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MBG 4110 Final Exam > Non- coding RNA, chromatin, and transposons > Flashcards

Non- coding RNA, chromatin, and transposons Flashcards

1
Q

Explain the process of miRNA biogenesis?

A

IN NUCLEUS
1) RNA Pol II transcribes primary miRNA : miRNA folds to form double stranded region
2) Nuclear double stranded RNA endoribonuclease, Drosha, and double stranded RNA binding protein, DGCR8 bind pri-miRNA double stranded regions
3) Drosha cleaves pri-miRNA which generates a 70 nucleotide pre-miRNA
4) Exportin-5 transports pri-miRNA to the cytoplasm
5) Dicer with double stranded RNA binding protein, TRBP, processes pre-miRNA into a double stranded miRNA with a two base single stranded 3’ end

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2
Q

What does Drosha/ DGRC8 do?

A

Removes hairpins in double stranded RNA

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3
Q

What does Exportin do?

A

Transports processed miRNA into the cytoplasm,

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4
Q

What does DICER do?

A

Cuts the double stranded RNA into small pieces (21-28 bp)

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5
Q

What is the Argonaut (RISC) protein?

A

A RNA helicase
Removes one of the strands of the double stranded RNA
Leads the trimmed siRNA to the target mRNA

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6
Q

What does RISC complex binding cause?

A

Causes the bound mRNPs to associate with P bodies leading to mRNA degradation

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7
Q

How does mRNA translation inhibition occur?

A

miRNA RISC complexes associate with target mRNPs by base pairing between Argonaute bound mature miRNA and complementary 3’ UTR of the target mRNAs
The more RISC complexes bound to the 3’ UTR of mRNA, the greater the repression of translation

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8
Q

How was it discovered that RNA was involved in the control of gene expression?

A

Wanted to make flowers more purple, by adding an extra copy of the anthocyanin gene but they ended up turning white, suggesting that mRNA was not translated
There was loss of activity of both the transgene and the genome gene leading to co-supression

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9
Q

What do lin-4 and let-7 do? How do lin-4 and let-7 work?

A

Lin-4 and Let-7 encode RNA molecules of about 70 bases
Lin-4 and Let-7 form stem and loop structures, which means they form dsRNA
These short temporal RNAS (stRNAs) from the dsRNA bind to the 3’ UTR of many target mRNAs and inhibit translation

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10
Q

What does EGO-1 encode?

A

RdRP: RNA-dependent RNA polymerase

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11
Q

What does DCR-1 encode?

A

DICER: Double stranded RNA endonuclease, which cuts inside RNA to make many small RNA pieces

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12
Q

What does AGO-1 encode?

A

SLICER: targets the siRNA to the mRNA to be silenced

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13
Q

Explain the process of RNA interference

A

1) dsRNA is cleaved to small (21-28 bp) RNA fragments by a dsRNA endonuclease called DICER
2) Small dsRNA binds to RISC to form an inactive RISC complex
3) Argonaut in RISC is a RNA helicase, which unwinds dsRNA, and active RISC is formed
4) RISC guided by a single stranded siRNA, cleaves target mRNA, which is the SLICER activity
5) Amplification of RNAi
–> The cleaved target RNA and RISC are used by RdRNP to produce more RISC- ssRnA to target more long RNA molecules
6) RNAi can inadvertently target RNAs that share short stretches of homology with the main target

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14
Q

How is the dsRNA signal spread and amplified?

A

RISC (argonaut activity) presents the anti-sense ssRNA to a new molecule of the target sense RNA
RdRP uses the siRNA as a primer and the target RNA as a template to make long dsRNA
This long dsRNA is then presented to DICER to continue the cycle
–> Image

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15
Q

What is the random degrative PCR model?

A

Photo

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16
Q

What are the 2 mechanisms for mRNA silencing?

A

1) Degradation
2) Hairpin Precursor

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17
Q

How does the hairpin precursor mechanism for mRNA silencing work?

A

Hairpin Precursor -> stRNA –> RISC like complex –> Translational inhibition
RNA is targeting the translational level

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18
Q

How does the degradation mechanism work for mRNA silencing?

A

dsRNA silencing trigger –> siRNA –> RISC like complex –> Degradation

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19
Q

What is the overall pathway for mRNA silencing?

A

1) dsRNA
2) Then dicer comes in to make siRNA
3) siRNA can either go through mRNA degradation via RISC and PTGS OR
4) Translational inhibition via the formation of stRNA

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20
Q

Where does dsRNA come from?

A

1) Synthetic Transgenes or Transposons
2) RNA viruses: dsRNA genomes or replicate through dsRNA intermediates
3) Failed transcription: prematurely terminated or unprocessed mRNA
4) Bi-directional transcription
5) Normal transcription of long non coding RNA

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21
Q

What can transgenes and transposons do?

A

Their transcription can produce both sense and anti-sense RNA leading to dsRNA

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22
Q

How do transposons or synthetic trangenes insert into the genome?

A

1) Transposon inserts in the introns of two unrelated genes
2)Transposon is inserted between two genes transcribed in opposite directions

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23
Q

What happens when transposons are inserted in the introns of two unrelated genes?

A

When transposons insert in more than one position in the genome, this could produce dsRNA
These unrelated genes are transcribed in opposite directions and eventually produce dsRNA
Both these unrelated genes get inactivated by RNAi amplification

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24
Q

What happens when a transposon is inserted between two genes transcribed in opposite directions?

A

The 3’ UTRs of the RNAs now contain complementary RNA sequences
Both genes get inactivated by RNAi amplification

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25
Q

What does S. pombe has that makes it a good model organism?

A

1) DNA in heterochromatin is methylated
2) Has a homologue of HP1, called SWI1
3) Has a homologue of Su(var)-9 called CLRK
4) Has homologues of AGO, DICER, RdRNP

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26
Q

Why is S. pombe a good model organism?

A

Similar to that of mammals and Drosophila
Has four chromosomes and silenced chromatin

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27
Q

What does the deletion of argonaut, dicer, and RdRNP result in?

A

Accumulation of complementary transcripts from centromeric heterochromatin repeats
This is accompanied by transcription de-repression, loss of histone H3 lysine 9 methylation, and impairment of centromere function

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28
Q

How did the researchers delete argonaut, dicer, and RdRNP?

A

A reporter gene (URA4) is inserted at three different positions next to the centromere, which is the heterochromatic locus
URA4 is silenced by the peri-centric heterochromatin
A screen was performed for genes that compromise the silencing of URA4 at these positions
Screen relieved that mutations in DICER, ARGONAUT, and RDRP polymerase relieve the silencing of URA4

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29
Q

How was URA4 inserted into S. pombe?
How was expression for URA4 measured?

A

URA4 was inserted in the centromere, and the internal right repeat and the outer right repeat
Expression was measured by RT-PCR relative to the expression of a mutant URA4 DS/E gene

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30
Q

Why was URA4 used for the DICER, AGO, and RDRP screen?

A

URA4 confers culture growth of uracil
Strains with peri-centric URA4 have been used to screen for genes that regulate the peri-centric heterochromatin

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31
Q

What is peri-centric DNA?

A

Contains inner repeats and outer repeats
Normally, no mRNA encoding genes in pericentric DNA
Peri-centric DNA repeats are wrapped in heterochromatin

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32
Q

What happened in the genetic screen in which URA4 was inserted in the S. pombe centromere? Be sure to explain wild type and mutation results, but general results not specific.

A

Functional URA4 gene has been inserted at three different positions
In the wild type, URA4 is silenced by peri-centric heterochromatin and the cells cannot grow in the absence of uracil
Mutations in the genes involved in heterochromatin formation will un-silence URA4 and allow growth in absence of uracil
Deletion mutations do not affect the centromere

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33
Q

What is special about DCR1, AGO1, and RDRP1 in the genetic screen with URA4?

A

Dcr1, Ago1 and Rdrp1 were pulled out of the screen which means that these three regulate peri-centric heterochromatin

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34
Q

What did the first experiment in the URA4 genetic screen reveal?

A

There was loss of repression of URA4 by mutations in DCR, RdRP, and AGO

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35
Q

What did the first experiment in the URA4 genetic screen show?

A

URA4 at otr and imr is not expressed in wild type cells
URA4 at otr and imr is expressed in dcr1, rdp1, and ago1 mutant cells
Centromeric silencing is relieved in ago-, dcr-1, and rdrp- mutant strains compared to wildtype
The control is euchromatin, and therefore no mutation expressed URA4 silencing
In the centromere, the control shows URA4 activity, but the centromeric transgene does not.

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36
Q

What did the second experiment in the URA4 genetic screen show?

A

They detected bi-directional transcription at native otr repeats and that transcripts accumulate in dcr1, rdp1, and ago1 mutants

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37
Q

How was the second experiment in the URA4 genetic screen conducted?

A

Strand specific RT- PRC analysis
Samples were incubated with primers from the dh repeats that were complementary to either the forward or reverse transcripts in first strand cDNA synthesis
Strand specific control reacts were conducted using primers specific for act1 sense and act1-anti sense transcript

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38
Q

What did the second experiment results show for the genetic screen of URA4? Be specific

A

No bands, indicated heterochromatin
There is a diagram to help illustrate this

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39
Q

What model was suggested to maintain peri-centric heterochromatin?

A

dsRNA is expressed from peri-centric heterochromatin
This dsRNA is processed by RISC/Dicer/RdRp
This process is necessary for the maintenance of peri-centromeric heterochromatin

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40
Q

What does transcription of peri-centric repeats give rise to?

A

Give rise to long non coding RNAs that are processed into primary small RNAs by Dicer dependent and independent pathways

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41
Q

How does RITS- dependent Gene Silencing work?

A

1) Dicer, RdRP and RITS amplify the dsRNA signal
2) RITS binds to the elongating RNA, transcribed by RNA pol II via the unzipped single stranded RNA
3) RITS binds to RNA via Ago1/siRNA and to the methylated H3K9 via Chp1 (RTS contains a protein called Chp1)
4) RTS communicates with CLRC
5) CLRC is a H3K9 methyl transferase
6) The methylated H3K9 recruits SWI6 (HP1) and provides binding to RITS
7) SWI6 (HP1) initiates the cascade of building heterochromatin and eventually shuts of the synthesis of RNA
8) If heterochromatin is compromised, RNA synthesis resumes and initiates the rebuilding of heterochromatin

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42
Q

Elaborate on the first part of transcriptional gene silencing

A

A small RNA loaded onto the RITS complex targets nascent non coding RNA by base pairing interactions, this leads to the recruitment of RDRC and conversion of the targeted RNA into dsRNA, which is diced into siRNAs by Dicer
RITS also recruits the CLRC H3K9 MT complex to chromatin via interactions with Rik1 subunit of CLRC and Stc1, an adaptor protein

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43
Q

Elaborate on the second part of transcriptional gene silencing

A

H3K9 methylation stabilizes the association of RITS with chromatin and also provides binding sites for HP1 proteins (Swi6 and Chp2).
Swi6 facilitates the recruitment of RDRC and degradation by the exosome

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44
Q

Elaborate on the third part of transcriptional gene silencing

A

Chp2 recruits the SHREC complex containing the Clr3
HDAC promotes TGS by mechanisms that remain to be defined

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45
Q

Elaborate on the fourth part of transcriptional gene silencing

A

In addition to TGS, efficient silencing requires co-transcriptional RNA degradation (CTGS) by RNAi-dependent (A, dicing and slicing) and RNAi-independent (C, TRAMP/exosome degradation) mechanisms. Dicer-independent priRNAs contribute to low levels of H3K9 methylation

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46
Q

What did Barbara McClintock do?

A

Correlated the phenotype of kernels with the structure of chromosomes
Studies led to amazing discoveries:
Some chromosomes always break at the same position
This fragility is caused by a mobile (un-mappable) DNA element
The same “genes” cause hyper-mutability of a known gene
Barbara McClintock found transposable elements

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47
Q

Why did no one believe B. McClintock?

A

Genetics before DNA and gene discovery
Said there were not mutations, but chromosomes are actually moving around, which is not explained by Mendelian Genetics

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48
Q

What famous experiment did Barbara McClintock do?

A

She mapped the C Sh Wx loci on chromosome 9
She could read the genotype of each kernel and score the frequency of recombination between C Sh Wx
She mapped the chromosome in corn kernels and looked at colour, size, and starchy vs waxy phenotypes
Concept of genes not well known at this point

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49
Q

Explain what should be observed for the breaks in chromosome 9

A

If chromosome 9 breaks between C and Sh, there would be a colourless sector in the purple kernel
If it breaks between Sh and Wx, we see a colourless shrunken sector
If it breaks between Wx and the centromere, we will see a colourless shrunken waxy sector

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50
Q

Explain what was observed for the breaks in chromosome 9

A

McClintock discovered peculiar phenotypes:
On a specific chromosome (Chromosome 9S) a frequent breaking point is always at the same position, so that C, Sh and Wx are always lost together
The breaking point was at a locus called Ds (dissociation)
The theory is that chromosome structure is responsible for the observed phenotype, and the phenotype depends where the chromosome breaks

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51
Q

What allele did McClintock discover?

A

If gene C looses activity, we will see a colourless spot on a purple kernel
If a mutant gene C, re-gains activity, you will see a purple spot on a colourless kernel
McClintock discovered a strain with a highly unstable C allele, called Cm (C-mutable)

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52
Q

What else did McClintock discover?

A

It turned out that the phenotypes caused by the Cm and Ds are both dependent on another gene, called Ac, the activator
Barbara attempted to map the Ds and Ac loci, but they seemed un-mappable; they were present at multiple positions and not always at the same position
She proposed that Ds and Ac move around the genome and that Ac is responsible for this mobility, which is why the gene was unmappable

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53
Q

What is the theory regarding the Cm allele?

A

Changes between active and inactive phenotypes, so you often see spotted kennels
Proposed she couldn’t map the genes because they keep moving around

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54
Q

What is an activator? (Ac)

A

Transposon containing a transposase gene

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55
Q

What is a dissociation? (Ds)

A

Defective Ac element (no transposase)

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56
Q

What is a transposase?

A

An enzyme capable of existing and moving an element to a different position
There are 2 mechanisms transposases can use to move transposons

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57
Q

What are the 2 mechanisms transposases can use to move transposons?

A

1) Cut and Paste Mechanism: Cut and paste transposons are flanked by inverted repeats which are targeted by the transposase
2) Copy and Paste Mechanism

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58
Q

What is critical in a transposon?

A

Need Ac and Ds or the transposon cannot move around the genome

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59
Q

Where are transposable elements/transposons found?

A

In genomes of all organisms
In many species, including humans, transposons constitute a large fraction of the total DNA

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60
Q

Why do we have transposons?

A

Transposons are known to harm genome, yet they are tolerated by the genomes which mean transposons have evolved mechanisms to keep them at bay, so transposons contributed to evolution by introducing more genetic diversity

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61
Q

What suppresses transposons?

A

Chromatin Structure

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62
Q

Where are transposes in the genome?

A

Transposases are encoded in one particular location, but can act on any transposon in any location
Can happen all over the genome
Cm: hypermutable locus

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63
Q

How are transposons classified?

A

Cut and Paste Transposons
Copy and Paste Transposons, Retro transposons (LTR transposons)
Retrotransposons (Non LTR)

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64
Q

What are the cut and paste transposons?

A

Move as excised DNA fragments and need transposases
Ac, Ds, Mariner, Sleeping Beauty

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65
Q

What are the LTR retro-transposons?

A

Use copy and paste mechanism
Contain a reverse transcriptase gene, which means transposon is transcribed into mRNA and then made into cDNA, so the cDNA of the transposon is placed in some place on the DNA
Move through an RNA intermediate and insertion via recombination
Have a common origin with retroviruses
Examples: Ty, copia, Gypsy, ERV

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66
Q

What are the non LTR transposons?

A

Move via an RNA intermediate and insertion via a polyA sequence
LINES, SINES,

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67
Q

What are examples of some transposons?

A

Mariner: Inactivated by mutations in transposase gene, 100 million years
Sleeping Beauty: Inactive for 15 million years, Recently re-activated and active in human cells, screen for mutations that cause or suppress cancer
Penelope(Athens): Ancient copy and paste transposons that do not have transposases, but gene similar to telomeres and reverse transcriptase, moves in a different way

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68
Q

What is the transposon invasion?

A

Seen in “ugly” flies
The P+ genotype in Drosophila is a striking developmental disorder that produces progeny with multiple abnormalities (dysgenesis)

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69
Q

How is the P+ phenotype inherited?

A

P+ phenotype is inherited through P+ males when they are mated with naive (P-) females

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70
Q

How does P+ cause dysgenesis?

A

Caused by the activation of a cut and paste transposon (P transposons)

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71
Q

What is the brief history of the P transposon?

A

Invaded flies in the 20th century, and now most flies carry the P- transposon
Naive strains are found in labs, that have established their collections before 1950
P+ is only expressed in males, which cause dysgenesis leading to a wide variety of serious developmental effects
This indicates that transposons can be good for some organisms, but they are capable of de-stabilizing the genome

72
Q

How does the P element promote dysgenesis?

A

This is easiest explained by a diagram found in the notes

73
Q

What are important details on dysgensesis?

A

Drosophila labs trains established before 1950 have No P elements
15% of the genome of wildtype Drosophila consist of P-transponsons
50% of mutant phenotypes are due to transposons
New transposons tend to invade and amplify in the genome and to cause mutations

74
Q

What are important details about why P+ is inherited only via males?

A

P strains carry P elements buy experience no dysgenesis, suggesting that P strains have some repressor of P mobility
This repressor is present in the egg, but not the sperm, which is why the phenotype is transmitted only through males

75
Q

What is seen in the mating of flies with P+ strain?

A

Male (native strain) * Female (P+) : no mobility of P transposons
Male (P+) * Female(native strain): Show dysgenesis

76
Q

What do piRNAs do?

A

Encoded by heterochromatic clusters and induce heterochromatin formation

77
Q

What does P element invasion trigger?

A

Heritable changes in genome structure that appear to enhance transposon silencing

78
Q

Explain Cut and Paste Transposons

A

Cut and Paste transposons share significant homology
When mobile, they tend to insert at positions occupied by other transposons
Multiple insertions generate loci of transpose clusters where transposons can insert in opposite orientations

79
Q

How are transposons silenced?

A

piRNA is synthesized over the clusters of transposons, clustering in heterochromatic regions, a very long RNA over the locus with transposons is produced, making a non-coding RNA
Basically, P elements tend to insert near one another at the same point in the genome, so piRNA is synthesized over these clusters of transposons to induce heterochromatin formation, leading to the silencing of transposons

80
Q

What is a resident transposon? What is the benefit?

A

Reside in the genome, but inactive
The more resident transposons, the better defence there is against the transposons

81
Q

How can double stranded RNA be produced by transposons?

A

Picture to illustrate
Transposon inserted in one loci in one direction (sense RNA)
Transposon inserted in one loci in the opposite direction (anti-sense RNA)
The opposite directions are complementary to each other, and therefore can hybridize, producing double stranded RNA

82
Q

What happens with all cut and paste transposons in the P strains?

A

All cut and paste transposons, including the resident maternal transposons are mobilized in the progeny of crossed P+ males and naive P females
Soon after fertilization the mobility of all transposons is dramatically reduced
The reduction in mobility is dependent on the PIWI proteins and their synthesis of piRNA
This suggests that all cut and paste transposons are surpassed by the same mechanism and involves lncRNAs

83
Q

How was the P+ Drosophila experiment conducted? What was seen?

A

Males carrying P- elements mated with naive females
1) P- elements mobilize, activating resident transposons (which were originally silenced/inactivated transposons)
2) Resident elements transpose into clusters
3) piRNA from cluster insertions trans-silence matching resident elements
The invasion of P elements causes expression of all other transposons, in addition to P elements to become active to produce a dysgenic phenotype
There is a drawing –> Confusing need clarification

84
Q

What does the arrival of P elements cause? How? What happens in the genome?

A

Arrival of P elements in a naive zygote cause a wide spread insertion/removal of all kinds of cute and paste transposon (resident transposons) in the female genomes
P elements express transposases which activate all other transposons in the genome
This is caused by the expression of transposase by the P element
The genome treats these insertions and and removals of DNA fragments as DNA damage and attempts to repair them to suppress the mobility

85
Q

How are sites of DNA repair highlighted?

A

By the phosphorylation of H2A (a key epigenetic mark of chromatin on DNA) damaged DNA
yH2A, yH2Av are symbols of chromatin remodelling associated with DNA repair (phosphorylation)
Phosphorylation of H2A indicates DNA damage repair

86
Q

Explain the results observed in the Drosophila P+ strain experiment regarding RNA?

A

Transposons are mobilized for 2-4 days after P+ males are crossed with naive females, which can be seen by the high expression of P element RNA, repressed by day 21 after fertilization
Transposon mobility coincides with high level of expression of a special class of non-coding RNA from the site of transposons
During these 2-4 days, there is a high peak of RNA not found in wild types, so the hypothesis is that this RNA is responsible for silencing the P element
Lots of RNA production is employed towards the silencing of P elements in females

87
Q

Explain the results observed in the Drosophila P+ strain experiment regarding DNA damage?

A

P+ females crossed with naive males, 2-4 days post fetilization show that a repressor prevents dysgenesis, no DNA damage seen
P+ males crossed with naive females, 2-4 days post fetilization show dysgnesis because there is no repressor for P elements, and as such there is DNA damage
P+ males crossed with naive females, 21 days post fetilization, show no more DNA damage
Naive females do not have P element repressors
P+ males have P elements, cut and paste transposons

88
Q

What are piRNAs? What do they associate with? When are they expressed? Explain what the piRNAs/PIWI proteins do

A

piRNAs are a large class of lncRNA with versatile functions
piRNAs associate with a group of proteins called PIWI
piRNAs are constantly expressed at low levels
piRNA/PIWI proteins are the cytoplasmic repressive factor transmitted through the eggs and not the sperm
piRNAs/PIWI proteins are intercepted by the AGO/RdRP/DICER system to amplify the piRNAs and to pair them with other piRNAs

89
Q

How are piRNAs generated?

A

1) precursor long piRNA is targeted by the PIWI initiator piRNA complex
2) This PIWI initiation piRNA complex (PIWI protein) cleaves and trims at the 3 end of the precursor RNA
3) Remainder of precursor RNA is targeted by additional PIWI proteins without the initiatior piRNAs
4) A new set of initiatior piRNA/PIWI proteins target other long piRNAs to generate a massive defence against all transposons in the genome
These multiple piRNAs/PIWI complexes carry new initiator piRNA sequences complementary to the other transposons in the piRNA clusters throughout the genome
These piRNA/PIWI complexes eventually build up a suppressive heterochromatin in these piRNA loci
There is constant cycling of precursor long piRNA and PIWI proteins

90
Q

Explain the PIWI proteins

A

They cover about 25 bp of DNA
Distinguish between PIWI proteins by how many base pairs they cover, one covers 25 base pairs, and one covers 21 base pairs
PIWI cleaves piRNA into small pieces

91
Q

What is the Ping Pong Cycle?

A

Enhances response to invading transposons and involves piRNA, PIWI and siRNA response

92
Q

How does the Ping Pong Cycle work?

A

Upon the encounter of a transposon RNA, the PIWI/leader piRNA complex detects it and presents it to AGO3/RISC and then to RDRP and DICER to diversify and amplify the counter response to the newly arrived (or inappropriately activated transposons)
piRNAs from transpose clusters are constantly expressed at low levels
piRNAs are processed by PIWI proteins, AUB and ZUC, into leader piRNAs as previously described

93
Q

What does a piRNA cluster do?

A

Keeps producing piRNA precursor

94
Q

How does PIWI/RITS direct silencing of the transposon locus?

A

The goal is to generate non coding piRNA to suppress expression of transposons
The loci containing transposons are identified by the Ping Pong System
1) AGO3/RISC and AGO/RITS target the synthesized piRNA in the transposon cluster using other piRNAs to anneal and to present them to RdRp and Dicer to diversity and amplify the counter response to the newly arrived piRNAs from transposon clusters
The non coding RNA is transcribed over the locus we want to shut off

95
Q

What does AGO3/RITS do?

A

AGO3/RITS recruit heterochromatin factors that repress transposons
If the locus gets de-repressed, piRNA is generated that represses it back

96
Q

What happens in the nucleus?

A

HP1 is the reader and SUV3,9 methylates H3K9, which then becomes a suppressor for transposons

97
Q

What happens in the nucleus?

A

HP1 is the reader and SUV3,9 methylates H3K9, which then becomes a suppressor for transposons

98
Q

What is important to know at the Flamenco locus?

A

PIWI/AGO3 guide RNAs recognize the transcribed RNAs at the locus and recruit H3K9MT and LSD1- H3K4me demethylases
H3K4me indicates active transcription

99
Q

What is the Flamenco locus?

A

Major source of piRNAs in the follicle cells of Drosophila
Flam has a promoter that is decorated with H3K4me3 marks and is controlled by transcription factor CI
RNA pol II transcribes the flam locus, and the resulting RNA is capped and shows splicing signatures
The body of flam is marked with H3K9me3

100
Q

What does the FLAM locus have?

A

H3K4me3, which activates transcription, pol II (Promoter)
H3K9me3, which supresseses transcription

101
Q

Explain the biogenesis of piRNAs (2 mechanisms)

A

Following the export of precursor piRNAs from the nucleus, piRNA biogenesis is initiated through Ping Pong Loop
1) Precursor transcripts are recognized and cleaved by Ago3 in a complex with a trigger piRNA
2) This silencing event gives rise to a responder piRNA which is loaded into Aub
3) Remaining 3’ portion of cleaved transcript serves as a substrate for further piRNA production
Endonuclease Zuc, will generate trail piRNAs approximately every 25 nucleotides

1) The loading of piRNA depends on Shu and Hsp90, this causes a conformational change in Piwi, allowing its nuclear translocation
2) Hen1 methylates 3’ end of mature Piwi loaded piRNAs prior to import to the nucleus

102
Q

What are the main steps of the Ping Pong Cycle?

A

1) Aub bound to anti-sense piRNA recognizes and cleaves a transposon mRNA
2) Resulting product is converted into a new sense piRNA that associates with Ago3
3) Ago3 associated with sense piRNA can recognize and cleave cluster transcriptions
4) The product of this splicing event re-initiates the cycle, becoming an Aub bound piRNA and the remaining 3’ splicing product is processed into PIWI loaded piRNAs via Zuc mediated biogenesis

103
Q

Explain what H3K4me2 and H3K9me3 do in a locus

A

H3K4me2 is the promoter and methylation is reduced here. LSD1 reduces the methylation of H3K4me3, converting into H3K4me2, where it can be a promoter an activate transcription
H3K9me3 is the transposon body and SUV 3,9 methylates H3K9.
H3K9me3 is increased in the transposon body by SUV 3,9 and there is a build up of heterochromatin here, repressing transcription

104
Q

What is important about transposons?

A

Eukaryotic genomes have massive transposons in the genome
In humans, transposons comprise 40% of the genome
Sometimes, active transposons can become active or cross a species border and cause genetic disorder and disease, like P elements
To prevent further activation of transposons, transposons are kept silenced by heterochromatin
Under certain circumstances transposons are mobilized to fulfil yet unknown functions

105
Q

How is dsRNA processed in S. pombe and A. thaliana?

A

dsRNA is processed by the RNAi machinery and AGO- RITS activate H3K9- methyl transferases rebuild heterochromatin

106
Q

How is dsRNA processed in Drosophila, mice, and humans?

A

dsRNA is processed by a different system including PIWI (MIWI, HIWI, MILI, HILI) proteins that communicate with AGO- RITS like complexes
PIWI activity is triggered by a special class of RNA called piRNA and activate H3K9 methyl transferases to rebuild heterochromatin
piRNA and PIWI/MILI linked processes are critical for the suppression of transposons

107
Q

What do PIWI/MILI like proteins do?

A

They activate a PRC like H3K27 methyl transferase to instate the H3K27 epigenetic mark
H3K27 is a heterochromatin protein
Enzymes carry Enhancer of Zeste (EZ) or Enhancer of Zeste Homologue (EZH)

108
Q

What are the Mice and Human PIWI proteins?

A

Mice: MIWI
Humans: HIWI

109
Q

What do mutations in PRC2 cause?

A

PRC2 mutations were identified in Drosophila
They cause a peculiar phenotype where the morphology of many organs and features resemble something else, hence polycomb
Phenotypes produced by PRC genes indicate a flawed development and reiterate the significance of gene silencing for development, health and many other key processes in multi cellular organisms

110
Q

What are the variations and similarities of the Ago/RITS mechanisms? (S. pombe, plant, Drosophila, mouse)

A

S. pombe: Ago1
Plant: Ago4
Drosophila: Piwi and gene repression through H3K9me and SUV3,9
Mouse: MILI, MIWI2

111
Q

Compare miRNA and piRNA processing in various species?

A

First of all, miRNA and piRNA are classified as short interfering RNAs
Majority of PIWI, MIWI and HIWI proteins target transposons
Human RISC: miRNA –> AGO1-4, esiRNA –> AGO1-4, piRNA –> HIWI2, PIWIL3, HILI
Mouse RISC: miRNA –> AGO1-4, esiRNA –> AGO1-4, piRNA –> MILI, MIWI2
Drosophila RISC: miRNA –> AGO1, esiRNA –> AGO2, piRNA –> Aub, AGO3

112
Q

How can AGO and PIWI be distinguished?

A

AGO binds on 21-22 base pairs
PIWI binds on 23-25 base pairs

113
Q

What else does PIWI/piRNA contribute too?

A

Oocyte development, brain function, wound healing, metabolic functions, muscle and tissue regeneration, tissue morphogenesis, sex determination, organogenesis, embryogenesis, spermatogenesis
Regulatory System: IMPORTANT
When PIWI proteins are mutated, lots of things can be affected

114
Q

How is non coding RNA processed? Why is it important for non coding RNA to be processed?

A

Processing of non coding RNA communicates with epigenetic machinery (readers, writers, erasers) to set up and maintain heterochromatin structures
These functions are mediated by a various classes of proteins that directly interact with these RNAs: AGO, PRC2, SUV39, LSD1, SET, and SET2

115
Q

What does PRC2 do?

A

Methylate H3K27
It is the major enzyme working in heterochromatin formation, therefore associated with heterochromatin

116
Q

What does LSD1 do?

A

Demethylates H3K9

117
Q

What does SET1 and SET2 do?

A

Methylate H3K4
Act as HATS

118
Q

What is an LTR?

A

LTR: Long terminal repeat (“junk DNA”)
Most of our genome is junk DNA

119
Q

What do both mechanisms for copy and paste transposons have in common?

A

Both have RNA intermediates but use different mechanisms

120
Q

Explain how an LTR retrotransposon, the Ty1 element of budding yeast gets inserted into our DNA

A

1) The Ty1 element is transcribed to produce RNA
2) The RNA is reverse transcribed into DNA by an enzyme encoded by the TyB gene, which is a reverse transcriptase enzyme
3) The DNA is inserted into a chromosome, creating a new copy of the Ty1 element

121
Q

What does an LTR contain?

A

An LTR contains promoters

122
Q

What are Ty elements?

A

Ty elements are copy and paste transposons in yeast
They are present in telomeres, where they are suppressed and they encode for 2 proteins:
TyA: structural protein
TyB: reverse transcriptase enzyme
When reverse transcriptase is producing DNA from RNA, it requires a primer, so LTR transposons use tRNA with correct sequence bound to mRNA

123
Q

Explain LTRs and how the Ty and solo LTRs are excised

A

This is how transposons with LTRs move
The flanking LTRs are about 100-300 base pairs long and are homologous
They can recombine and form a Holiday junction
The resolution of the junction will expel the ORFs
A single LTR will remain in the genome
These are Called solo LTR or omega elements
More than 500 solo LTRs are found in the yeast genome
Similar remnants of LTR transposons are found in other eukaryotes

124
Q

What is the process for the excision of Ty and solo-LTRs

A

1) DNA with an LTR transposon
2) Homologous (can hybridize)
3) LTR can combine and there will be homologous recombination
4) Then the solo LTR is inserted, expelled, and then remnant remains
Homologous recombination allows the LTRs to move
There is a figure to help explain this

125
Q

What is LINE1 in humans? How does it move within the genome?

A

LINE1 is a non LTR retrotransposon in humans
LINES are transposons that do not have LTRS
LINEs encode a polyAtail which finds the stretch of the genome with a polyT stretch, which it uses as a primer for reverse transcriptase
Basically, LINES encode polyAtails which they use as a primer to move

126
Q

How are non LTR transposons expressed?

A

Non-LTR transposons are expressed as RNAs with an encoded polyAtail
The polyAtail anneals to a polyTstetch in the genome and inserts the LINE1 via the reverse transcriptase as the polyTstretch, which acts as the primer

127
Q

Explain the process of mobility of LINE1 in humans
There is a diagram to explain this process

A

NOTE: LINES and SINES have different mobility
1) A complete L1 element inserted into a chromosome is transcribed into L1 RNA
2) The L1 RNA is polyadenylated in the nucleus
3) The polyadenylated L1 RNA moves into the cytoplasm
4) The L1 RNA is translated into two polypeptides corresponding to each of its ORFs. These polypeptides remain associated with the L1 RNA
5) The L1 RNA and its associated polypeptides moved into the nucleus
6) The ORF2 polypeptide nicks one strand of a chromosomal DNA molecule, and the 3’ end of the polyAtail on the L1 RNA is juxtaposed to the 5’ side of the nicked DNA
7) The ORF2 polypeptide exercises its reverse transcriptase function to synthesize a single strand of DNA using the L1 RNA as a template. The 3’ end of the nicked chromosomal DNA serves as the primer for this DNA synthesis
8) The newly synthesized single strand of DNA swings into place between the two sides of the nicked chromosomal DNA. Simultaneously, the L1 RNA is eliminated, and the other strand of chromosomal DNA is nicked to allow for synthesis of a second strand of DNA, complementary to the L1 sequence. All the nicks are repaired to link the newly inserted L1 element to the chromosomal DNA

128
Q

What does reverse transcriptase do?

A

Reverse transcriptase cuts a site with a polyT stretch, which becomes a primer for reverse transcriptase

129
Q

What does the ORF2 polypeptide do?

A

ORF2 polypeptide proteins bring mRNA into nucleus

130
Q

What is the difference between LINES and SINES?

A

LINES and SINES have different mobility

131
Q

Why is LINE1 important?

A

LINE1 mobility is important in early development and is expressed in early development that will be silenced in later development by heterochromatin and LINe1 is helping to establish heterochromatin in some locations

132
Q

What does TALE-L1-VP64 show?

A

Prevents LINE1 silencing after 2 days
The development rate is low, and the chromatin is decondensed

133
Q

What does TALE- L1-DEL show?

A

Decondenses chromatin, does not activate transcription
Developmental rate is delayed

134
Q

What does TALE- L1- KRAB show?

A

Premature silencing of LINE-1 at the zygote stage
Decondenses chromatin, does not activate transcription
Developmental rate is low

135
Q

What happens to promoters of unwanted RNA?

A

Promoters of unwanted RNA are silenced once detected by the cell, it is a negative feedback loop (LINE is unwanted RNA)
But, if the cell is cancerous or something else is wrong with it then you will see LINE expression
There is a diagram to show mechanisms to ensure LINE1 is silenced, because LINE1 needs to be silenced

136
Q

What does MDAS do?

A

1) Senses endogenous dsRNA
2) Initiates immune response
3) Binds to the LINE-1 5’ UTR
4) Supresses its promoter activity

137
Q

What is suppressing Ty transposon mobility in yeast?

A

S. cerevisiae has 51 Ty retrotransposons
Most of them are positioned in the sub-telomeric regions
The expression of Ty RNA is suppressed by heterochromatin
Elevated expression of Ty elements is observed upon ageing and stress
Expressed transposons tent to insert into solo-LTR remnants in the genome

138
Q

What is repressing LINE mobility in mammals?

A

Humans have about 516000 LINES
Most LINEs and SINEs are mutated and are inactive
Only a very small number (80-100) are active in humans
These LINEs and SINEs are mostly heterochromatic and inert in somatic cells, but are mobile in early embryos and stem cells

139
Q

What is suppressing cut and paste transposon mobility?

A

The majority of cut and paste transposons are nutated and do not move
They reside in large clusters in the genome
These clusters are wrapped in heterochromatin
These clusters are needed to inactivate newly arriving transposons

140
Q

What are long non coding RNAs?

A

lncRNs are non coding RNAs 200 bases or longer
lncRNA can be tens of thousands bases long
piRNA is also a long non coding RNA

141
Q

What are the functions of lncRNAs?

A

Formation of heterochromatin
Supression of transposon mobility
Various functions in regulation of gene expression

142
Q

Explain Convergent (bi-directional) transcription

A

Transposon or transgene insertion
If a transposon (or transgene) is inserted between two genes that are transcribed in opposite directions, dsRNA is produced between the two genes (this can also apply in any situation of convergent transcription)
Transposons are inserted between two genes in opposite directions
Bi-directional transcription is causing the set up of heterochromatin through a process similar to the one at the centromere of S. pombe (RITS, CLK, SWI1, Shrek, etc…)
dsRNA will also be processed via DICER, RISC, RdRp to target other RNAs that contain the sequences complementary to the ones in the region of convergent transcription –> this process has major significance in the suppression of mobile genetic elements (transposons)

143
Q

Explain R Loops

A

R loops mop a promoter or enhancer and re-settle chromatin structures
Basically, R loops get rid of promoter or enhancers of transposons

144
Q

What does the synthesis of lncRNA do?

A

Synthesis of lncRNA over promoters or enhancers can inactivate these elements
The lncRNA has no function, but it is the process of its transcription that is important
Transcription can over run a promoter or an enhancer and displace transcriptional activators
lncRNA also gets rid of transcription factors
Transcription of the lncRNA can also lead to the restructuring of chromatin in these regions, either heterochromatin or chromatin that in refractory to binding of activators

145
Q

What happens in FLO11?

A

Mopping of the promoter of FLO11 in S. cerevisiae in preparation for heater chromatinization and gene silencing

146
Q

What is FLO11?

A

FLO11 is encoding a cell surface leptin type of protein

147
Q

Where is FLO11 expressed? Where it is found?

A

FLO11 is expressed in a subset of cells, and similarly to genes in sub-telomeres, it occasionally converts to a repressed state and remains repressed for multiple generations
FLO11 is close to the sub-telomeres, but not in the subtelomeres, it is about 40 bp away from sub-telomeres

148
Q

If FLO11 is not in the sub-telomeres, why does it switch between on and off?

A

If FLO11 is not in the sub-telomeres, it should not have position affection variegation phenotype, but it still does and switches between on an off as if it is variegated and this is due to the long non coding RNAs, so it acts as if it is variegated, but it really is not

149
Q

What regulates FLO11’s expression between an on and off state?

A

Its alternative expression is regulated by the synthesis of two lncRNAs called PWR1 and ICR1

150
Q

Why does this variegation phenotype appear in FLO11 even though it is not in sub-telomeres?

A

Variegation phenotype happens because of lncRNAs that are encoded upstream of FLO11

151
Q

What is FLO11 responsible for?

A

FLO11 is a gene responsible for coagulation phenotype in yeast, which causes the cells to clump together, and this responds to external environments

152
Q

What is the process of promoter mopping of FLO11?

A

1) Several transcriptional activators and repressors bind to the promoter of FLO11 and regulate its expression in response to various signals
2) Two lncRNAs, PWR1, and ICR1, are encoded upstream of the FLO11 promoter
3) The expression of the anti-sense PWR1 represses the expression of the sense ICR1. Flo8p stimulates the expression of PWR1, Rdp3p, which represses the expression of ICR1
4) Occasional alternative binding of Sfl1p leads to the displacement of Flo8p and Rdp3p leads to the repression of PWR1 and the expression of ICR1
5) The transcription of ICR1 displaces the transcriptional activators and repressors. ICR1 produces an R loop, which removes any transcription factors and recruits factors to induce formation of heterochromatin over the FLO11 locus

153
Q

Overall, what happens when the promoter of FLO11 is mopped?

A

Activators and/or repressors bind to FLO11 promoter to activate/deactivate in the gene
Upstream of the promoter the 2 lncRNAs are encoded, PWR1 and ICR1

154
Q

In the process of mopping the promoter in FLO11 what are the factors?

A

Rpd3p: Histone deacetylase prevents expression of ICR1, silences ICR1
ICR1: Sense
PWR1: Anti-sense
When PWR1 is expressed, ICR1is not expressed
FLO11 is the promoter, which is active or inactive
Slf1: A transcription factor, when Sfl1 is bound, PWR1 is not expressed, and IRC1 is expressed
IRC1 produces an R loop and removes any transcription factors and recruits factors to induce formation of heterochromatin over the FLO11 locus.

154
Q

In the process of mopping the promoter in FLO11 what are the factors?

A

Rpd3p: Histone deacetylase prevents expression of ICR1, silences ICR1
ICR1: Sense
PWR1: Anti-sense
When PWR1 is expressed, ICR1is not expressed
FLO11 is the promoter, which is active or inactive
Slf1: A transcription factor, when Sfl1 is bound, PWR1 is not expressed, and IRC1 is expressed
IRC1 produces an R loop and removes any transcription factors and recruits factors to induce formation of heterochromatin over the FLO11 locus.

155
Q

How is the silencing of E2F target genes in senescing human cells regulated?

A

1) Early in G1, pRB binds the E2F family of transcription factors
2) The bound E2F proteins are unable to stimulate transcription of their target genes
3) Cyclin/CDK complexes phosphorylate pRB when transcription needs to occur, which is after origin firing, so the cyclin/CDK complexes will phosphorylate pRB after origin firing
Cyclins will come in and phosphorylate pRB proteins when the cell is about to go through the cell cycle
4) Phosphorylated pRB releases the bound E2F proteins, which activate their target genes
5) Proteins encoded by the targets of the E2F transcription factors are involved in cell cycle progression

156
Q

How is the role of pRB in the progression of cell cycle regulated?

A

Through pRB’s negative interaction with E2F transcription factors, pRB stalls the cell in G1 phase
Phosphorylation of pRB by the cyclin/ CDK complexes frees E2F proteins to activate their target genes, which encode proteins that are instrumental in moving the cell past the START checkpoint into the S phase
Once E2F is active, by the phosphorylation of E2F, the cell will go through the cell cycle

157
Q

How is piRNA synthesized in the cell cycle?

A

piRNA is synthesized over the promoters of the E2F target genes
The synthesis of piRNA occludes the promoter
The piRNA/AGO2 complex recruits histone methyl transferases (HMTs) and deacetylases (HDACs)

158
Q

Why is piRNA synthesized over the promoters?

A

piRNA is synthesized over the promoters to induce the formation of heterochromatin and to prevent expression of genes that would allow the cell to continue through the cell cycle

159
Q

What can lncRNAs do?

A

They provide a scaffold for the formation of higher order RNA complexes

160
Q

What are higher order RNA complexes roles?

A

1) Formation of chromatin loops in conjunction with CTCF
2) Bridging enhancer-promoter complexes
3) Recruiting an epigenetic modifier
Two examples: HOTAIR and TERRA

161
Q

What is HOTAIR?

A

2000 base lncRNA expressed from the HOX C locus

162
Q

What is HOTAIR required for?

A

Required for the silencing of the HOX-C locus in differentiated somatic cells

163
Q

What is the importance of HOTAIR and its expression?

A

HOTAIR expression correlates with the formation of many cancers
HOTAIR is a lncRNA that is expressed in early development and needs to be shut off in differentiated somatic cells

164
Q

What does HOTAIR RNA form? What does it interact with?

A

HOTAIR RNA forms extensive secondary stem loop structures
One of the loops targets the DNA in the promoters of early developmental genes
Two stem loops interact with modifiers of chromatin: PRC2 and LSD1/REST

165
Q

What is PRC2 and what is it built of?

A

PRC2 is built of SUZ12, EZH and EED
Writer of trimethylating H3K27
H3K27me3 is a major heterochromatin mark, associated with inactive transcription
PRC2 methylates H3K27 (trimethylates)

166
Q

What is LSD1/REST?

A

It is a H3K4 demethylase
H3K4me is a histone mark associated with active transcription
IS a euchromatin mark

167
Q

What does HOTAIR do?

A

HOTAIR RNA represses gene promoter activity (LSD1) and generates heterochromatin (PRC2)

168
Q

What is TERRA?

A

TERRA is a very long lncRNA expressed from sub-telomeres in many eukaryotes
Heterochromatic, so TERRA is heterochromatinized
Therefore plays a role in silencing telomeres

169
Q

Why is TERRA required?

A

Required for the silencing of subtelomeric genes
Necessary for telomere production and elongation

170
Q

Where is TERRA found?

A

In the sub-telomeres

171
Q

What is important about TERRA’s expression?

A

The over-expression correlates with the formation of many cancers
Do not want TERRA to be expressed, but need TERRA to keep TERRA locus silenced, so it works both ways
Works very similarly to HOTAIR

172
Q

What does TERRA associate with?

A

TERRA associates with and unwinds subtelomeric DNA to expose G4 sequences and potentially causes pausing of replication forks
TERRA associates with LSD1/REST and SUV39H
When TERRA associates with G4 sequences, it recruits factors to create heterochromatin, which are the factors responsible for fork pausing and/or fork arrest in subtelomeres
REMEMBER: SUV39H is a histone methyl-transferase for H3K9 and LSD1/REST is a H3-K4 demethylases

173
Q

What does TERRA produce?

A

STEM loop structures

174
Q

What does TERRA do?

A

Represses promoter activity and generates heterochromatin

MBG 4110 Final Exam (3 decks)

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