Non-coding RNAs & their Applications (11)

Question 1

how do you determine the function of a newly discovered gene?

Answer 1

create a loss-of-function mutant & look for a change in the phenotype

Question 2

antisense RNA

Answer 2

RNA complementary to the mRNA

when binding to mRNA will prevent ribosome from binding

Question 3

why does the cell signal DS RNA as an invasion?

Answer 3

some viruses have DS RNA genomes

Question 4

RNAi

Answer 4

silencing of a gene by destroying the mRNA from a dsRNA

Question 5

RNAi steps

Answer 5

1. dsRNA enters cell
2. Dicer cuts dsRNA into siRNAs
3. RISC binds to siRNAs, unwinds siRNAs & keeps one strand
4. RISC & SS of siRNA scans mRNA in the cell & looks for any mRNA that match the siRNA
5. Once a match is found, the splicer enzyme in RISC splices the mRNA. mRNA pieces are then attacked by exonucleases & destroyed

Question 6

What are some exs of short-term gene silencing?

Answer 6

1. Make dsRNA in vitro (in a test tube, will polymerize DS RNA)
2. Liposomes (lipid bubbles) – transfect cells
   o Micelle, DS RNA gets packaged up, micelle fuses with membrane dumping DS RNA into the cell
3. Inject
4. Shoot
5. Feed –> ants eating DS RNA
   o Enables DS RNA to spread

Question 7

What are some exs of long-term gene silencing?

Answer 7

genetic transformation

Question 8

applications for gene silencing in animals & humans?

Answer 8

turn off disease-causing cancer genes & restore cells to its original type

turn off genes of invading pathogens & boost immune sys

Question 9

applications for gene silencing in pests?

Answer 9

turn off genes in reproduction

turn off genes that allow mosquitos to spread disease

Question 10

what is a new anticancer therapy in liver tumour of mice? not on midterm

Answer 10

siRNAs injected into mice

dsRNA targeted the BCI-1 gene mutant & wild-type remained

liver tumour size reduced & normal livers remained

Question 11

how does siRNA effect HIV? not on midterm

Answer 11

cells treated with siRNA specific for the CD4 gene prevented HIV infection

Question 12

how can we make transgenic indivs?

Answer 12

place a promoter in front of the gene

combine a gene with a certain promoter

Question 13

transposon (not on midterm) & what is its major use?

Answer 13

DNA fragments capable of squeezing themselves into c’somes

parasitic molecule

enable the addition of a promoter to genetically modify an organism

Question 14

how do we introduce DNA into a new host organism?

Answer 14

o	Injection 
o	Gene gun
o	Electric shock 
o	Lipid capsules 
o	Viruses

Question 15

how do we detect transgenic indivs?

Answer 15

use a reported gene (ex: green fluorescent PRO)

Question 16

gain of function & what type of genotype would display the trait? & when does this method work best

Answer 16

mutants add gene & ask what is the change in phenotype

homozygous recessive

looking at dominant genes

Question 17

loss of function & what causes the complete loss?

Answer 17

mutants remove or turn off the gene & ask what is the change in the phenotype

homozygous recessive

Question 18

RNA interference technology makes it much easier to create ____ -of-function mutants:

Answer 18

loss

Question 19

natural functions of RNAi

Answer 19

o Defense mechanism against dsRNA virus

o Regulate development

Question 20

drosha

Answer 20

nuclear enzyme that processes pre-miRNAs

also processes long DS RNA

Question 21

processing of miRNA

Answer 21

Drosha processes pre-miRNAs & dicer processes pre-miRNAs into miRNAs

Question 22

where do miRNAs bind?

Answer 22

3’ UTR (non-coding sequence)

Question 23

RISC scans for what type of sequence?

Answer 23

exon sequences

Question 24

miRNA effect in plants

Answer 24

Match to the exon sequences of genes –> RNA will make miRNAs typically bind to 3’UTR & be degraded in plants

Question 25

miRNA effect in animals

Answer 25

miRNAs don’t bind to their target with 100% complementarity

Translation is blocked ( mRNA isn’t degraded)

Question 26

what is the diff in miRNA among plants & animals?

Answer 26

plants –> binds perfectly to 3’ UTR
animals –> binds imperfectly to 3’ UTR

plants –> mRNA degraded
animals –> blocks translation

Question 27

what enables miRNA to bind to more than one gene?

Answer 27

imperfect matches of the 3’ UTR

Question 28

multiple layers of regulation of a gene is caused b/c of

Answer 28

more than one miRNA can target a single gene

Question 29

what helps determine tissue type

Answer 29

miRNAs expressed in a tissue specific manner

have miRNAs to a specific tissue type

Question 30

gene expression can differ in tissues due to:

Answer 30

diffs in transcription factors

tissue-specific alternative splicing

diffs in miRNAs

Question 31

in a recent study, one human’s c’some was predicted to have 700 genes but had how many transcripts?

Answer 31

7000 diff transcripts

many were non-coding –> miRNAs? or other functions?

Question 32

what are the dsRNAs in the body?

Answer 32

siRNA & miRNA

Question 33

siRNA vs. miRNA

Answer 33

o miRNA attacks 3’ UTR & blocks translation
o siRNA is used by RISC to find a mRNA match
o Both: processed by dicer
o miRNA can bind to multiple genes
o siRNA is specific to an mRNA
o siRNA come from outside the cell & miRNA is made within the cell
in animals, miRNA binds imperfectly & siRNA binds perfectly to their
both: involved in gene silencing