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M2M1 > Non Coding Regulatory RNA > Flashcards

Non Coding Regulatory RNA Flashcards

1
Q

miRNAs

A

up to 30% of genes may be regulated by miRNAs
translational repression of mRNA
mRNA degradation

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2
Q

siRNA (endogenous)

A

cleavage of mRNA
transcriptional silencing / heterochromatin formation
mobile element silencing

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3
Q

piRNA

A

mobile element silencing

transcriptional repression

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4
Q

microRNAs

What do they function in? (2)

A 
function in mRNA degradation 
function in translation repression
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5
Q

microRNAs

what are they derived from?

A

Derived from hairpin precursor

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6
Q

microRNA

imperfect match =

A

RNA degradation or translational repression

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7
Q

microRNA

perfect match =

A

RNA degradation

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8
Q

microRNA

what cleaves pre-miRNA

A

Dicer

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9
Q

Once dicer cleaves the hairpin precursor of micro-RNA…forming the miRNA/miRNA* duplex… what protein does the duplex now interact with

A

Argonaute

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10
Q

After argonaute and micro-RNA duplex is formed, one of the strands is lost… leaving argonaute and the guide strand… what is this now called?

A

The RISC complex (RNA-induced silencing complex)

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11
Q

How are mRNA targets of miRNAs identified in the cell?

A

Base-pairing with target mRNA 3’ UTR serves to guide effector complexes to particular mRNAs

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12
Q

How many binding sites in the 3’ UTR mRNA does a single miRNA have?

A

A single miRNA may have multiple mRNA binding sites in the 3’ UTR for a sinlge small RNA and also several different miRNA target sites may be present within a 3’ UTR

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13
Q

How many target mRNAs does a single miRNA have?

A

a single miRNA may target many different mRNAs

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14
Q

what percent of mammalian genes are regulated by miRNAs

A

approximately 50%

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15
Q

How many miRNAs are though to regulate over 50% of genes?

A

1000

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16
Q

What determines the fate of the miRNA target mRNA

A

Perfect of imperfect base pairing determines the fate of the target mRNA in general
perfect = destruction
impefect = degradation or translational repression

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17
Q

what defines target for miRNA?

A

nucleotides 2-8 (seed region)

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18
Q

can miRNA target other regions… aside from 3’UTR?

A

Yes, miRNAs can also target the coding region or 5’UTR

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19
Q

What is key in regulation of miRNA activity

A

mRNA target site access

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20
Q

Aside from mRNA target site access, what else is key for regulation of miRNA activity?

A

miRNA can also be sequestered by other RNAs including lncRNA and circular RNA

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21
Q

What could limit access of miRNA to mRNA?

A

protein bound to target site - or if the structure is folding up limiting access of the miRNA

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22
Q

mechanisms of miRNA mRNA silencing (2)

A

mRNA decay

Translational repression

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23
Q

how does miRNA silence via mRNA decay?

A

could internally cleave and degrade or could start from either or both ends (decapping and deadenylation)

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24
Q

how does miRNA silence via mRNA translational repression? (2)

A

could repress initiation - block recruitment of the ribosome

could repress elongation - block translation via translation blockers

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25
Q

siRNA … what does it function in?

A

functions in mRNA degradation
functions in chromatin formation
functions in transcriptional silencing
functions in mobile element repression

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26
Q

siRNA - what makes it different from miRNA targeting?

A

siRNA - perfect match with target

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27
Q

what is siRNA derived from?

A

long double stranded RNAs

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28
Q

siRNA is it always exogenous?

A

no!
exogenous (virus/experimental)
endogenous (secondary endo siRNA (C. elegans)

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29
Q

what system does siRNA play a role in ?

A

defense/immunity

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30
Q

siRNA strictly functions in

A

degradation - perfect base pairing

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31
Q

which of the small noncoding RNAs is though to have arisen as a defense mechanism to coutract viruses? What makes this make sense?

A

siRNA
BECAUSE…
Many viruses are dsRNA and siRNA comes from dsRNA :)

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32
Q

where could dsRNA come from (cellularly)?

A

convergent transcription
read through transcription of transposons in inverted orientation
bidirectional transcription
trans interaction between founder and pseudogene
duplicated and inverted pseudogene copies
BASICALLY
Anything that can fold back on itself can be a substrate for dicer

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33
Q

siRNA pathway

A 
dsRNA -->
dsRNA processing via Dicer (leaving 3'OH overhangs)-->
Argonaute (RISC) loading -->
Passenger strand elimination -->
2'O methylation at 3' terminus -->
Target recognition and cleavage
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34
Q

who cleaves hairpin pre-miRNA in the nucleas?

A

DROSHA

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35
Q

pre-miRNA duplex exits nucleas and is bound by who?

A

DICER

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36
Q

What does dicer do to the pre-miRNA duplex?

A

cuts it to yield miRNA/miRNA* duplex which has 2 3’OH nucleotide overhangs

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37
Q

how many nucleotides are miRNA?

A

usually 21 nucleotides

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38
Q

how many nucelotides are siRNA

A

22 nucleotides

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39
Q

what’s secondary siRNA?

A

amplified siRNA in plants and worms

40
Q

Methods of RNAi Knockdown in Mammalian Cells (3)

A

Synthetic molecules
Vector-based expression of shRNA or miRNA
In-vitro synthesized dsRNA
REGARDLESS –> All three methods lead to siRNA in the mammalian cell which can then target mRNA for destruction

41
Q

How long are piRNAs?

A

24-27 nt

42
Q

What is unique about piRNA derivation

A

derived from single stranded precursors

43
Q

What is the job of piRNA?

A

Control mobile elements (uber important in gametogenesis)

44
Q

What are the 4 main groups of protein associated with small RNA silencing?

A

DICER
ARGONAUTE
AUXILLARY PROTEINS IN RISC
DROSHA

45
Q

The ribonuclease of the RNase III family that cleaves miRNA precursor (pre-miRNA) and double-stranded RNA molecules into 21-25 nucleotide long dsRNA with a two-base overhang on the 3’ ends

A

DICER

46
Q

How long of 3’ overhang does DICER leave?

A

2 base

47
Q

RISC component and effector of small RNAs

A

Argonaute protein
Ago
Piwi

48
Q

TRC18 (GW182)

A

Axillary proteins in RISC or other complexes - required for miRNA silencing

49
Q

The RNAse III enzyme that is implicated in processing newly transcribed primary miRNA in the nucleus

A

DROSHA

50
Q

What determines the 5’ and 3’ ends of the Dicer substrate

A

Drosha cleavage

51
Q

So what is the effector in RISC?

A

The argonaute and associated proteins

52
Q

What is the target-er in RISC?

A

The small RNA

53
Q

Are there different argonautes?

A

Yes, there are many different argonautes in the cell, and depending on what argonaute binds to the small rna determines what it is going to do

54
Q

Are all argonautes slicers?

A

No… not all argo have slicer activity

55
Q

Ago2

A

mammalian slicer argonaute

56
Q

Argonaute functions

A

Argonautes with small rna play a role in just about everything - from DNA retention, elimination, silencing, etc.

57
Q

Some comparing and contrasting…
Where does miRNA come from vs.
Where does siRNA come from vs.

A

miRNA is transcribed from the nucleus and then folds back on itself to form hairpin - we need drosha then to cut the hairpin

siRNA is double stranded from the get go - where it comes from can vary - but does not use Drosha because already ds… also uses Dicer

58
Q

What makes Pi-RNA processing unique

A

it is already single stranded so it does not use DICER

59
Q

Main function of miRNA vs. siRNA vs. piRNA

A 
miRNA = reg. mRNA stability and translation...only sometimes perfect match
siRNA = reg transposon and protect from virus... usually perfect match
piRNA = reg. transposon and unknown fx
60
Q

How do we define long non coding RNAs?

A

RNA that is longer than 200 nucleotides but has less than 25 amino acid codons to make a protein (open reading frames less than 25aa)

61
Q

Do lncRNAs exhibit cell type specific expression?

A

yes

62
Q

do lncRNAs exhibit localization of subcellular compartments?

A

yes

63
Q

are lncRNAs associated with human disease

A

yes

64
Q

lncRNAs

Secondary structure?

A

extensive secondary structure through intramolecular base pairing.

65
Q

What is crucial for lncRNAs to assume their biological functions?

A

presence of distinct structural motifs

66
Q

what do lncRNAs regulate?

A

virtually every process in cell

some examples include regulation of skeletal and cardiac muscle development, and of the immune response

67
Q

what kinds of regulatory complexes do lncRNAs form to control gene expression adn function

A

lncRNA - protein
lncRNA - mRNA
lncRNA -miRNA

68
Q

how do lncRNAs modulate genome activity (3)

A

affecting DNA methylation
post-translational histone modification
chromatin structure

69
Q

lncRNAs have domains that function like___

A

proteins

70
Q

4 ways that lncRNAs function

A

subcellular localization signals
molecular scaffold
nucleic acid interaction and targeting
RNA catalysis

71
Q

What allows lncRNA to target a particular region of the genome

A

Base pairing with DNA

*Note that if lncRNA is bound to a protein - this will allow it to carry protein to that region of the genome :)

72
Q

what transcribes lncRNA

A

Pol II

73
Q

lncRNAs are serving key regulatory roles that were previously assumed to be the domain of ______

A

proteins

74
Q

can lncRNAs act as precursors for small RNAs?

A

yes

75
Q

do lncRNAs effect other RNAs?

A

yes, affect the splicing and processing of other RNAs

76
Q

example of lncRNA serving as nuclear scaffold

A

nuclear paraspeckle assembly

lncRNA colocalizes with paraspeckle (a nuclear protein)… if we knockout the lncRNA we don’t see the protein either

77
Q

lncRNA can sequester miRNA

how?

A

well, we know that normally miRNA targets mRNA…However, if our lncRNA also has binding sites for the miRNA, it can bind the miRNA and “Compete” for the miRNA - thus miRNA can no longer block the message

78
Q

lncRNA can regulate via DNA mimicry…

example

A

lncRNA can sequester transcription factors that would normally bind to and transcribe genes…example is GAS5 and the glucocorticoid receptor - GAS5 can bind to and sequester GR by mimicking DNA

79
Q

What’s the advantage to using non-coding reglatory RNA targeted therapeutics over targeting protein?

A

You have to know very specific molecular details of proteins 3D structure to be able to target - but all we need to know for RNA is the sequence

80
Q

what is the advantage of using non coding regulatory RNA over ab?

A

can get inside the cell

81
Q

we also have some mRNA therapeutics…e.g.

A

vaccines

82
Q

4 classes of antisense oligonucleotide (ASO) therapeutics and their mechanism of action

A
  1. RNase H-dependent ASOs
  2. Exon skipping ASOs
  3. siRNAs
  4. Anti-miRs and miRNA mimics
83
Q

What type of molecules are antisense oligonucleotides?

A 
They can be 
DNA
RNA
DNA-RNA hybrids 
or 
Other chemically similar molecules
84
Q

Current limitation of ASO therapy

A

Need to be able to easily target site of action … i.e. liver and eye

85
Q

Exon skipping ASOs

A

Single stranded, chemically modified ASOs that target intron exon junctions (splice sites) or splicing regulatory elements

86
Q

How do exon skipping ASOs work?

A

binding to the target site (splice site usually) inhibits (blocks) splicing at this site and forces the choice of an alternative splice site

87
Q

What is the consequence of exon skipping?

A

changing splice site leads to the translation of an alternative protein isoform that can restore stability or function to the mutated gene product (i.e. Duchenne’s muscular dystrophy)

88
Q

siRNA ASO therpeutics

A

siRNA = dsRNA with an antisense active strand that is exactly complementary to a sequence anywhere in the target mRNA
siRNA is taken up by cytosolic RISC, which ejects one strand, leaving the antisense strand to bind to the target mRNA and mediate it sequence specific cleavage by the RISC RNase Argonaute 2
Once cleaved, the target mRNA is rapidly degraded

89
Q

ASO therapeutics

Anti-miRs

A

oligonucleotides that antagonize microRNA - single standed, chemically modified ASOs that can bind to miRNA and block activity

e.g. because miRNA-122 stabilizes the hepC virus genomic RNA, anti-miRNAs targeting miR-122 are being investigated for treatment

Similarly, if miRNA is repressing something that we don’t want it to, we can introduce a complimentary RNA (anti-miR) that binds it and represses its function

We can also introduce micro-RNA into cell to repress message we don’t want translated

90
Q

ASO therapeutics

miRNA mimics

A

mimic the function of endogenous microRNA
e.g. miRNAs are globally deplete in many cancers with poor prognosis. Therefore, their replacement could be used as a form of cancer therapy

91
Q

How are we getting siRNA into people (3)

A 
lipid nanoparticles (endosomes)
glycoconjucated (endocytosed)
virus (with gene that expresses as RNA that folds back on itself)
92
Q

what targets lipid particle to particular cell?

A

based on what is attached on the outside (ligand)

93
Q

what targets glycoconjugates to specific cell type?

A

the glycoconjugate itself is targeting to specific cell type

94
Q

Problem with viral therapy

A

Once you start it is difficult to stop

95
Q

How do we address the instability of oligonucleotides

A

modify

change phosphodiester to morpholino or other modification so that enzymes cannot target

96
Q

Barriers to siRNA therapeutics (7)

A 
distribution and targeting 
avoiding excretion 
avoiding degradation 
avoiding immune response (we are programmed to respond to dsRNA (viruses!!!))
extravasation 
uptake 
endosomal release

M2M1 (7 decks)

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