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PHAR211 > Gene Silencing > Flashcards

Gene Silencing Flashcards

1
Q

What is RNAi?

A

RNA interference is an accurate and potent gene silencing method
silences RNA by breaking it down or stopping translation

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2
Q

What is a key requirement of RNAi?

A

dsRNA

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3
Q

What are the different types of silencing RNAs?

A

small interfering RNAs (siRNAs)
microRNAs (miRNAs)
shRNA
CRISPR

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4
Q

What is the key enzyme in RNAi?

A

DICER
-long dsRNA “diced” into small fragments

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5
Q

What is the role of RNAi in the cell?

A

RNAi is a natural cellular process that silences gene expression by promoting the degradation of mRNA
it plays an important role in gene regulation and innate defense against invading viruses

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6
Q

Where does long dsRNA come from?

A

following sources:
-hairpin
-complementary RNAs
-RNA dependent RNA polymerases

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7
Q

What is the role of dicer in the siRNA pathway?

A

dsRNA is processed by specialized ribonucleases enzyme named dicer in the cytoplasm into a smaller dsRNA molecule
-this short dsRNA molecule is known as the siRNA, which has 21-23 nucleotides with 3’ two-nucleotide overhangs
cleaves long dsRNA into siRNA 21-23nt dsRNA

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8
Q

What happens when siRNA interacts with dicer?

A

activates the RNA-induced silencing complex (RISC)
the endonuclease argonaute 2 (AGO2) component of the RISC cleaves the passenger strand (sense strand) of the siRNA while the guide strand (antisense strand) remains associated with the RISC
subsequently the guide strand guides the active RISC to its target mRNA for cleavage by AGO2

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9
Q

What does the guide strand of siRNA bind to?

A

only binds to mRNA that is fully complementary to it
-siRNA causes specific gene silencing

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10
Q

What is siRNA?

A

small interfering RNAs
bind to argonaute proteins

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11
Q

What happens once siRNA binds to RISC?

A

one strand of the dsRNA is removed, leaving the remaining strand available to bind to messenger RNA target sequences
once bound, the argonaute protein can either cleave the targeted messenger RNA - destroying it, or recruit accessory factors to regulate the target sequence in other ways

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12
Q

What is miRNA?

A

class of small RNA molecules that negatively regulate gene expression

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13
Q

How is miRNA gene transcription carried out?

A

by RNA polymerase in the nucleus to give pri-miRNA with a double-stranded stem-loop structure

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14
Q

How is pre-miRNA formed?

A

pri-miRNA is cleaved by a microprocessor complex comprising of Drosha to form pre-miRNA which is a duplex that contains 70-100 nucleotides with interspaced mismatches and adopts a loop structure

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15
Q

What is the role of Exportin 5?

A

transports pre-miRNA from nucleus to cytoplasm, where it is further processed by dicer into a miRNA duplex of 18-25 nucleotides
miRNA duplex then associates with RISC forming a complex called miRISC

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16
Q

What happens when miRNA duplex is unwound?

A

releases and discards the passenger strand
-unlike in processing of siRNA, in which AGO2 of the RISC causes cleavage of the passenger strand of siRNA

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17
Q

What does the mature single-stranded miRNA do?

A

guides the miRISC to the target mRNAs
the miRNA binds to the target mRNAs through partial complementary base pairing with the consequence that the target gene silencing occurs via translational repression, degradation, and/or cleavage

18
Q

Differentiate siRNA and miRNA based on target recognition.

A

siRNA is usually fully complementary to the coding region of its target mRNA
miRNA is partially complementary to its target miRNA

19
Q

How many targets does miRNA have?

A

one miRNA strand can recognize an array of mRNAs and hence miRNA has the characteristic of having multiple targets

20
Q

Describe RNAi therapeutics.

A

can overcome the major limitation of traditional small drug molecules, which can only target certain classes of proteins
even for protein-based drugs including mabs that are highly specific, their targets are mainly limited to cell-surface receptors or circulating proteins
siRNAs and miRNAs can downregulate the expression of virtually all genes and their mRNA transcripts

21
Q

Differentiate siRNA and miRNA.

A

prior to dicer processing:
-siRNA: dsRNA that contains 30 to over 100 nucleotides
-miRNA: pre-miRNA contains 70-100 nucleotides with interspersed mismatches and hairpin structure
structure:
-siRNA: 21-23 nucleotide RNA duplex with 2 nucleotides 3’ overhang
-miRNA: 19-25 nucleotide RNA duplex with 2 nucleotides 3’ overhang
complementary:
-siRNA: fully complementary to mRNA
-miRNA: partially complementary to mRNA, typically targeting the 3’ untranslated region of mRNA
mRNA target:
-siRNA: one
-miRNA: multiple
mechanism of gene regulation:
-siRNA: endonucleolytic cleavage of mRNA
-miRNA: translational repression, degradation of mRNA, endonucleolytic cleavage of mRNA
clinical applications:’
-siRNA: therapeutic
-miRNA: drug target therapeutic agent, diagnostic and biomarker tool

22
Q

What are RNAs extremely vulnerable to?

A

serum nucleases

23
Q

What is the half-life in vivo of RNAs?

A

short
-although dsRNA is more resistant to nuclease degradation than ssRNA, naked RNAs in their unmodified forms are degraded rapidly following administration by nucleases in bloodstream

24
Q

What is one of the major obstacles of successful application of siRNAs and miRNAs as therapeutic agents?

A

stability
poor delivery

25
Q

What is the solution to stability issues of siRNA and miRNA?

A

chemical modification of RNA

26
Q

Are siRNA and miRNA permeable across biological membranes?

A

poorly permeable across biological membranes
-hydrophilic nature, negative charge, high molecular weight

27
Q

What is the primary role of a delivery system?

A

facilitate the uptake of siRNAs or miRNAs to their target sites
can also protect the nucleic acids from premature nuclease degradation

28
Q

True or false: siRNA and miRNA have different delivery technologies

A

false
similar delivery technologies
-similar physicochemical properties and intracellular site of actions

29
Q

Describe viral delivery systems for siRNA and miRNA.

A

viruses (lentiviruses, adenoviruses, and aden-associated)
-high transduction efficiency
-genetically engineered to remove their virulence
-can integrate into the host genome

30
Q

What are the limitations of viral delivery?

A

serious safety concerns (immunogenicity)
risk of insertional mutagenesis
high production costs
non-viral vectors have become attractive

31
Q

What are delivery systems that are non-viral vectors?

A

polymer-based and lipid-based systems

32
Q

What are the advantages of non-viral vectors?

A

good safety profile and low production cost
versatile and easily modified to improve delivery efficiency

33
Q

What is genome editing?

A

group of technologies that give scientists the ability to change an organisms DNA
-allow genetic material to be added, removed, or altered

34
Q

What is CRISPR-Cas9?

A

genome editing technology
-targets a specific section of DNA
-makes a precise cut/break at the target site

35
Q

What can CRISPR-Cas9 do?

A

make a gene nonfunctional
replace one version of a gene with another
base editing

36
Q

How was CRISPR-Cas9 adapted?

A

from a naturally occurring genome editing system in bacteria
the bacteria capture snippets of DNA from invading viruses and use them to create DNA segments known as CRISPR arrays
the CRISPR array allows the bacteria to “remember:” the viruses
if the virus attacks again, the bacteria produce RNA segments from the CRISPR arrays to target the viruses DNA
the bacteria then use Cas9 or a similar enzyme to cut the DNA apart which disables the virus

37
Q

How does CRISPR-Cas 9 work?

A

small piece of RNA with a short guide sequence attaches to a specific target sequence of DNA in a genome
the RNA also binds to the Cas9 enzyme
the modified RNA is used to recognize the DNA sequence and the Cas9 enzyme cuts the DNA at the targeted location
once the DNA is cut, researches use the cells own DNA repair machinery to add or delete pieces of genetic material or to make changes to the DNA by replacing an existing segment with a customized DNA sequence

38
Q

What are the features of CRISPR-Cas9?

A

simple to make components: guide RNAs, Cas9, and DNA templates
simple to introduce into cells and early embryos
highly specific
highly efficient
versatile

39
Q

What is sickle cell disease caused by?

A

mutation in Hg beta gene found on chromosome 11

40
Q

What is BCL11A?

A

transcription factor that represses y-globin expression and fetal HgB in erythroid cells

PHAR211 (16 decks)

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