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BIOL 361 EXAM 2 > Small Regulatory RNAs > Flashcards

Small Regulatory RNAs Flashcards

1
Q

what are 4 reasons c elegans are a good to work with in lab genetic studies?

A

1) Small (~1mm) free-living nematode
2) Self-fertilizing hermaphrodites making it easy to obtain homozygous lines
3) Can easily be grown in the lab
4) Large array of tools and techniques available.

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2
Q

what are 5 reasons why c elegans are good model system?

A

1) Relatively simple anatomy
2) Fate of each cell is known and can be monitored microscopically
3) Easy to generate mutants, easy genetics
4) Easy to manipulate genetically
5) Complete genome

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3
Q

What two mutations in C. elegans lead to repeated early developmental processes (usually found in early larval stages) leading to the absence of adult structures?

A

1) Lin-4 (recessive)
2) Lin-14 (dominant)

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4
Q

What mutation in C.elegans causes the developing larvae to skip early developmental processes leading to a premature differentiation of adult structures?

A

lin-14 (recessive)

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5
Q

what does heterochronic mean?

A

a developmental change in the timing or rate of events, leading to changes in size and shape

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6
Q

from an evolutionary perspective, what can small heterochronic changes do?

A

small heterochronic changes can have profound impacts on morphology

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7
Q

What is the major morphological difference between larval stage 3 and 4 of C. elegans?

A

In larval stage 3 no alae form and seam cells divide where as in stage 4 alae is formed and seam cells stop dividing.

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8
Q

What was deduced to be how LIN-14 acts based on the gain of function and loss of function mutations?

A

LIN-14 must be an activator of early developmental programs (larval stage 3) and the absence of LIN-14 induces the later larval stage 4.

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9
Q

What was determined to be the function of LIN-4 after double mutants were produced?

A

LIN-4 is a repressor of LIN-14, a activator of early developmental programs.

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10
Q

What happens to C. elegans when both LIN-4 and LIN-14 are operating normally?

A

Wild type. Activation of LIN-14 causes early processes and LIN-4 represses LIN-14 allowing progression to the late processes.

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11
Q

What happens to C. elegans when LIN-4 is missing?

A

What happens to C. elegans when LIN-4 is missing?

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12
Q

What happens to C. elegans when LIN-14 is constitutively active?

A

There is a reiteration of early stages

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13
Q

What happens to C. elegans when LIN-14 is constitutively active and LIN-4 is missing?

A

There is a reiteration of early stages

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14
Q

What happens to C. elegans when LIN-14 is missing?

A

The early stage is skipped.

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15
Q

What happens to C. elegans when both LIN-14 and LIN-4 are missing?

A

early stage is skipped

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16
Q

When looking at the F2 segregation’s of C. elegans LIN-4 and LIN-14 mutants what did researchers observe?

A

1:3 segregation of LIN-4 and 3:1 segregation of LIN-14 suggesting they were both single genes and LIN-4 is recessive while LIN-14 is dominant.

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17
Q

When LIN-4 and LIN-14 were mapped what was the recombination frequency to a RFLP marker?

A

0.1%

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18
Q

After finding a marker with a recombination frequency of 0.1% or LIN-4 and LIN-14 what did researchers do to generate a DNA code?

A

Used chromosome walking and complementation of phenotypes to find the gene region

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19
Q

Using chromosome walking and complementation of phenotypes to rescue the LIN-4 mutants how large of a genomic fragment was found sufficient to rescue the phenotype?

A

a 5kb genomic fragment was found sufficient to rescue the LIN-4 phenotype.

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20
Q

What did researchers discovered happened to the lin-4 mutant that causes the mutant phenotype that changes C. elegans developmental patterns when they used a pVT2D as a probe for genomic Southern Blots?

A

Several kb of genomic DNA including large parts of pVT2S are deleted in lin-4

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21
Q

After discovering that a deletion event had occurred and was the source of the lin-4 mutant phenotype in C. elegans, researchers narrowed to a small genomic fragment that was causing the phenotype. What was the size of the fragment needed to rescue the phenotype?

A

0.7kb genomic fragment is sufficient to rescue the lin-4 phenotype.

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22
Q

What type of mutation causes the lin-4 mutant phenotype in C. elegans?

A

lin-4 is a complete null, loss-of-function mutant.

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23
Q

After discovering what caused the lin-4 mutant in C. elegans, researchers wanted to know if there is a transcript corresponding to lin-4. They used pVT2D as a probe to screen a cDNA library, what did they find?

A

They identified only one type of cDNA that hybridized with the probe, but the 0.7kb region containing lin-4 is not part of the mRNA.

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24
Q

Why when researchers identified only one type of cDNA that hybridized with the probe, the 0.7kb region containing lin-4 was not part of the mRNA?

A

The genomic region downstream of the lin-4 fragment contains the splice junction causing lin-4 to be spliced out.

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25
Where did researchers discovered lin-4, the gene that causes mutant developmental phenotype in C. elegans, to reside?
lin-4 resides in an intron of another gene so no mRNA corresponding to lin-4 can be identified.
26
The lin-4 locus contains a few potential open reading frames (ORF) and is highly conserved between Caenorhabditis species, but they contain base pair indels and no putative ORF are conserved even though mutated version can still complement lin-4. What does this suggest about lin-4?
The lin-4 locus does not encode a protein.
27
When the lin-14 gene was map based cloned, what did researchers discover?
It encodes a protein but has no similarity to other known gene and homologs are only present in other nematodes. The protein is localized to the nucleus and has DNA binding activity.
28
What does the lin-14 gene product (protein) do?
lin-14 activates early developmental processes via transcriptional activation.
29
What are the two alterations that occur in the lin-14 mutant alleles and what do they do?
1) Null mutant (complete gene is deleted), this leads to a loss of function mutant 2) A small deletion in the 3' untranslated region leaving the protein coding region perfectly fine leading to a gain of function mutant
30
The structure of the lin-14 gene has some regions in its 3' untranslated regions (UTR) that are conserved across Caenorhabditis species, what region is complementary to lin-4?
There are 7 short imperfect repeats (22 bp) within the conserved UTR regions that are complementary regions also present in the lin-4 locus.
31
What was lin-14 discovered to be?
lin-14 is an activator of early developmental processes acting as a transcription factor regulating growth hormone gene expression.
32
Describe the lin-14 gain-of-function mutation in C. elegans.
This is a dominant gain of function mutation causing lin-14 to be constitutively active but the protein coding region is not mutated instead there is a short deletion in the 3' untranslated regions
33
Describe the function of lin-4 gene.
lin-4 is a repressor of lin-14. The lin-4 locus is located in the intron of another gene and does not encode a protein.
34
There are no large mRNA found corresponding to lin-4 but what was found?
Two very small RNA's that are expressed where the long version of the small RNA's, lin-4L, can form a hairpin structure.
35
What structure does the small RNA lin-4L form?
A hairpin structure.
36
How does lin-14 interact with the small lin-4 RNA?
lin-4 small RNA is complementary to the small (22bp) region on lin-14 3'untranslated region.
37
Does binding of the small lin-4 RNA to lin-14 mRNA degrade lin-14 mRNA or prevent the production of lin-14 protein?
This leads to the inhibition of lin-14 translation (mRNA levels are not affected)
38
What is the 3 step process that leads to the inhibition of lin-14 translation?
1) lin-4 is expressed as a primary transcript and processed to a precursors known as pre-miRNA 2) The pre-miRNA is processed into single stranded RNA and is now known as micro- or miRNA 3) miRNAs binds the 3'-untranslated region of lin-14 leading to inhibition of lin-14 translation.
39
The lin-4/lin-14 model was the first time what was shown?
The first time shown that small RNA's can mediate regulation of translation.
40
When looking at let-7, another 22 bp miRNA heterochronic mutant causing the reiteration of C. elegans larval stages, what did researchers discover?
A single miRNA can regulate translation of multiple target genes.
41
miRNA genes have been identified in many eukaryotic lineages, there are 1424 miRNA genes in humans. How much protein coding genes do they regulate?
Regulate up to 60% of protein coding genes.
42
Why is it believed that the miRNA gene regulation mechanism likely existed in a common ancestor within the animal lineage?
Because at least one miRNA is conserved across all eumetazoa and 34 common miRNA's exist in bilateria.
43
What is the process of creating a miRNA?
a miRNA gene is transcribed by RNA Pol II into a pre-miRNA with a cap and a polyA tail, these ends are cropped off. The transcript is the exported from the nucleus and the pre-miRNA is diced at the terminal loop creating a miRNA-miRNA duplex. this is then degraded into two miRNA which target mRNA and create translation inhibition.
44
What are the four was that miRNA can exist in the genome?
1) solo miRNA genes 2) Clustered miRNA genes 3) Intronic miRNA genes 4) Mirtron genes
45
How are solo miRNA genes processed?
They are transcribed into pre-miRNA and then cropped into pre-miRNA
46
How are clustered miRNA genes processed?
They are transcribed into pre-miRNA and then cropped into pre-miRNA
47
How are intronic miRNA processed?
Either they are transcribed the same as solo miRNA or they are transcribed and the cropped and spliced into pre-miRNA.
48
How are mirtron genes processed?
They are transcribed and then spliced into pre-miRNA
49
When an over-expression construct is introduced into plants it sometimes leads to down-regulation of both trans- and endogenous mRNA, what is this termed?
Post-Transcriptional Gene Silencing (PTGS) or Sense Suppression.
50
When an over-expression construct is introduced into Neutospora crassa (red bread mold) it sometimes leads to down-regulation of both trans- and endogenous mRNA, what is this termed?
quelling
51
Artificial introduction of either sense or antisense RNA into C. elegans results in down-regulation of the corresponding endogenous mRNA levels, what is this termed?
RNA interference (RNAi)
52
Once RNA interference (RNAi) is initiated in one cell of C. elegans what can happen?
The RNAi can spread through the organism and can be inherited to the next generation.
53
What is the 4 step procedure to understanding what is produced by a specific RNA interference (RNAi) event?
1) generate sense and anti-sense RNAs for genes with well characterized mutant phenotypes 2) Purify single stranded RNA's thoroughly 3) Inject each strand separately and both strands together into the body cavities of C. elegans 4) Observe the phenotypes of the next generation (F1)
54
What is the fate of endogenous RNA when RNA interference (RNAi) occurs?
The complete target mRNA is degraded, not just the part targeted by dsRNA used in RNAi.
55
Exogenous application of double stranded RNA leads to what in C. elegans?
This leads to efficient degradation of the corresponding endogenous mRNA.
56
What happens to the complete double stranded RNA when present in a cell?
is cut into ~ 22nt pieces
57
Both RNAi (RNA interference) and miRNA involve 22nt intermediates but the mechanism to disrupt target mRNA's are different, what are they?
1) RNAi leads to target mRNA degradation, but requires perfectly matched RNA's 2) miRNA leads to inhibition of translation of target mRNA's
58
what is ACO?
an enzyme involved in ethylene biosynthesis
59
How was Post-Transcriptional Gene Silencing first observed?
In an experimental phenomena in plants when the introduction of anti-sense constructs lead to a down regulation of an endogenous gene of interest mRNA levels. Also observed that in some cases sense constructs lead to silencing.
60
Why are plants able to produce a post-transcriptional gene silencing effect?
This allows the over-expression of RNA virus derived genes in normally susceptible plants leading to degradation of viral transcripts during infection and enhanced resistance to that virus.
61
What happened when potato virus x (PVX), a single stranded RNA virus, was used to infect Nicotiana benthamiana (tobacco)?
The viral RNA was copied into double stranded RNA (as dsRNA is part of the viral replication cycle) and the dsRNA was cut into 25 nt pieces (siRNA)
62
Small RNA's are post-transcriptional regulators of gene expression, how do they achieve this?
Small RNA (20-25 nt) can lead to translational inhibition or mRNA degradation of complementary target mRNA's
63
What are the origins of long dsRNA's, needed to produce siRNA?
Exogenous origin from RNA viruses, the viral genome (dsRNA) is origin of host generated siRNA's and also their target.
64
What are the three experimental exogenous origins of dsRNA in a cell?
1) dsRNA injection 2) RNAi transgenes (plants or animals) 3) Anti-sense transgenes.
65
What are the three possible endogenous origins of dsRNA?
1) Endogenous anti-sense 2) Duplicated pseudo-genes 3) Aberrant RNA's (lack of CAP or polyA tail)
66
what is RdRP?
RNa dependent RNA polymerase
67
In what organisms is RdRP (RNA dependent RNA polymerase) found?
RdRP is present in plants, fungi, and some animals (nematodes), but not in insects or mammals.
68
What is the primary role of RNA interference (RNAi)
Its primary role appears to be in defense against viruses
69
How does RNAi (RNA interference) disrupt the viral life cycle?
The siRNA's produced from the viral RNA target degradation of any kind of dsRNA present.
70
What are the two novel regulation systems that can be controlled by RNAi (RNA interference)?
1) Target mRNA turnover (siRNA's) 2) Translation from target mRNA (miRNA)
71
How is RNAi (RNA interference) in plant and nematodes long lasting, being inherited to subsequent generations?
It involves an amplification/maintenance loop. The initiated dsRNA is processed into primary siRNA that initiate the degradation of target RNA. This produces aberrant RNA's from the target that are recognized and turned into dsRNA via RNA dependent RNA polymerase (RdRP). This new dsRNA are process to for secondary siRNA's. This keeps going as long as the target RNA's are expressed.
72
Transposable elements (TE) may be beneficial in some cases but are mainly considered deleterious. TE's may be expressed both in sense and anti-sense orientation, why?
non-LTR transposons need to be expressed from an existing promoter and the chance of integration behind a promoter is equal for sense and anti-sense orientation.
73
What does the creation of long dsRNA from transposable elements (TE) result in?
Results in production of endogenous si-RNAs and the degredation of TE-transcripts.
74
What are the two large protein complexes involved in RNAi (RNA interference)?
1) Microprocessor Complex which recognizes dsRNA and generates small RNA's 2) RNA induced Silencing Complex (RISC) which binds small regulator RNA's and guides them to the target for silencing via cleavage or translational inhibition.
75
What is the central component of the microprocessor complex in RNAi (RNA interference)?
dicer
76
What is the central component of the RNA induced Silencing Complex (RISC) in RNAi (RNA interference)?
Argonaute
77
What are the three main domains of the Dicer protein?
1) Two RNaseIII domains 2) PAZ domain 3) Spacer domain
78
What do the two RNaseIII domains do in the Dicer protein?
They form the internal dimer that resembles the homeodimer of RNaseIII from bacteria. They cleave dsRNA producing two-nucleotide overhang at 3' end
79
What does the PAZ domain do in the Dicer protein?
It binds to the 3'-overhand of the dsRNA produced by the two RNaseIII domain and anchors dicer to the end of the dsRNA
80
What does the Spacer domain do in the Dicer protein?
It positions RNaseIII domains ~25 nucleotides from the end and acts as the internal ruler.
81
Dicer is encoded by multiple (2-4) genes and has two isoforms that differ in their specificity. what are the isoforms and what do they bind?
1) DCR-si binds to perfect-match dsRNA (resulting in siRNA) 2) DCR-mi binds to imperfect dsRNA (like pre-miRNA's)
82
what is drosha?
A nucleus localized protein that can be considered a Dicer variant that also contains two RNaseIII domains but lacks the spacer and the PAZ domain.
83
What does Drosha interact with and what does this do?
It interacts with DGCR8 (Pasha) which binds to the base of the pri-pre-miRNA (flanking the stem of the hairpin) and positions Drosha to cleave just upstream of the beginning of the hairpin.
84
Does Drosha cleave in a site-specific manner?
No, isolated Drosha cleaves dsRNA in a unspecific way. It relies of Pasha to guide its activity.
85
In RNA induced Silencing Complex Argonaute is the central component, what does it do?
It obtains the dsRNA generated by dicer in the microprocessor complex and ejects one strand of the dsRNA while the other strand remains bound.
86
What are the three domains of the Argonaute protein (AGO)?
1) PAZ domain 2) MID domain 3) PIWI domain
87
What does the PAZ domain in the Argonaute protein (AGO) do?
It binds the 3'overhand of the dsRNA generated by dicer and positions dsRNA into argonaute.
88
What does the MID domain in the Argonaute protein (AGO) do?
It binds the 5'nucleotide of the guide strand and positions the guide RNA to allows pairing with the target RNA
89
What does the PIWI domain in the Argonaute protein (AGO) do?
it contains RNAseH fold and mediates RNA cleavage, it is active in only some of the AGO classes.
90
A single species of the Argonaute subfamilies may contain up to how many argonaute-like protein encoding genes?
up to 50
91
What is unique about AGO-mi (Argonaute species)
It has lost slicer activity in the PIWI domain and accepts imperfect dsRNA's. It will eject the passenger strand uncut (may require an 'unwindase' to separate the strands) allowing the binding to imperfect targets to form miRNA duplex. It then keeps bound to the target leading to inhibition of translation (mechanism widely unknown)
92
What are the three core protein components of miRNA mediated inhibition of target transcript translation?
1) Drosha/Pasha 2) Dicer-mi (MCR-mi) 3) Argonaute-mi (AGO-mi)
93
What does the si-related class of Argonaute, known as RNA-induced silencing complex (RISC) do?
It binds to target RNA allowing Argonaute-si slicer activity to cleave the target RNA exactly in the middle of the complementary siRNA. Following this RISC is released from the target RNA and can be recycled.
94
what are the two core protein components of siRNA mediated target transcript degradation?
1. DCR-si 2. AGO-si

BIOL 361 EXAM 2 (3 decks)

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