Molecular Biology of the Cell Flashcards
(167 cards)
1
Q
How can the formation of the nucleus be explained as an extension of the hydrogen hypothesis (formation of mitochondria through Archaeal cell engulfing a symbiotic α-Proteobacterium)?
A
After the α-Proteobacterium was engulfed, the nucleus formed in order to protect the host genome by either:
- Partitioning translation from transcription, so α-Proteobacterium introns which recombine into host genes can be removed.
- Preventing reactive oxygen series produced by the mitochondria from attacking host DNA.
2
Q
Endosymbiosis is usually understood to be the result of an incomplete phagocytosis, how does the alternative ‘inside-out’ hypothesis explain this instead? How does this model explain nucleus formation?
A
- Instead of phagocytosis, the mitochondrion was engulfed via cell protrusions -blebs- which grew out around the bacterial cell wall.
- The base of these protrusions share homology with the nuclear pore complex, suggesting the original cell formed the nucleus whilst the protrusions became the rest of the cell.
3
Q
Why are Asgard archaea a compelling extant model for the formation of eukaryotic cells?
A
Obligate syntrophy- impossible to culture Asgard archaea without another archaeon and a bacterium present.
4
Q
How does the Martin and Lane hypothesis explain large genome size in eukaryotes?
A
- Endosymbiosis of mitochondria results in increase in bioenergetic membranes in parallel with mitochondrial genome reduction.
- Greater ATP availability permits massive increase in the number of genes that can be expressed allows innovation of new protein folds
5
Q
How does DNA organisation differ between Domains?
A
- Bacteria- Circular, no histones, stable maintenance of chromosomes (SMC) proteins maintain ‘bottlebrush’ shaped nucleoid.
- Archaea- histones and SMC proteins
- Eukaryotes- histones and SMC proteins
6
Q
How are Chromosomes organized in Interphase?
A
- Chromosome territories (CTs) occupy distinct but variable nuclear positions
- The interchromatin compartment (IC) contains non-chromatin domains with factors for transcription, splicing, DNA replication and repair.
- The transcriptional status of genes correlates with gene positioning in CTs- genes near centre of nucleus are more highly expressed.
7
Q
How does Chromosome conformation capture help reveal the 3D organization of Chromosomes?
A
- DNA is fragmented using restriction enzymes.
2.DNA closely associated fragments are ligated, producing small loops.
- Loops are sequenced, and compared to a reference genome.
- Loops containing sequences from two distant points of the chromosome indicate those regions are topologically associated.
8
Q
What are the 4 levels of chromosome organization hierarchy?
A
Level 1: Chromsome territories
Level 2: Chromosome compartments of transcriptionally active and inactive chromatin regions
Level 3: Topologically and Lamina associated domains (TADs and LADs) and
Level 4: Loops.
9
Q
What are the key features of stable maintenance of chromosomes (SMC) proteins?
A
- Hinge domain- binds two proteins together to form ring-like complex (cohesin) around chromatids , allows ring to open and close.
- ATPase domain- may act as motor to extrude DNA through cohesin ring
- Ring is large enough to allow nucleosomes to pass through it.
10
Q
How do SMC proteins form loops of DNA, and how is the size of these loops determined?
A
- Cohesin is loaded on the chromatin by Nipbl
- ATP-dependent cohesin movement pulls out a loop
- Antiparallel (convergent) CTCF binding sites stall movement and cohesin is unloaded by Wapl (Loops are dynamic)
11
Q
What function(s) do TADs and the loops which make them up perform?
A
TADs provide spatial control, grouping multiple promotor/enhancer/repressor complexes into co-regulatory units
Cohesins may also act as a molecular ‘comb’ to untangle supercoiling by pushing to the boundaries where topoisomerases can resolve coils
12
Q
Apart helping to organize the chromosome into TADs, what other function may cohesins perform when they loop DNA?
A
Cohesins may also act as a molecular ‘comb’ to untangle supercoiling by pushing to the boundaries where topoisomerases can resolve coils
13
Q
What did Peter Cook controversially suggest based on the observation of discrete foci of transcription within the nucleus?
A
- Rather than DNA remaining stationary, and polymerases moving, DNA moves and polymerases are stationary.
- Polymerases are organized into discrete factories of 8 RNA polymerases, creating a higher effective concentration of transcriptional machinery.
- Different transcription factories specialize in different subsets of genes.
14
Q
What evidence is there for fixed polymerases and DNA movement?
A
- Following nuclease digestion, nascent mRNA, transcribed DNA sequence and the polymerase all remain associated, suggesting they are parts of a single, stationary unit.
- During transcription of a short and long gene separated by 50 Mbp but co-regulated by TNFα, TNFAIP2 (10 kbp) and SAMD4A (221 kbp) remain in contact, this association makes sense if they are both marked for transcription at a specific factory due to being coregulated.
15
Q
What are the key differences between prokaryotic and eukaryotic DNA replication?
A
- Prokaryotes have one replication origin, whilst Eukaryotes have many.
- Prokaryotic DNA is circular, whilst Eukaryotic chromosomes are linear, requiring telomeres for replicating ends.
- Prokaryotic replication can be continuous, whilst Eukaryote chromosomes are only replicated during S phase.
16
Q
What are the stages of licensing (pre-replicative complex formation) in Eukaryotic DNA replication?
A
- Origin recognition complex (ORC) binds to DNA origin and Cdc6 combines with it to form ORC/Cdc6 complex
- ORC/Cdc6 complex recruits 2 Cdt1/Mcm2-7 complexes, which are loaded onto a single strand of the dsDNA.
- Regulatory proteins (Cdc45, Sld3, Sld2) bind to the Mcm2-7, forming a pre-replicative complex which can function as a helicase.
- Pre-loading complex, made up of Dpb11, GINS, and Pol ε, which replicates the leading strand, is also added.
17
Q
How do Protein kinases CDK and DDK control activation of DNA replication in Eukaryotes
A
- CDK phosphorylates Sld3 and Sld2, allowing the Pre-loading complex (Dpb11, GINS, Polε) to bind to the pre-Replicative complex.
- DDK phosphorylates Mcm2-7 complex, allowing Cdc45 to bind
18
Q
After licensing, what are the stages of initiation in eukaryotic DNA replication?
A
- Helicase generates ssDNA , then a single strand binding protein (RPA) binds, temporarily stabilising the ssDNA.
- DNA Pol α-primase produces RNA primers before handing over to Pol ε for replication of the leading strand.
19
Q
Why does Pol α have a lower fidelity than other DNA replication associated polymerases?
A
Pol α lacks a proofreading function.
20
Q
What is the role of Ctf4 in DNA replication?
A
Ctf4 facilitates interactions between various proteins on the leading and lagging strands
21
Q
How are adjacent Okazaki fragments ligated to form a continuous sequence?
A
- When Pol ε meets an existing Okazaki fragment, it continues to extend sequence, displacing a small portion of it.
- Fen1 removes small ‘flap’
- DNA ligase 1 ligates fragments
22
Q
During termination of eukaryotic DNA replication, how is the replication complex removed?
A
Mcm7 is ubiquitylated (but not degraded) and entire complex is removed from chromatin by p97 ATPase
23
Q
Why do metazoan Origin Recognition Complexes use epigenetic markers and G4 structure to determinine the origin during DNA replication, rather than specific binding sequences like those found in Yeast?
A
Lack of a specific binding sequence allows greater flexibility- S phase can vary in length during different developmental stages, and different regions of the genome are transcribed throughout an organism’s life. Flexibility of replication origins allows conflicts between replication and translation to be prevented.
24
Q
Why is it necessary to regulate DNA replication
A
- Some cell types (i.e. oocytes) must remain in a non-proliferative state and not replicate DNA for long periods.
- Chromosome origins must not fire more than once per cell cycle, to ensure each region is replicated only once.
25
How is DNA replication prevented in quiescent (G0) cells?
1. Quiescent cells do not contain key proteins needed for pre-RC formation
2. DDK and CDK not active in quiescent cells
26
How is it ensured each DNA region is only replicated once per cell cycle?
1. Factors needed for pre-RC formation are degraded during initiation.
2. The formation of the replication fork also results in Cdt1 being proteolyzed: when it is bound to DNA, PCNA (associated with Pol𝛿) acts as a focal point, allowing ubiquitin ligase to proteolyze Cdt1
27
What is the bifunctional role of CDK in DNA replication control?
1. Low CDK in G1 permits pre-RC formation
2. High CDK in S activates replication, but blocks pre-RC
28
Why is it necessary to have a defined timing programme during DNA replication?
1. Fewer copies of replication proteins needed than if all origins fire simultaneously
2. Reduce demand on dNTPs (low dNTPs can lead to genomic instability)
3. Timing of replication may influence chromatin modification
4. Genes replicated early in S phase will have (transient) higher copy number
29
What are the two forms of chromatin found in eukaryotic cells?
1. Euchromatin (loosely packed, genes active).
2. Heterochromatin (densely packed, genes inactive).
30
What is a nucleosome?
1. DNA (approx 150 bp) wrapped around globular domain composed of H2A, H2B H3 and H4 histone proteins.
2. Stabilised by electrostatic interaction- DNA backbone is -, histones are +
31
The H2A, H2B, H3 and H4 histones form the core nucleosome; what role does H1 play
Phosphorylated H1 interacts with linker DNA and is involved in higher order packing, allowing for chromatin to be compacted.
32
How do typical textbook illustrations of higher order nucleosome packing oversimplify the reality?
Show packing as highly organised, with regular structure. Higher order packing is actually likely quite irregular and disorganized as shown in this diagram.
33
What three structures/mechanisms allow for epigenetic regulation of chromatin
1. Variant H2A and H3 histones
2. Histone PTMs
3. DNA methylation
34
How do variant histones such as H2A.Z differ from their canonical counterparts?
Variant or non-canonical histones differ from canonical histones in that they are DNA replication-independent, meaning they are transcribed throughout the cell cycle, not just during S phase.
35
What functions do variant histones perform?
Variant histones are involved in conferring local properties to specific chromatin regions. These properties often facilitate transcriptional regulation and processes such as DNA repair.
36
The enzymes associated with histone post translational modifications fall into which three functional groups?
1. “Writers”- modify specific residues in histones (Usually residues like Arginine, Serine, Lysine, Threonine due to their side chains)
2. “Erasers”- remove modifications
3. “Readers”- interact with the histone modification and change the local properties of chromatin
37
How do histone post translational modifications impact chromatin function?
1. Negatively-charged modifications (acetylation, phosphorylation) may weaken histone- DNA interaction.
2. Large modifications e.g. ubiquitylation may change the structure of the nucleosome
3. Modifications can be ‘read’ by proteins with specific binding domains for modification, allowing enzymes etc to be recruited which alter the properties of local chromatin.
38
What does this histone PTM do?
Acts as a ‘memory’ of recent transcription- Compass complex methylates it upon initiation of transcription. H3K4me3 keeps genes active by recruiting a chromatin remodeller NURF, which destabilises the nucleosomes
39
How does the H2K26me3 histone PTM prevent the accidental initiation of further transcription within the gene body whilst a gene is being transcribed?
1.SetD2 moves with RNA pol II, methylating H3K36.
2. Histone deacetylases bind to H3K36me3, deacetylation increases the positive charge of the histones, causing them to associate more strongly with surrounding DNA.
40
How does DNA methylation regulate transcription?
1. DNA methyltransferases (DNMTs) establish and maintain methylation of cytosine.
2. methylation can then either:
i. Directly block transcription factors.
ii. Be recognized by proteins (MBDs or DNMTs) which recruit transcription inhibitors.
41
How are DNA methylations removed?
TET oxidises base, causing it to be read as damaged by base excision repair system, which removes and replaces it.
42
How does DNA methyltransferase 1 (DNMT1) ensure both strands of DNA remain fully methylated during semi-conservative replication?
DNMT1 only methylates hemimethylated DNA- where one strand is methylated and the other isn't. This creates two fully methylated sets of dsDNA.
43
How are histone modifications retained after DNA replication?
1. PRC2 complex binds to modification on parental nucleosome and becomes active.
2. PRC2 modifies neighboring nucleosomes, spreading modification along chromatin molecule.
3. Blocking domains prevent PRC2 from extending too far
44
How are sister chromatids held together after replicating in the pre-meiotic S phase?
Meiotic cohesin ensures they remain associated.
45
What two different alignment mechanisms do eukaryotes use to bring homologous regions into close proximity during recombination in meiotic prophase I?
1. Synaptonemal complex: compares sequences of chromosomes and aligns a small matching region, before moving down chromosome and aligning the rest based on that initial alignment.
2. Horsetail movements: telomeres are attached, then chromosomes are ‘shaken out’ aligning them. Found in a few eukaryote species, such as fission yeast (S. pome)
46
What is the mechanism of homologous recombination meiosis prophase I?
1. Nuclease(Spo11) makes double strand break in one of the homologous chromosomes.
2. 5’-3’ nuclease (MRA) resects ends leaving 3’ overhang
3. 3’-strand invades the other homologous chromosome, extended by a polymerases (Rad51) forming D-loop (displacement loop)
4. Homologous chromosome catches other side of initial double strand break forming
5. Ends are religated to form double Holliday junction
6. Holliday junction resolvases nick and religate DNA
7. Depending on process of nicking and ligating product can be either crossover or non-crossover
47
How do chromosomes attach correctly to the meiotic spindle during meiosis I?
Cohesin maintains association of homologous chromosomes after recombination is completed.These links are called chiasmata.
48
In meiosis there are potentially four kinetochores, which could potentially result in incorrect attachments. Which proteins prevent this from occuring?
Monopolins- modify sister kinetochores so they behave as a single unit
49
In meiosis there are potentially four kinetochores, which could potentially result in incorrect attachments. Which proteins prevent this from occuring?
Monopolins- modify sister kinetochores so they behave as a single unit.
50
Total loss of cohesin during meiosis I would result in the sister chromatids separating, and so not being configured correctly for meiosis II. How is this prevented?
1. Shugoshin (guardian spirit) protein protects cohesin in vicinity of centromere by recruiting PP2A phosphatase, which makes cohesin resistant to cleavage
2. Cohesin in chromosome arms allows homologous chromosomes to separate, whilst cohesin maintained at centromere keeps sister chromatids linked.
51
What prevents DNA replication between meiosis I and II?
1. High CDK activity is maintained throughout meiosis via the limitation of cyclin B proteolysis.
2. High CDK activity prevents DNA helicases from loading onto origins of replication
52
Which two RNA polymerase-catalysed reactions are necessary for transcription?
Transcription encompasses two opposite reactions: nucleotide addition and excision of incorrect nucleotides.
53
Outline the process by which DNA is guided into an RNA polymerase, and then transcribed into RNA during the elongation stage of RNA transcription.
1. DNA enters the RNA polymerase through the jaws and is guided along the bridge until it meets the wall.
2. Rudder and clamps secure DNA and allow strands to be separated.
3. Nucleotides enter through secondary channel and bind to ssDNA.
4. RNA is then guided out of the enzyme by the rudder.
54
What components of RNA polymerase are likely retained from a common ancestor in Bacteria, Archaea and Eukaryotes?
Fe-S cluster, the catalytic centre of the enzyme, and accessory proteins associated with elongation
55
RNA polymerase initiation factors evolved independently in Bacteria, Archaea and Eukaryotes, in response to what shared problem?
Initiation factors likely evolved as a way of more tightly regulating where RNA polymerases bound to DNA.
56
What are the 6 subunits of bacterial RNA polymerase, and what are their functions?
Subunits: α, α, β, β’, σ, ω
* β and β’ - responsible for catalysis
* σ (and α)- make contact with DNA
* ω and α- scaffold other subunits
57
How does bacterial RNA polymerase make contact with the right sites on the DNA?
1. 1D Hopping- continuous association and dissociation, moving along the DNA
2. 1D Diffusion- sliding along DNA
3. Intersegmental transfer: movement from one DNA site to another that is nearby due to DNA looping.
4. 3D diffusion- passive diffusion whilst dissociated- MOST IMPORTANT DRIVING FACTOR
58
How can a protection assay be used to identify RNA polymerase binding sites in DNA?
1. DNA is incubated with RNA polymerase.
2. Endonuclease which cuts at random sites is added
3. Electrophoresis will produce bands associated with cuts at all sites, except those where the RNA polymerase is bound.
59
How does the σ subunit of bacterial RNA polymerase change the DNA affinity of the entire complex when it makes contact with a DNA binding site?
1. Specific domains of the σ subunit bind to discrete regions in the promoter
2. σ is primarily composed of alpha helices, every 3rd amino acid is a specific residue which is able make contact with the exposed nucleotides in the dsDNA binding site.
3. σ subunit sigma factor contains DNA binding domain, which is usually masked, but when it makes contact with a DNA bind site, it becomes exposed, changing the affinity of the entire RNA polymerase.
60
How do σ factors (subunit of prokaryote RNA polymerase) help to regulate gene expression?
1. Organisms possess multiple different σ subunit variants, with different promoter preferences. Allows control of where RNA polymerases bind depending on evironmental/developmental conditions.
3. anti-σ factors bind to σ subunits to prevent them binding to DNA, can be dependent on environmental conditions such as nutrient availability.
61
What are the three forms that the bacterial RNA polymerase-DNA complex moves between during initiation?
1. Closed: When DNA has only partially engaged with RNA polymerase and is still double stranded
2. Intermediate: DNA bends, and σ region 1.1 makes contact, helping to bring it into the channel.
3. Open: DNA enters the channel and the strands are separated (again by σ subunit), allowing first dNTP to bind.
62
What is' scrunching' during bacterial RNA polymerase initiation?
Once the DNA strands have been separated, the RNA polymerase produces short RNA oligos. When a fragment >10 nt is produced (usually after about 5 cycles0, the Sigma factor is released and elongation initiates.
63
How does the σ subunit of bacterial RNA polymerase separate the DNA strands during initiation of transcription?
σ subunit flips two nucleotides (adenine at -11 and thymine at -7), helping to break the two strands apart.
64
What are the topological consequences of transcription, and how are they resolved?
Transcription results in supercoiling either side of the transcription site. It would be problematic for the transcriptional machinery to encounter these coils, so Gyrase and topoisomerase resolve these structures.This results in heavily transcribed regions having very low levels of supercoiling/DNA-DNA interaction
65
How does Rho independent (intrinsic) transcription termination occur?
Terminator region of DNA containing two-fold symmetry is transcribed, producing an RNA sequence which binds to itself to form a terminator hairpin, which induces conformational change in RNAP, displacing it.
66
How can Rho-independent termination be prevented under certain circumstances, and why is this desirable?
In certain conditions (i.e. exposure to certain metabolites) mRNA can instead form an antiterminator hairpin, which does not induce RNAP conformational change.
This is desirable as it allows for the transcription of certain coding regions (occurring after the terminator) to be dependent on the presence of specific environmental conditions.
67
How does Rho-dependent termination occur?
1. DNA sequence is transcribed to produce high cysteine content rho-utilisation (RUT) RNA sequence
2. Rho termination complex binds to RUT and moves along RNA towards RNA polymerase. through helicase activity.
3. Rho termination complex reaches RNAP and displaces it.
68
What are the three main RNA polymerase complexes found in eukaryotes and what are their functions?
1. RNA polymerase I- transcribes 18S/28S rRNA
2. RNA pol II- transcribes mRNA and some sRNAs.
3 RNA pol III- transcribes tRNAs, 5S Ribosomal RNA, and some sRNAs
69
Plants possess two extra RNA polymerases in addition to those found in other prokaryotes, Pol IV and Pol V. What is their role and how did they evolve?
They are involved in the production of non-coding RNAs. They probably evolved from Pol II
70
What is the role of basal transcription factors in the initiation of transcription in eukaryotes?
Basal transcription factors are auxiliary proteins which bind to promoters surrounding the transcription start site to generate a docking site at which RNA polymerase can bind.
71
What is the role of basal transcription factors in the initiation of transcription in eukaryotes?
Basal transcription factors are auxiliary proteins which bind to promoters surrounding the transcription start site to generate a docking site at which RNA polymerase can bind to form.
72
How do basal transcription factors recruit RNA pol I during transcription initiation?
1. Upstream binding factor (UBF) binds to Upstream promoter element, and then rolls up DNA so it is in proximity with the core promoter
2. UBF recruits complex including TBP, which recruits Pol I
73
How many different types of promoter can bind RNA Pol III?
3- they consist of conserved elements arranged in alternative configurations
74
Outline the initiation mechanism broadly followed by all RNA polymerases during eukaryotic transcription initiation.
1. Polymerase is recruited by basal transcription factors, forming a pre-initiation complex (PIC)
2. Complex enters intermediate open (unstable) state
3. polymerase scrunches, and is then released from the initiating complex.
75
How is RNA pol II-dependent transcription in eukaryotes initiated?
1. TF IID moves along DNA and binds to TATA-box, bending the DNA
2. TF IID recruits TF IIA (which it holds in place by protein-protein interactions) and TF IIB (which binds to its own downstream binding site)
3. RNA pol II, in complex with additional factors inc. TF IIF, associates with the bound complex and recruits TF IIH
76
What two roles does TF IIH perform?
1. Helicase: breaks apart dsDNA allowing scrunching.
2. Kinase: phosphorylates RNA Pol II, weakening association with DNA due to -/- repulsion between DNA and phosphate groups. This allows Pol II to escape the initiation complex and begin elongation.
77
What is the role of TFII S recruited to eukaryotic RNA pol II during elongation?
Responsible for quality control of mRNA transcript.
78
How is mRNA capped to protect against exonuclease during eukaryotic pol II-dependent transcription?
1. Guanilyl transferase adds guanosine monophosphate to 5' end
2. Methyltransferase methylates guanine component of guanosine monophosphate at position 7
79
How is eukaryotic pol-II dependent transcription terminated?
Two factors are recruited to pol II:
CstF (Capping specification termination factor) and CPSF (Capping and polyadenylation specification factor)
1. CstF acts as endonuclease to cleave transcript from pol II after termination sequence is transcribed.
2. CPSF recruits PolyA polymerase which adds the 3' polyA tail. Poly A tail recruits PolyA binding proteins which protect against exonucleases.
80
How is histone organization impacted during transcription?
As first pol II moves past H2A/2B dimer is ejected, whilst H3/4 tetramer is maintained with a lower affinity for DNA. If no more pol II pass that point in rapid success, the histone will reassemble.
At high level of transcription, additional pol II will pass the lower affinity tetramer in rapid succession, resulting in the entire histone octamer being ejected.
81
Other than protecting against endonucleases, what function do the 5' cap and polyA tail of mRNA perform?
1. Help with the assembly of the ribosomal complex during initiation of translation.
2. polyA tail serves as basis of reverse transcription: polyT oligonucleotide binds to it.
82
What benefits does RNA splicing provide?
1. Makes genes modular, allowing sections to be reordered or included/excluded in order to produce multiple different proteins from a single gene.
2. Splicing adds markers to mRNA which facilitate subsequent transport and quality control.
83
Introns are highly variable in size and sequence apart from 3 highly conserved sites necessary for splicing. What are these sites?
1. Left (5') site
Major (U2): GT
Minor (U12): ATATCCT
2. Internal (branch) site-
Major (U2): AC
Minor (U12): CCTTAAC
3. Right (3') site-
Major (U2): AG
Minor (U12): AC
84
How are splice sites paired during splicing to ensure exons are not unintentionally removed?
C terminal domain of pol II recruits splicing apparatus, so transcribed RNA is immediately spliced.
As such, transcription provides an order of splicing (first-come, first-served), where sequences surrounding the splice sites (both introns and exons) act as regulatory elements. C terminal domain of pol II recruits splicing apparatus, so
85
Describe the lariat (lasso) mechanism of RNA splicing and how it ensures external energy is (theoretically) not required for the reaction.
1. A lariat is formed when the intron is cleaved at the 5′ splice site and the 5′ end is joined to a 2′ position at an A in the branch point sequence.
2. The intron is released as a lariat when it is cleaved at the 3′ splice site, and the left and right exons are then ligated together.
3. Number of phosphodiester bonds is maintained during the reaction, meaning the process is not reliant on external energy.
86
Describe the composition of the U2 spliceosome
U2 spliceosome is composed of:
1. 5 snRNAs.
2. 41 proteins which associate directly with snRNA to form snRPs.
3. 70 proteins necessary for assembly of the spliceosome (bind to RNA to construct RNA-based centre for transesterification reactions).
5. ~30 proteins which recruit factors involved in processes such as regulation and quality surveillance.
These are organized into 5 small nuclear ribonucleoprotein (snRP) complexes.
87
U1 is the first snRP to contact pre-mRNA during U2 intron splicing. Explain how U1 recognises its target.
The snRNP U1 recognises the 5’ splice site (left) AG of the pre-mRNA by base pairing of its SnRNA.
88
Describe the assembly of the U1 and U2 complexes during U2 spliceosome formation
1. The first snRP, U1, recognises the 5’ splice site (left) AG of the pre-mRNA by base pairing of its snRNA.
2. U2 binds to the branch point with assistance of 3 auxiliary factors: U2 auxiliary factors U2AF65 and U2AF35 (bind at Polypyramidine tract and 3' splice site respectively) and branch point binding protein (BBP).
3. U2 displaces adenine at branch site, making it protrude from intron and so more susceptible to nucleophilic attack.
89
After U1 and U2 have bound to intron, how is the rest of the U2 spliceosome assembled, and how does it become catalytically active?
1. Super complex composed of snRNPs U4/U5/U6 is incorporated. U4 and U6 snRNA interact by base pairing, while the U5 snRNP associates by means of protein-protein interactions.
2. Completed spliceosome undergoes rearrangement: U1 and U4 are ejected, causing U6 to pair with U2 via snRNA interaction, forming the catalytic spliceosome.
90
How do transesterification reactions form the lariat during U2 splicing?
91
Despite ATP not being necessary for the transesterification reactions (due to the number of phosphodiester bonds remaining the same), U2 splicing still requires ATP. Why is this?
ATP is necessary for the activity of several structural and regulatory proteins which perform functions such as:
1. linking snRPs together
2. rearranging RNAs
3. facilitating the release of components during complex transitions.
92
Introns are highly variable in length. What two mechanisms involving SR proteins (composed of RNA Recognition Motif and Arg-Ser rich domain) are used to define intron size within an mRNA molecule?
Two similar mechanism in which SR proteins form a chain which is associated with RNA:
1. Intron definition: SR associated RNA region is defined as intron
2. Exon definition: regions flanking SR associated RNA are defined as introns
93
In organisms with large introns, such as mammals, backsplicing can occur. How is exon backsplicing (in which two ends of an exon are accidentally ligated, forming a ring) typically avoided during splicing?
Steric encomberance: RNA molecule is large and complex enough (and in the case of exon-definition associated with SR proteins) to prevent complex from assembling correctly if two ends of an exon are loaded, forcing the complex to rearrange.
94
How can backsplicing still sometimes occur, and why can it be beneficial?
1. Very long (or multiple connected) exons can form a large enough loop to accomodate the spliceosome, making steric encumbrance no longer an issue.
2. Backspacing generates circRNAs, which are exonuclease resistant and can perform a range of cellular roles by interaction with RNA and proteins, for example, serving as sponges which 'soak up' miRNAs.
95
What is the key way in which the alternative (U12) spliceosome differs from the U2 spliceosome
Recognises and is assembled by variants of U1 and U2: U11 and U12 which are part of a single complex, unlike U1 and U2.
96
How do Prokaryote pre-mRNAs undergo splicing without a spliceosome?
They have self-splicing introns, provided by conserved secondary structures within the intron which perform catalytic function. (some additional proteins assist in vivo).
97
How does mRNA splicing inform its transport and (in the case of non-sense mRNA) degradation?
1.Spliceosome recruits Exon junction complex to splice point. This complex directs the mRNA to the nuclear pore.
2. In functional mRNA, stop codon occurs after ECJs, meaning ribosome will move past and displace ECJs during translation. If ECJs are maintained, mRNA is non-sense due to premature stop, and is degraded.
98
How does alternative splicing occur?
Specific RNA-protein interactions mask or unmask intron/exon regions which repress or enhance local spliceosome assembly.
99
What are small RNAs?
1. Functional RNA molecules that are not translated into a protein
2. Many types including miRNA and siRNA
100
What are microRNAs
1. Small non-coding ssRNAs which negatively regulate gene expression post-transcriptionally.
2. Action RNA:RNA
3. Transcribed as a 18-25 nucleotide hairpin structure (miRNA stem-loop) with a region containing the mature miRNA
101
How do miRNAs regular gene expression?
1. miRNA, is assembled into a RISC complex in which the short RNAs are bound by an Argonaute protein.
2. miRNA-RISC complexes associate with target mRNAs by base pairing between the miRNA and complementary regions of target mRNAs
3. RISC complexes bound to miRNA that base-pairs extensively with its target mRNA, induce target mRNA cleavage.
4.RISC complexes associated with a short RNA which base pairs less extensively inhibit translation and induce a slower form of target mRNA degradation
102
What are the key differences between plant and animal miRNAs?
1. Number of biosynthesis enzymes: 1 in plants, 2 in animals
2. Location of biosynthesis: nucleus in plants, cytoplasm and nucleus in animals
3. Location of miRNA-binding motif: 5’-UTR and ORF in plants, 3’-UTR in animals
103
How does miRNA processing differ between plants and animals?
1. In animals Drosha removes tail to form Pre-miRNA and Dicer removes loop structure to form miRNA duplex. In plants, DCL1 removes tail to form Pre-miRNA and removes loop structure to form miRNA duplex- one enzyme performs two modifications.
2. In animals, nuclear export occurs before loop is removed, whereas in plants it occurs after
3. In animals, mature miRNA ends in -OH group, whilst in plants it ends in -OCH3 methyl group
104
How can mRNAs become miRNA resistant?
Mutations can make mRNA too divergent, preventing miRNA from recognising it
105
What is the role of miRNA in establishing the meta/proto-xylem gradient in plants?
1. SHORT ROOT (SHR) transcription factor is made in central vascular tissue, it then moves into the endodermis where it enters the nucleus.
2. SHR-transcription factor activates a gene encoding a miRNA called 165/6 in endodermal nuclei.
3. miR165/6 moves into adjacent cells, forming a ‘morphogen’ gradient (high abundance in dark green cells, low in light green cells).
4.An mRNA encoding the PHABULOSA (PHB) transcription factor is down-regulated by miR165/6 in a dose-dependent manner
5. High levels of PHB specify metaxylem, low levels specify protoxylem.
This produces a Meta/Proto- xylem gradient
106
What are small interfering RNAs?
1. Around 18-25 bp long derived from dsRNA originating from: Viruses, Heterochromatin and Transposons (jumping genes)
2. siRNAs cleave mRNAs
3. 100% complementary to the target -> very high target specificity
4. Reduce transposon activity and combat viral infections
Also, regulate genes via ‘epigenetics’
107
What role does siRNA gene silencing play in developing an immune response towards viruses?
1. Sometimes, viral ssRNAs are improperly capped, RDR binds and makes the RNA double stranded.
2. Dicer detects this dsRNA and cleaves it into siRNAs
3. siRNAs can then act as primers, binding to fully capped viral ssRNAs, amplifying gene silencing.
108
What are the two classes of siRNA-directed epigenetic modification?
1. RNAi-directed DNA methylation
2. RNAi-mediated histone methylation
109
RNAi-directed DNA methylation
1. In the nucleus, dsRNA can be produced by transcription of inverted DNA repeats or by RDR2 (RNA-dependent RNA polymerase 2) activity on ssRNA templates -> cleaved by DCL3
2. siRNA signals (wavy red lines) directs DNA methylation enzymes to targets:
- MET1 for CGs and
- DRM2 for primarily non-CGs
3. Maintenance of methylation of CGs and CNGs can be maintained in the absence of the RNA trigger through the activity of MET1 and CMT3 with the help of other proteins.
4. DNA glycosylase proteins ROS1 or DME are needed for loss of methylation — and therefore reactivation
110
Describe RNAi-mediated histone methylation
1. Tandem repeats or transposable elements in histones can be converted to a double-stranded RNA through the activity of RNA- dependent RNA polymerase (RdRP)
2. The dsRNA is cleaved by Dicer to produce small interfering RNAs (siRNAs)
3. These siRNAs guide histone methyltransferases (HMTs) to the chromatin to modify histone H3 on lysine 9 (H3K9)
4. The methylated form of H3 is bound by proteins to maintain the silenced state
m = methyl group
111
What are the components of the nuclear membrane?
1. Inner nuclear membrane
2. Outer nuclear membrane
3. Perinuclear space
112
What is the Nuclear lamina?
Nuclear cytoskeleton attached to inner nuclear membrane via intermediate filaments (lamins)
113
The nucleus and cytoplasm are topologically continuous, what are the implications of this for nuclear transport?
Transport between the two doesn’t require crossing a membrane.
114
Describe the overall structure of a nuclear pore complex.
Large- 500-1000 proteins, assembled from multiple copies of ~30 nucleoporins.
Two main features:
1. Symmetric core:
- 3 rings
- central pore
2. Asymmetric nuclear and cytoplasmic peripheries:
-Nuclear basket
-Cytoplasmic filaments
115
Describe the structure of a nuclear pore complex's symmetric core.
3 rings:
1. Inner ring- spans inner and outer nuclear membranes
2. cytoplasmic and nucleoplasmic rings
Rings are composed of scaffold NUPs which form the structural foundation of the NPC
Central pore:
1. Contains highly disordered domains formed from FG (phenylalanine-glycine) repeat proteins
2. FG-repeats have weak hydrophobic interactions with each other, creating a gel-like structure which acts as a sieve.
116
What is the role of transport receptors in the active transport of macromolecules across the nuclear membrane?
Transport receptors are slightly hydrophobic, allowing them to interact with, and pass through, FG NUPs permeability barrier along with cargo.
117
What types of transport receptors are used in the active transport of proteins and RNA across the nuclear membrane?
Proteins:
1. Karyopherins, e.g. Importin β and Exporting
RNA:
1. NXT1/NTF2 heterodimer
2. Karyopherins (less common)
118
Describe the structure of an Importin β transport receptor and the role this plays in its function.
HEAT repeat protein- 30-40 residues forming amphiphilic α-helices.
Free Importin β is hydrophilic, but hydrophobic regions are exposed once cargo is bound, facilitating interactions with FG NUPs.
119
How do cargo proteins bind to receptor proteins in order to be transported across the nuclear membrane
1. Cargo proteins contain sorting signals called NLSs
2. Two types of NLS: Classical and PY-NLS
3. NLS binds either directly to the receptor or via an adaptor, e.g. Importin α.
4. PY-NLS usually found in proteins which bind to Importin β without an adaptor.
120
How is unidirectional transport of cargo protein-importin complexes maintained across the nuclear membrane during nuclear import?
1. When Importin is bound to cargo, it is able to interact with hydrophobic FG repeats to due to hydrophobic regions being exposed ], allowing it to pass through the NPC.
2. Once inside nucleus, Ran GTPase adds Ran-GTP to importin, resulting in the cargo dissociating.
3. Importin-Ran-GTP leaves through NPC
4. Cytoplasmic RanGAP induces GTP hydrolysis, causing complex to dissociate.
5. Ran-GDP is imported into nucleus by NTF2 protein
6. Chromatin-bound RanGEF induces RanGTP reformation.
121
What stage of the nuclear import of proteins is energy dependent
Energy is required for RanGTPase mediated control
122
What is the the key difference between nuclear export and import by Exportin and Importin respectively?
Key difference is type of binding between Importin/Exportin and RanGTP
122
What is the the key difference between nuclear export and import by Exportin and Importin respectively?
Key difference is type of binding between Importin/Exportin and RanGTP
123
What are the 2 forms of RNA export?
1. Bulk mRNA transport- 95%
2. Ran-dependent RNA transport- 5% (select mRNA, tRNA, small RNAs etc.)
124
What are the stages of bulk mRNA transport?
1. Assembly of an export‐competent mRNA-containing ribonucleoprotein (mRNP) complex
2. Receptor binding and translocation of mRNP through the NPC
3. Dissociation of receptor from the translocated mRNP complex
125
Describe mRNP complex formation
1. 5’ methyl guanosine cap binds (Cap-bind proteins) CBP complex; CBPs promote splicing
2. After splicing Exon junction complex (EJC) and Serine-Arginine (SR) proteins are deposited in the coding region. First EJC and CBC together recruit TREX and Dbp5 DEAD-box RNA helicase.
3. Exon interactome (green) helps folding and packaging the coding region, including ATP-dependent DEAD-box proteins.
4. After 3’ UTR cleavage and polyA synthesis, PolyA tail binding Protein complex ( PABPC) forms
126
After the formation of the mRNP complex, how is mRNA exported from the nucleus via bulk mRNA transport?
1. Diffusion of mRNP from the site of transcription to NPC is a rate limiting step (seconds up to minutes).
2. TREX and SR proteins recruit NXF1-NXT1 receptor ( Nuclear RNA export factor 1/ NTF2-like export factor 1). Interaction with TREX induces the affinity of NXF1 to RNA binding.
3. Docking of mRNP-receptor complex to the nuclear basket
4.Translocation is very quick (~20 ms)
127
How does the nuclear basket act as quality control during bulk mRNA export
If the mRNP-receptor complex binds to the nuclear basket for too long, this indicates a fault with the complex, and is correlated with RNA degradation.
128
Describe Ran-dependent RNA export
1. Uses exportin1/ CRM1 receptor and Ran GTPase
2. CRM1 cannot bind RNA directly, it needs NES containing adaptor proteins
3. Rare and sequence specific- export a subset of mRNAs that encode proteins involved in cell cycle ( cyclin D1), proliferation, survival (MYC), metastasis and invasion
129
Describe nuclear envelope budding as an alternative form of mRNA export
1. RNP granules can exit nucleus by budding
2. Local lamina disassembly allows membrane coat molecules to distort inner nuclear
membrane and induce docking and budding
3.Vesicles in perinuclear space fuse to outer nuclear membrane or ER membrane
130
Describe how pulse-chase analysis can be used to determine how proteins move through a secretory pathway.
1. Radioactively labelled amino acids are added to the tissue- these will be incorporated into proteins- 'pulse'.
2. Add excess of unlabelled amino acids- 'chase'.
3. As radioactive proteins move through cells, the proteins that take their place will be unlabelled due to chase.
4. Prepare tissue for electron microscopy at various times.
In each microscopy image the radioactive proteins can be identified, allowing their progress to be tracked between images
131
What is a polyribosome? How might these inform the folded structure of the the endoplasmic reticulum?
1. Group of ribosomes all translating the same mRNA
2. Folds of ER might serve to increase its surface area to allow polyribosome formation.
132
What were two key findings of early experiments with regards how proteins are selectively delivered to and through the ER membrane?
1. Cytosolic and ER-bound ribosomes are identical and inter-changeable in vitro
2. Discovery of signal peptides in secreted proteins - 1972
133
What key findings of Cesar Milstein and George Brownlee's experiments with Immunoglobulin light chains led to the discovery of the Signal Peptide?
1. IgG light chains translated initially in a higher molecular weight form
2. This is because IgG chains have N-terminal extensions
3. These are enzymatically removed after synthesis
4. Removal requires translation in the presence of microsomes (homogenized ER)
134
Describe the structure of a signal peptide
1. Polar, hydrophilic ends
2. ~20 amino acid hydrophobic region.
3. Hydrophilic/Hydrophobic regions allow peptide to span membrane
135
Describe how the Signal Recognition Particle targets proteins to the ER membrane.
1. ER proteins have a hydrophobic signal peptide of ~20 residues at the N terminus
2. The Signal Recognition Particle (SRP) binds the signal peptide as it emerges from the ribosome and arrests translation
3. The SRP docks the ribosome onto protein translocator in the ER membrane, this opens the pore in the translator.
4. SRP then leaves and translation reinitiates so translocation is co-translational
136
Describe the features of the signal recognition particle and their functions.
1. SRP-54: Recognises signal peptide:
-features methionine bristles
- SRP54 binds ~20aa hydrophobic sequence on signal peptide
- Affinity for SP is relatively low (sub-nM KD)
2. SRP-9 and SRP-14: Arrest translation
- Arrests translation via competition with elongation factor binding on the ribosome
3. SRP-68 and SRP-72: Dock with ER
137
How is the specificity of the Signal Recognition Particle improved via competition with NAC (nascent polypeptide associated complex)?
1. NAC associates with essentially all emerging nascent polypeptides
2. NAC has higher affinity for hydrophilic sequences (usually associated with cytoplasmic proteins) than for Signal Peptides
3. All polypeptides will be bound by one or other complex
4. Competition can increase the fidelity of even weak discrimination
138
What is the translocon and what is its role in transport of proteins across cellular membranes?
1. Universally conserved protein-conducting channel, referred to as the Sec61 channel in eukaryotes and as the SecY channel in prokaryotes.
2. Single copy of the heterotrimeric Sec61 or SecY complex forms the channel, resulting in a narrow pore
3. Positively charged region of signal peptide interacts with negatively charged residues on cytosolic side of pore
3. signal sequence is intercalated into the lateral gate and the following segment passes through the actual pore as a loop. This results in displacement of the plug domain of the channel.
139
How are transmembrane domains defined by the translocon (translocation channel)?
140
How are transmembrane domains incorporated into the membrane by the translocon
Lateral gate opens in the translocon, allowing the TMD to move out into the cytoplasm where it is held via hydrophobic interaction.
141
Beginning in the 1980s, there was a debate as to how proteins exited the endoplasmic reticulum, the two main hypotheses were Bulk Flow and Signal Mediated Export. What were the different assumptions and predictions of these hypotheses?
142
What early evidence for the Bulk Flow hypothesis was discovered during the 1980s?
1. Bacterial proteins can be targeted to the ER of animals, plants, and fungi by addition of a signal peptide (SP)
2. Bacterial proteins which enter the ER are usually exported and secreted
3. It is unlikely that prokaryotic proteins contain any essential export signal, therefore consistent with default bulk flow
143
What early evidence for the Signal-Mediated Export hypothesis was discovered during the 1980s?
1. Not all proteins are secreted at the same rate
2. The rate determining step is export from the ER
3. Proteins are not exported in proportion to their ER abundance
4. Artificially introduced bacterial proteins are exported at the rate of the slowest native proteins:
- slowest rate is the bulk flow rate
- faster rates are signal mediated
144
What were two early candidates for the export signal predicted to exist by the Signal Mediated Export hypothesis, and why were they initially rejected?
1. N-glycans?
- Specific to proteins that enter the ER
- Tunicamycin inhibits all N-linked glycoproteins and blocked export of some glycoproteins
Rejected at the time:
- Some secreted proteins are not glycosylated
- Some ER-resident proteins are glycosylated
- Tunicamycin doesn’t block secretion of all glycoproteins
2. Peptide sequences?
Rejected at the time:
- no obvious uniquely conserved sequence motifs
- mutational studies of secreted proteins failed to identify any common necessary sequence
- many secretion-inhibiting mutations found
- mutations turned out to be too numerous and scattered to define any clear export signals
145
How did the debate surrounding the Signal Export Mediated and Bulk Flow hypotheses become more nuanced over time?
Both now accept that export is the default.
146
Describe how the QC glycocode selectively retains misfolded proteins within the ER/
1. Oligosaccharide transferase N-links a high mannose glycan to asparagine residue in the nascent polypeptide
2. The two terminal glucoses are removed by Glucosidase I and Glucosidase II,
3. Calnexin (CNX) or Calreticulin ((CRT) soluble lumenal form of CNX) bind to remaining glucose (Glu1)
4. Glu1 is removed as the protein folds
5. The rate of dissociation from CNX or CRT governs the export rate
6. UDP-glucose:glycoprotein glucosyltransferase 1 (UGT) selectively adds glucose to unfolded proteins.
7. How does it discriminate?
a. exposure of hydrophobic sequences
b. accessibility of the glycan core
8. Adding of glucose to unfolded causes proteins to continually re-associate with CNX and CRT and be retained
147
Most proteins retained in the ER by the QC glycocode are eventually folded and released, but this sometimes requires the trimming of the mannose core of the N-glycan, reducing it from Man9 to Man8. How does this facilitate export?
Conversion to Man8 reduces the interaction with CRT/CNX and UGT, preventing the re-adding of glucose to the protein
148
Some proteins retained in the ER by the QC glycocode cannot be folded, and are fed into the ERAD pathway to be degraded. Describe the ERAD pathway.
1. ER mannosidase I converts unfolded proteins to Man-8
2. Reduces interaction with CRT/CNX and UGT
3. If the protein is not folded – Man8 glycans become substrates for ER mannosidase-II
4. EDEM = ER Degradation Enhancing Mannosidases
5. Man-7 associates with specific ‘ERAD’ lectins
6. The unfolded protein is fed back through the Sec61 pore
7. It becomes ubiquitinated and targeted to the 26S proteasome
149
Why was Bulk flow believed to be triumphant by the early 1990s?
1. Influential Rothman and Weiland experiment seemed to demonstrate that synthetic glycosylated tripeptides were exported at maximum rate- Bulk flow can account for maximum rates of export so no need for export signals
2. Hugh Pelham and colleagues discover K/HDEL retrieval signal and receptor system for ER resident proteins
3. Different export rates are accounted for by quality control- fast folding proteins leave at fastest rate, this is maximum, bulk-flow, rate. o Slow folding proteins fail to achieve this rate.
150
How did an investigation of cargo protein concentration by Bill Balch call the Bulk flow hypothesis into question?
Bulk flow hypothesis predicts that cargo will be most concentrated in the Golgi apparatus (post-export). Using quantitative immuno-electron microscopy Balch determined cargo was in fact most concentrated at export sites (prior to export).
151
How are ER resident proteins retained?
1. The ER is mainly occupied by resident proteins (>90%) termed ‘reticuloplasmins’
2. Reticuloplasmins interact extensively but loosely with each other
3. Use calcium cross-bridges via Ca2+-binding domains (hence Calnexin and Calreticulin)
4. This reduces their diffusion into COPII vesicles - RETENTION
5. K/HDEL and COPI retrieval is a failsafe that prevents slow leaking-away of residents
152
How did the exclusion model attempt to reconcile Bill Balch's findings regarding cargo concentration with the Bulk Flow hypothesis?
1. Exclusion of cargo by residents may passively ‘squeeze’ them into the void space, including ER export sites.
- cargo molecules do not participate in calcium cross-bridges
2. Accumulate passively in areas depleted of reticuloplasmins. e.g. export sites
3. Further concentrated by CopI retrieval of escapees
4. May apply also to membrane proteins excluded by resident protein complexes
Provides a passive mechanism for the high concentration of cargo proteins around export sites. Removes need for an export signal actively driving cargo to accumulate at export sites.
153
The observations regarding cargo molecule concentration by Bill Balch are, in fact, the result of an active mechanism, the COPII Sec24p receptor system, making them incompatible with the bulk flow hypothesis. Describe the COPII Sec24p receptor system.
1. Sec12p activates Sar1p by exchanging GDP for GTP
2. Sec12p is resident in the ER and ensures Sar1p is only activated there
3. Sar1p-GTP embeds in the membrane
4. The Sec24p/Sec23p component is recruited to the membrane by Sar1p-GTP
5. Membrane cargo protein bind via their cytoplasmic tails
6. Sec13p/Sec31p complex binds to Sec24p/Sec23p to form the coat
154
How were export signals in cargo proteins finally discovered?
1. Trick was to start with membrane cargo with short cytoplasmic tails and look specifically at the tail
2. Numerous such export motifs found
3. Tested by deletion and transplant experiments to reduce and increase export rates, respectively
4. Short, diverse so previously hard to spot
155
What are extracellular vesicles?
1. Intralumenal vesicles formed in the late endosome and expelled through fusion of MVBs with the PM
2. Non-replicating structures delimited by a lipid bilayer
3. Contain cargoes specific to producing cell (RNA, proteins, lipids, metabolites)
4. Membrane proteins retain their plasma membrane topology- the face the same direction as they do on the PM, rather than being inverted like in the early endosome.
5. All living cells across all kingdoms produce and release EVs into the extracellular milieu
156
What early evidence for the existence of extracellular vesicles was unintentionally uncovered by Chargaff and West's research into blood clotting in the 1940s?
1. Discovered that platelet-free plasma exhibited clotting properties
2. Found that after a high- speed centrifugation of 31,000 × g, the plasma’s ability to clot was suppressed significantly
3. Human plasma possesses a coagulation component that sediments into a small pellet during highspeed centrifugation.
4. Had unknowingly isolated EVs
157
How did Ernst Ruska's work with Transmission Electron Microscopy in the 1960s help to build the case for the existence of Extracellular vesicles?
1. Exosomes were directly observed for the first time, and appeared in Micrographs from many organisms.
2. At this stage they were assumed to be inert cellular debris or even artifacts of electron microscopy itself
158
How did Rose Johnstone and Philip Stahl's research into reticulocytes (immature red blood cells) in the 1980s accidentally demonstrate that the release of extracellular vesicles was a regulated process?
1. Reticulocytes contain Transferrin Receptors (TfR) at their PM that bind di-ferric-Transferrins (Tf) via their Extracellular Domain. These receptors are lost during maturation.
2. Both were interested in visualizing the path taken by TfR-Johnstone used fluorescent and radio-labelled TfR, whilst Stahl used radio-labelled Tf
3. Found that prior to maturation, TfR/Tf complex dissociated in the early endosome and components were recycled.
5. Determined that, during maturation, TfR was instead expelled from the cell via an exosome- this demonstrated the release of these vesicles was a regulated process.
159
How did Graça Raposo's research into the Major Histocompatibility Complex Class II prove that extracellular vesicles were biologically active, rather than inert?
1. Demonstrated that EVs could possess MHC Class II proteins on their membranes,
2. Demonstrated MHC Class II proteins were functional in EVs by incubating them with T cells and showing that they stimulated an immune response.
160
What function of extracellular vesicles was demonstrated by papers such as Valadi et al. (2007)?
1. EVs contain sRNAs and mRNAs, many of which are specifically packaged exclusively into exosomes
2. Cells can take up exosomal RNAs produced by other cells- suggests role in cell-to-cell communication.
161
How are RAB5-GTPase (small GTPase) labels added to multivesicular bodies in order to prepare them for transport?
162
How are the RAB5-GTPase labels on multivesicular bodies utilized during their transport?
1. RAB5-GTPase binds to kinesin, tethering the multivesicular body to microtubule.
2. Different RABs specify destination, e.g. RAB27b directs towards plasma membrane.
163
What role is performed by SNARE (Soluble NSF Attachment REceptor) proteins in exosomal trafficking?
1. SNAREs are proteins on the surface of the multivesicular bodies (V-SNAREs) and the interior of the plasma membrane (T-SNAREs).
2. When MVB reaches the plasma membrane T and V-SNAREs form a complex along with SNAP25, tethering the vesicle to the membrane and allowing it to fuse with it.
164
How does the ESCRT-dependent pathway generate intralumenal vesicles in multivesicular bodies?
1.ESCRT-0 binds Ubiquitinated cargo, PtdIns3P and Clathrin
2. ESCRT-I binds to ESCRT-II via TSG101 and recruits ESCRT-III monomers (Vps20, Snf7, Vps24, Vps2)
3. ESCRT-0, ESCRT-I ESCRT-II- cluster cargo into nanodomains at site of vesicle formation
4. ESCRT-3- plays a role in invagination
165
What 4 pathways can generate intralumenal vesicles in multivesicular bodies?
1. ESCRT-dependent pathway
2. Syndecan-Syntenin-ALIX pathway
3. Tetraspanin (TET) pathway
4. Ceramide pathway
166
How does the Syndecan-Syntenin-ALIX pathway generate intralumenal vesicles in multivesicular bodies?
