • system to revise mistakes in transcription
  • enhancement of genome/transcriptome plasticity
  • once evolved as defense system to inactivate retroviral mRNA/retrotransposons
43
Q

ribozymes

A

[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
44
Q

intron rna has catalytic activity - ribozymes

A

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

45
Q

RNAse P has nuclease activity - ribozyme

A
  • 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)

46
Q

types of ribozymes?

A
  • 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

47
Q

ribosomes

A

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

48
Q

hammerhead ribozyme

A

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?

49
Q

ribozyme therapy

A

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

50
Q

processing of rRNA

A

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

51
Q

chemical modification of bases - ribosome synthesis

A

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

52
Q

regulation of nuclear export of mRNAs

A

[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

53
Q

miRNA

A

[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
54
Q

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

A

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

55
Q

what are microRNAs? (again)

A

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

56
Q

miRNA formation

A

LOOK AT DIAGRAM IN L15 S35

57
Q

miRNA function in gene knock out or knock down

A

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

58
Q

miRNAs are specific in what ways?

A

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

1. MOLECULAR CELL BIO (6 decks)

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