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Bio 2520 > Lecture 11 > Flashcards

Lecture 11 Flashcards

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1
Q

What is miRNA? Describe their shape

A

double stranded RNA.

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2
Q

Explain how pri-miRNA form their signature shape.

A

pri-miRNA is transcribed by RNA-POL II into long primary transcriptions that fold back on themselves to form a “hairpin” shape.

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3
Q

Define Drosha. Explain how it operates.

A

An RNase protein found within the nucleus that processes pri-miRNA.
More specifically, Drosha cleaves the hairpin shaped, primary miRNA out of the long strand.

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4
Q

How is pri-miRNA processed?

A

Drosha, an RNase protein cleaves hairpin structure of primary miRNA. Thus pri-miRNA becomes pre-miRNA

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5
Q

What is the function of Exportin-5?

A

Exportin-5 binds to pre-miRNA and exits the nucleus via the nuclear pore.

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6
Q

What is Dicer? what is its function?

A

Another RNase protein.

It processes pre-miRNA in the cytoplasm by cutting the hairpin structure into small fragments (~22bp long).

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7
Q

Name the RNase proteins involved in miRNA processing.

A

Drosha - operates in the nucleus

Dicer - operates in the cytoplasm

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8
Q

What is miRISC? What is its function?

A

miRNA Induced Silencing Complex, a ribonucleic protein involved in silencing translation of targeted mRNA.

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9
Q

Generally, how does miRISC operate?

A

first RISC binds to miRNA and kills off one of the miRNA strands (this means miRNA is now single stranded).
miRISC guides miRNA to targeted mRNA.

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10
Q

Is pre-miRNA single stranded or double standed?

A

Double Stranded.

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11
Q

At what point does miRNA become single stranded?

A

When miRNA is bound to RISC.

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12
Q

Where do miRISC complex usually bind?

A

To the 3’ UTR of mRNA

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13
Q

How does miRISC complex bind to mRNA? Describe what happens.

A

-miRISC can bind to mRNA with an overhang (this means the strand is not fully complementary).
Translation is blocked or slowed down.
-miRISC binds to mRNA with full complementarity. The mRNA and miRISC (now double stranded RNA) will be cleaved and degraded by Argonatue protein.

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14
Q

What is Argonaute protein?

A

A cytoplasmic protein involved in miRNA processing.

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15
Q

Explain how miRNA are thought to control developmental processes. Provide Examples.

A

-miRNA can bind to mRNA without full complementary binding. This means a single mRNA can have multiple miRNA bind to it
-miRNA and mRNA interactions are thought to fine tune the levels of translation of mRNA
Example: miRNA thought to be involved in nervous system patterning, cell proliferation, cell death, differentiation, and leaf/flower development in plants

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16
Q

Describe Long-noncoding RNA (lncRNA). Provide examples.

A

-they are 200 - 5,000bp long
-they do not fit the categories of mRNA,rRNA,tRNA
-they can fold into complex 3D shapes (like rRNA)
Example: XIST and Telomerase RNA

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17
Q

What is the hypothesized function of lncRNA?

A

researchers think they may act as a guide/tether to bring protein complexes to specific sequences in the genome.

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18
Q

How could miRNA and lncRNA explain differences in organismal complexity? Why don’t genes or chromosome number effect organismal complexity?

A
  • miRNA and lncRNA have the ability to alternatively splice proteins (remove introns, different protein enhancer regions within the gene)
  • in one study their results predicted 700 genes, but 7,000 different transcripts.
  • Different species although very similar do not necessarily have the same number of genes (asian deer, kind of like a pronghorn).
  • humans and apes are only a few genes different from each other, yet we are fairly different
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19
Q

How do we study gene/protein function in a living organism?

A 
Forward genetics (classic method), mutate the genome and find the mutant phenotype. Uses miRNA
-Reverse genetics, begin with a single gene and knock-down the mRNA expression levels using RNAi, then look for the mutant phenotype
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20
Q

Explain how Forward Genetics can determine the function of a protein/gene.

A
  • using a large population of organisms
  • screen all the mutants for the phenotype you are interested in (mutant screen)
  • figure out which gene was altered/eliminated (causing the mutant phenotype) in each mutant
  • perform more experiments on the mutant to confirm how the mutated gene is functioning
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21
Q

What are the pros and cons to forward classical genetics?

A

Pros:
-phenotype leads you to the gene
-you can find the function of genes you have never encountered before
-mutations can become very informative
Cons:
-it can take a long time to figure out the mutation and fully understand the mutated gene (years)
-it can be expensive to keep your mutants alive (consider a population of 100,000 mice)

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22
Q

Name some mutagens commonly used in the lab to alter a genome.

A
  • Ethylmethyl Sulfonate -causes G:C bp to become an A:T bp
  • fast neutrons - knock out entire genes or large segments of a gene (usually create a null mutation)
  • X-rays - cause random nucleobase mutations (similar to EMS)
  • UV Light - cause random nucleobase mutations (similar to EMS)
23
Q

What is a nonsense mutation? How does it occur?

A
  • a point mutation that introduces an early stop codon

- the protein is not functional

24
Q

What is a null mutation?

A

-when a gene is either not transcribed or a protein from that gene is not translated at all

25
Q

What is a missense mutation? What can result from it?

A

when a base pair within the codon is altered, causing a completely different amino acid to be incorporated into the protein
-can result in a null mutation, altered function, or may have no change in the protein.

26
Q

When is a missense mutation more likely to occur?

A

a change in amino acids is more likely to occur when the alteration occurs in the primary or secondary location of the codon.

27
Q

What is PTGS?

A

Post Transcriptional Gene Silencing

28
Q

How did researchers first come about finding PTGS?

A
  • they were trying to enhance the expression of purple coloring in petunias.
  • had placed the gene behind 35S protomer, a strong viral promoter, to increase expression of the pigment gene.
  • Sometimes over-expression worked and a nice dark purple was expressed. Sometimes however, pigment production was completely eliminated in some areas in the flower.
29
Q

What is RNAi?

A

RNA Interference, the broad process of double stranded RNA regulating translation using Argonaute proteins.
miRNA and siRNA both operate as RNAi

30
Q

Describe another study that helped to conceptualize what RNAi is.

A

Fine and Mello (1998), injected short pieces of double stranded RNA (GFP) into nematodes. Double stranded RNA separated and then targeted homologous mRNA.

31
Q

What is siRNA?

A

Small Interfering RNA, small fragments of RNA that can alter the translation of a particular gene.

32
Q

What are some of the differences between siRNA and miRNA?

A

siRNA:
-pre-siRNA is double stranded contains 30 - 100nt
-full complementarity to mRNA
-singular mRNA target
-found in the nucleus or cytoplasm
-completely degrades mRNA
miRNA:
-pre-miRNA contains 70 - 100nt and hairpin structure
-partial complementarity to mRNA or full complementarity
-found only in cytoplasm
-translational repression, complete degradation of mRNA
-multiple mRNA targets

33
Q

What are some of the similarities between siRNA and miRNA?

A
  • both are double stranded until RISC or RIST (siRNA in nucleus) binds to them
  • both are similar in nucleotide/basepair length
  • both use argonaute proteins
  • Dicer process dsRNA turning it into siRNA, dicer processes pre-miRNA turning it into miRNA
34
Q

Describe the process of siRNA being processed in the cytoplasm and associating with mRNA.

A
  • dsRNA is detected in the cytoplasm and chopped by DICER into small db stranded framents (siRNA)
  • siRNA associates with argonaute protein and other proteins to form pre-RISC complex
  • one of the siRNA strands is degraded while the other remains bound to pre-RISC.
  • Pre-RISC becomes mature RISC, goes out and searches for specific mRNA
  • when RISC complex finds mRNA target, Argonature protein has SLICER activity which cleaves part of the mRNA
  • RISC dissociates from mRNA, and mRNA is degraded by nucleases
35
Q

What is RITS?

A

RNA Induced Transcriptional Silencing, an argonaute protein that binds with siRNA in the nucleus

36
Q

Explain briefly how RITS works.

A
  • siRNA bind to RITS in the nucleus (double stranded to single stranded)
  • RITS complex finds and binds to target mRNA
  • other proteins within the RITS complex recruit chromatin remodeling factors (factors involved in histone methylation and local heterochromatin formation)
37
Q

What are some hypotheses about why we have RNA silencing machinery?

A
  • RNA silencing is thought to have evolved as a type of “genetic immune system” to protect from foreign or unwanted genetic material
  • since some viruses are double stranded, it is thought eukaryotes use RNAi to stop viral replication
  • RNAi machinery Dicer and RISC are not found in prokaryotes (only some eukaryotes)
38
Q

What are the requirements of using RNAi in gene studies?

A
  • you must know the genetic sequence of the gene you are studying
  • it must be ~21nt long
39
Q

How would you introduce a short-term effect of gene silencing? List the possible methods.

A
  • make dsRNA and inserting it into the tissues of the organism
  • insert it into a vector/liposome and transfect the cells
  • inject the dsRNA into the organism
  • spray dsRNA onto plants as a fungacide
  • feed dsRNA to organism (nematodes will eat dsRNA)
40
Q

How would you introduce a long term effects of gene silencing?

A

-form a DNA construct (using inverted fragments)
(the 3’ end needs to be inverted so the hairpin structure will form when it is translated)
-insert construct into a chromosome
-organism will make dsRNAi

41
Q

Explain the method of reverse genetics.

A
  • begin with a gene you are interested in (must know its sequence)
  • knock-down mRNA expression of that particular gene using RNAi
  • look for mutant phenotype
42
Q

Does RNAi eliminate all mRNA?

A

No. Hence the term knock-down, rather than knock-out. Therefore some of the gene/protein will still be translated

43
Q

What are the pros and cons to reverse genetics?

A

Pros: faster and less expensive than forward genetics
Cons: you may not find the phenotype if you do not know exactly what you are looking for or where to find it.

44
Q

What does CRISPR-CAS9 stand for?

A

Clustered Regularly Interspaced Short Palindromic Repeats. CRISPR Associated Protein 9

45
Q

How did we figure out the function of CRISPR-Cas9?

A

-found viruses were trying to infect bacteria.

To protect themselves, bacteria produced a ribonucleic protein known as CAS9.

46
Q

What RNase molecule is similar to CAS9?

A

CAS9 acts like RISC in the sense that CAS9 binds to tracrRNA and crRNA, which then go out to “find” viral DNA.
crRNA is the sequence that guides the complex to the targeted gene.
When viral DNA is found, CAS9 complex cuts DNA, killing the virus.

47
Q

Describe in detail the process of assembling CRISPR-CAS9 and the process of binding to viral DNA.

A
  • CRISPR array found within the genome of the bacteria is homologous to DNA sequences of viruses that infect bacteria (this means bacteria have already encountered the virus, like memory B cells in the human immune system)
  • tracrRNA and pre-crRNA is transcribed. CAS9 is also transcribed and translated
  • pre-crRNA is edited –>crRNA
  • crRNA is loaded into CAS9 and basepairs with viral DNA with help from tracrRNA
  • CAS9 cleaves both strands of Viral DNA
48
Q

How is CRISPR-CAS9 used in the lab?

A
  • to cut single or double strands of DNA
  • introduce mutations
  • cut out a gene and introduce a new gene at a specific site
49
Q

Why is CRISPR-CAS9 used to introduce mutations? How do researchers prevent mutations after a cut?

A

Inducing mutations: when CRISPR-CAS9 cuts, DNA does not repair itself very well. thus endonucleases degrade the end of the DNA and therefore create a mutation.
Preventing mutations: include a homologous base pair to the cut site

50
Q

What part of CRISPR-CAS9 guides the complex to the cut site?

A

crRNA guides CAS9 to cut any sequence

-guideRNA is a piece of fabricated RNA that operates the same as crRNA

51
Q

Explain how CRISPR-CAS9 may be used in gene therapy.

A

with diseases such as MS, Sickle Cell Anemia, Cystic Fibrosis, etc, the genes are mutated in some way.
The idea is we can introduce “healthy genes” into the genome by using CRISPR-CAS9 to cut out the mutated gene and insert a “healthy” gene

52
Q

Is CRISPR a short term or long term effect?

A

It is long term/permanent. This is because CRISPR-CAS9 operates in the genome, directly effecting the genetic code.

53
Q

What are some cons to CRISPR-CAS9?

A

we are aware that targeted cutting is still a problem. The complex will cut where it is supposed to cut, but then it will wander off and cut other parts of the genome we don’t want disrupted.
Hence this could be disastrous for humans.

Bio 2520 (4 decks)

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