Edgell Final Flashcards
Are mRNAs usually stable? Why?
usually unstable due to the presence of destabilizing cis-elements
usually are protected by stabilizing RNA-binding proteins
What do trans acting factors bind to?
cis elements
When does mRNA “quality control” occur?
in the nucleus after transcription, polyadenylation and splicing but before export
What do most cis and trans acting factors for mRNA decay work by doing?
regulating the speed at which the 5’ cap and 3’ polyA tail are removed
What chemicals can be used to measure the half-life of an mRNA?
RNAP-II inhibitors
actinomycin D
alpha-amanitin
5,6-dichloro-1-beta-ribobenzimidazole
What are p-bodies?
cytoplasmic foci consisting of RNA-protien complexes or dynamic aggregates of mRNPs and p-body components
this is where mRNAs go when they aren’t needed anymore
can be degraded from the 3’ or 5’ end
may also be stored here
the number of p-bodies depends on the number of non-translating mRNAs
What are the 3 pathways of mRNA decay?
i.e. after translation
deadenylation-dependent
deadenylation-independent
endonuclease-mediated
What is the first step in mRNA decay? What does this?
shortening the polyA tail to open up the circularized mRNA
this is done by deadenylases
they shorten it to about 80 nts and then to a critical length of 20-25 nts
How does the deadenylation pathway of mRNA decay work?
after removal of the polyA tail
it can either be 5’-3’ or 3’-5’
5’-3’
recruits decapping enzymes
then a 5’-3’ exoribonuclease can degrade it
3’-5’
after the polyA tail has been degraded to its critical length the mRNA can be degraded by exoribonucleases i.e. the exosome
How does the deadenylation-independent pathway of mRNA decay work?
decapping then 5’-3’ degradation
How does the endonuclease-mediated pathway of mRNA decay work?
cleave in the middle then have both 5’ to 3’ and 3’ to 5’ degradation of the 2 resulting pieces
What is AU-rich-mediated decay for?
mechanism to rapidly degrade mRNAs that encode oncoproteins, cytokines and growth factors to prevent their overexpression
Explain how AU-rich-mediated decay works
mRNAs of oncoprotein etc contain a 3’ UTR that has segments that are rich in As and Us called AREs that cause them to be highly unstable unless circumstances call for their protein to be expressed
AREs are repeating AUUUA motifs
they are cis-acting factors that destabilize the mRNA and promote AMD
AUBPs can bind to the AREs and either stabilize OR destabilize them
How are ARE-containing mRNAs classified?
based on the number of AUUUA motif repeats in the 3’ UTR
How do destabilizing AUBPs work?
can interact with decaying, deadenylation or exosome proteins to promote decay
How do stabilizing AUBPs work?
thought to outcompete binding of destabilizing ones
can stabilize the circularization of mRNA by interacting with PABP
What are the 3 steps of nonsense-mediated decay?
detection
tagging
degradation
How are PTC detected in NMD?
exon junction complex is placed 20-24nts upstream of exon-exon junctions AFTER splicing
EJC contains many proteins, key ones are UPF1-3 (UPF = up-frameshift protein)
if the PTC is >50-55nts from the exon-exon junction, the EJC can interact with the 5’ cap and the ribosome
there is a “pioneer” round of translation in which the EJC would normally be bumped off that allows the ribosome to detect PTCs
How do tagging and degradation happen in NMD?
once the ribosome detects a PTC there are a series of phosphorylation and dephosphorylation events that result in the mRNA being de-capped and 5’ to 3’ degradation by exonuclease
What is non-stop decay?
used to eliminate mRNAs that do not have a stop codon
the ribosome stalls when it gets to the 3’ end
this recruits the cytoplasmic exosome
exosome has a number of 3’-5’ exonucleases which degrade the mRNA
How long are the molecules that are used in RNAi?
20-30 nts
Where in genes do siRNAs and miRNAs have their complements?
in the 3’ UTR of genes
siRNAs that are a perfect match to their target can be anywhere, RISC will cleave the target molecule
What does RISC stand for?
RNA-induced silencing complex
it is the effector in RNAi
it is an RNP i.e. contains both proteins and RNA
Traditionally where do siRNA and miRNA come from?
siRNA = from outside and have perfect complementarity miRNA = from inside (i.e. miRNA genes) and do not have perfect complementarity
Which polymerase transcribes miRNAs?
RNAP II
therefore they have a 5’ cap
(it is needed for them to be exported out of the nucleus)
What structure do miRNAs have?
a stem-loop structure
What is pri-miRNA? What is pre-miRNA?
pri-miRNA is the primary transcript generated by transcription
pre-miRNA has been processed by microprocessor and is what gets exported from the nucleus to the cytoplasm (is the precursor stem loop miRNA)
How is RISC assembled?
DICER cleaves ds pre-miRNA at the loop end into 20-30nt unit lengths
this generates a 22-nt darn product with a 2nt overhang at the 3’ ends
passenger strand gets degraded and the miRNA strand that is complementary to the target RNA is incorporated into RISC
Ago2/Slicer is what cleaves the target RNA molecule if there is perfect complementarity (is an RNaseIII type endoribonuclease)
if there is not perfect complementarity then RISC functions in translational repression and/or deadenylation of the mRNA
Note: the miRNA or siRNA can also be called the guide RNA/gRNA
How does Ago2/slicer cleave target RNA molecules?
perfect base pairing puts the phosphate of the target RNA into the active site of Ago2/slicer
it gets cleaved 10nts from the 5’ end of the guide RNA
Note: only the target RNA is cleaved
What is the “seed region” of an miRNA?
the first 8 nts at the 5’ end of the miRNA
it is required to have perfect complementarity
(the 3’ region can be variable)
Is it possible for more than one miRNA to bind to many different targets? Why?
yes because only the first 8 nucleotides i.e. the seed region need to have perfect complementarity
How do miRNAs repress translation?
prevent the assembly of the 40S ribosome and circularization of the mRNA
may also prevent the 60S subunit from joining the 40S
it is also thought that they may mediate an elongation block or catalyze proteolysis of the nascent polypeptide strand
miRNPs bind to them and can help mediate these sort of things
How does RISC know which strand of RNA to incorporate?
the molecule has different stabilities at each end because of base stacking
RISC has 5’ to 3’ helicase activity
the end with the less stable interactions will get pulled apart first
whichever 5’ end becomes free first will be docked in argonaut
What is Ds/dissociator?
a DNA transposon that move by cut-and-paste
What is RNAi thought to be an early mechanism for?
targeting mobile elements for degradation
How much of the human genome is derived from mobile genetic elements?
~50%
What are cut and paste transposons?
have a transposase in the middle with inverted repeats on either side
transposes cuts out the sequence and inserts it somewhere else in the genome
insertion can be specific or random, depends on the element
What are LTR retrotransposons?
are from viruses i.e. HIV
What kind of transposon is LINE-1? How much of the human genome is made of them? Are they all active?
a non-LTR retrotransposon
21% of our genome is made of them
however, 99.9% of them are inactive
Explain how LINE-1 retrotransposition works
there is an RNAP II promoter or 2 in the 5’UTR that drives transcription in either direction
transcript encodes ORF1 and ORF2, it is capped, polyadenylated and exported
ORF1 and ORF2 are translates
ORF1 coats the mRNA, ORF2 binds to the polyA tail
now have a RNP complex
Note: these are cis acting elements!
the entire thing is imported back into the nucleus
ORF2 endonuclease activity loosely targets AT-rich areas which allows the polyA tail to anneal to the T-rich area
ORF2 makes a ss nick generating a 3’OH
ORF2 then reverse transcribes itself i.e. makes a cDNA copy of itself
second strand synthesis and strand cleavage is not yet understood
What is ORF1?
has RNA binding and chaperone activity
What is ORF2?
has endonuclease and reverse transcriptase activity
Why are most LINE-1 elements not active?
reverse transcriptases don’t have sliding clamps and are thus not very processive so they fall off and will result in a truncated 5’ end often (synthesize DNA 3’ to 5’)
haven’t evolved to be more processive because they need to keep their host alive
Describe how exon shuffling could occur during transcription of LINE-1 elements
cis event
if RNAP doesn’t stop transcribing at the polyA tail and includes downstream info, this extra info will be exported, imported etc along with the LINE-1 element and could end up in other exon etc
Describe how exon shuffling could occur during export of LINE-1 elements
trans event
ORF2 could bind to the polyA tail of a different mRNA
this other mRNA could then be imported back into the nucleus, reverse transcribed and incorporated into a new region
Note: in this case ORF1 is not binding
How could LINE-1 elements be used in RNAi?
need dsRNA for RNAi
depending on which way LINE-1 is incorporated into the genome there can be a LINE-1 and an antisense strand
they can anneal
then you have darn that can go into RISC and can then go back and act on itself
What does CRISPR stand for?
clustered regularly interspersed short palindromic repeats
What are some ways besides CRISPRs that bacteria have to prevent phage infection?
RNases
restriction enzymes
abortive infection
blocking uptake
How do CRISPRs work to target phages?
when a phage infects a cell the bacterial cell transcribes the segments of DNA that match the virus
these little segments of RNA act in a way similar to siRNA and cause the cleavage of viral DNA
Note: this can also work on plasmids because technically plasmids are foreign DNA
Describe the layout of the CRISPR system
there are CRISPR associated genes (Cas genes)
they are the protein parts of the mechanism (have the catalytic activity)
adjacent to them there is a leader sequence that has a promoter etc and a series of alternating spacers and repeats
the repeats are identical, the spacers can be identical or different, the spacers are derived from phages i.e. are exact complements to parts of the viral genome
the spacers, once transcribed are the guide RNA (crRNA)
Why can repeats only be so long? i.e. why are the length of CRISPRs limited
because recombination is very efficient and if they get too long they will end up getting deleted
Explain CRISPR acquisition/immunization
viral DNA gets chopped up by proteins
NOTE: this dicing step is DNA not RNA!!
this DNA that is chopped up is called the protospacer, it needs to be adjacent to a PAM sequence
Cas proteins chop out the protospacers and integrate it into the CRISPR array
i.e. the protospacer becomes the spacer
Note: PAM is specific for each bacteria
What is tracrRNA?
it is a trans-activatin element for CRISPR
it is needed for the processing of CRISPR repeats
it is expressed separately from the rest of the CRISPR
part of it is identical to the repeats
Explain how type II Cas9-mediated DNA interference works
Cas9 is inactive until it binds tracrRNA and crRNA, which causes it to undergo conformational changes
Cas9 has 2 active sites, one on the HNH domain and one on the RuvC domain, they are endonuclease that will nick a single strand
crRNA (guide RNA) base pairs with the target and tracrRNA base pairs with the repeated part of the crRNA
this C-terminal of Cas9 recognizes the PAM motif in the target DNA
this complex is only stable enough for cleavage if the there is a PAM motif adjacent to where it is trying to bind
the endonuclease domains make nicks and cause a DSB
Why does the bacteria not cuts its own spacers? (i.e. in the CRISPR array)
because there isn’t a PAM motif beside them so the complex won’t be stable enough for cleavage
Can you have cleavage using CRISPRs if there are mismatches?
no, need perfect complementarity for the complex to be stable enough to be cleaved
How can homologous recombination for genome editing be made more efficient? Why?
by making a DSB
because when there is a DSB the DNA gets chewed back to make 3’ overhangs which are very good at strand invasion
however, it is hard to target a DSB to a specific location
What is the difference between classic NHEJ and alternative NHEJ?
in classic there is minimum DNA loss, it is repaired in cis and gives genome stability
in alternative there is DNA loss, can be translocation and causes genome instability
How is NHEJ used in genome editing?
can be used to knockout a gene
break in a protein coding region, some info is removed, causes change in reading from, mRNA stability, a non-functional protein etc
Note: you still need to target the DSB though
How do you do gene correction?
at the same time as DSB is introduced, provide a correct sequence on donor DNA template to be used for repair
How do you add a gene?
at the same time as DSB is introduced, provide donor DNA template that has flanking homology with extra genes in the middle
Explain how Zn-finger nuclease knockouts of CCR5 promote HIV resistance
CCR5 is a co-receptor for HIV entry into T cells
use a Zn finger nuclease that targets the human CCR5 gene and makes a DSB
rely on the NHEJ pathway to repair and knockout the gene
ex vivo
use CD4+ cells
transfect an adenovirus that expresses the nuclease
make sure the gene has been knocked out, expand the cells and then infuse them back into the patient
need to keep doing it
also very expensive
How do Zn-finger nucleases work? What are some issues with them?
they have a Zn-finger DNA binding domain and a non-specific DNA nucleus domain from a type IIS restriction enzyme called FokI
each Zn finger contacts 3bp of DNA, can put a few in a row to target more specifically
however, you can’t put too many or else the non-specific binding constant will become the same as the specific one
Foci functions as a dimer so you need to make Zn finger domains on each side which is hard (in order to generate a DSB)
also FokI can dimerize with one monomer that is floating in solution and still cleave DNA so you can get off-target DSBs
What are TAL proteins?
proteins that are made in bacteria that infect rice
they are made in the bacteria and imported into the nucleus of the plant cells
there they act as transcription factors for genes that make the plant more susceptible to infection by the bacteria
Describe the structure of TAL effectors
very repeated structure
30-35 AAs that are repeated
called the repeat variable domains
the repeated domains are identical except for 2 amino acids, these 2 amino acids specify contact to a specific base
each TAL repeat contacts a single base
can use them to target 18-20bps, are more specific than Zn fingers
What are TALENS? What are problems with them?
TAL repeats fused to FokI domains
a problem is that they are huge proteins and have a lot of repeated units, so for practical concerns it is difficult
How can CRISPRs be used for genome editing?
you can make a synthetic guide RNA (crRNA) and tracrRNA (on a single vector)
the guide RNA specifies where Cas9 goes
can cleave any sequence you want, just need to order an oligonucleotide for it
Are CRISPRs specific? Why?
no, they will cleave things that are 2, 3 or 4 nts different from the guide RNA with the same efficiency as the on-target sites (sometimes even more efficiently)
partially due to the high frequency of PAMs because they are so short
also because it is occurring at 37 degrees so off-target sites can have stability that is just as good or even better than the on-target sites
also it depends on whether or not the DNA is accessible to breakage and repair
What makes synthetic biology possible?
biology is hierarchical and uses small modular parts to create more complex systems
Explain the parts of the hierarchy of synthetic biology
DNA is used to make parts
parts are used to make devices
devices are like regulatory on/off circuits
systems use devices to make circuits can report things etc
Explain BioBrick parts/assembly
DNA sequences which conform to a restriction-emzyme assembly standard
i.e. there are promoters, terminators, coding sequences etc
are used to assemble synthetic biological circuits and then are transplanted into living organisms
“cut and paste cloning”
all plasmids have the same restriction sites, you cut out the parts that you want and ligate them into a destination plasmid
uses classic type IIP restriction enzymes and put things together using compatible overhangs
What is a problem with BioBrick assembly and other traditional cloning techniques?
when you put everything together you destroy some of the cleavage sites
would need another enzyme to cleave here
i.e. you are limited by the enzymes that you have available
you also need to cleave and ligate the reaction separatey which takes time
What is non-directional/uni-directional cloning?
both sides of the piece you want to insert use the same restriction sequence so it can be ligated in either direction
therefore you need to screen for the right orientation
What is directional/bi-directional cloning?
each side of the piece you want to insert is cleaved using a different restriction enzyme so that it can only be inserted in one direction
What are 4 methods that have changed in order to make synthetic biology possible?
PCR
type IIS restriction enzymes
Gibson assembly
in vivo cloning
What is in vivo cloning?
take DNA fragments that have overlapping sections of homology and put them in yeast
yeast will recombine them for you so you don’t need to do any cloning steps
Describe type IIP restriction enzymes
have a symmetric recognition site
cut within their recognition site
cut specific sequences
Describe type IIS restriction enzymes. Why are they useful?
assymmetric recognition site
cleavage site is distant from the binding site
do not cut specific sequences
binding site is destroyed so it can’t be cleaved again
ie Dsa1 and FokI
Describe Golden gate cloning
have a vector with BsaI sites on it and a PCR product with them as well
make it so that the overhangs that are generated are identical in both (but different on each side)
PCR product and vector are cut at the same time and since there is only one way for it to be ligated and the substrate is not regenerated, it can all happen in a one-pot reaction
Explain Gibson assembly
(either use PCR or restriction digest so that the DNA fragments have identical ends)
use DNA fragments that have overlapping/compatible ends
isothermally amplify them
5’ exonuclease cuts back the ends making an overhang so that they are compatible
they anneal
DNAP extends the 3’ end to put it back together
ligase seals the nick
do this 10-15 times
result is a longer piece of DNA that is sewn together at homology
can then put this together with another one etc
What is a pro for Gibson assembly?
don’t need restriction enzymes etc
reaction can be performed in a single reaction with all of the buffers, enzymes etc
can also use it for cloning i.e. put fragments into plasmids and then transform them into bacteria etc
no restriction “scar” sit left
What components are needed for Gibson assembly?
5’-3’ exonuclease
DNA polymerase
DNA ligase
DNA fragments with at least 15-40 bases of overlap
Describe the influenza virus’s structure
8 linear ssRNA fragments, each encoding a gene
NA and HA are the ones we care about (major antigenic determinants)
Describe how the new RNA influenza vaccine is produced
Day 1- get sequence
Day 2- get oligos and start synthesizing gene
Day 3- ship synthetic gene to NV&D and order PCR primers for cloning
Day 4- clone gene into SAM vector
Day 5- DNA scale up
Day 6- purify DNA construct and linearize it for transcription
Day 7- in vitro transcription/capping of SAM vector and transfect it into BHK cells
Day 8- confirm RNA integrity, expression of proteins etc
use Gibson assembly to make the HA/NA gene
correct errors
clone into a vector that has promoters, terminators etc needed for expression in mammalian cells
do large scale transcription with T7 RNA polymerase
mix sRNA with lipids and cholesterol
have a little RNA virus, this is what you use as the vaccine
Describe the virus used for the SAM vaccine
derived from an alpha virus
it contains the genes needed for viral replication but the structural genes have been replaced by NA and HA
What are 4 advantages to the SAM vaccine?
does not require cell culture and is amenable to automation
robust, generic process, can manufacture against any influenza strain reproducibly
small manufacturing footprint with standard, disposable equipment
you can stockpile the raw materials and equipment in a single facility, which allows you to rapidly makes the vaccine after getting the gene sequence
