pp 20

Question 1

What is RNA interference used for?

Answer 1

normal regulatory mechanism in a number of organisms, involving double-stranded RNA, can involve degradation of mRNA or prevention of translation

Question 2

What is RNA interference used for?

Answer 2

laboratory method for gene-specific inactivation (instead of making a gene deletion, you can often inactivate a gene using RNAi)

Question 3

When does RNAi occur?

Answer 3

when a cell encounters various types of double-stranded RNA from different sources: viruses, transgenes

Question 4

What can RNAi result from?

Answer 4

1. processing of small interfering RNA (siRNA)
2. processing of micro-RNA (miRNA)

Question 5

What is the experiments with nematodes about?

Answer 5

Mex-3 gene involved in embryonic development, embryos had the mRNA so ds and ssRNA were introduced inside. A probe that hybridized the mRNA. dsRNA completely silenced mex 3 mRNA. dsRNA is better at silencing and levels of mRNA transcripts are decreasing when treated with dsRNA or ssRNA

Question 6

What is the mechanism of siRNA?

Answer 6

1. dsRNA from exogenous source is cut into short interfering RNA’s by dicer nuclease
2. ds siRNA is separated into single strands: passenger and guide strands
3. they assemble with proteins in a complex called RNA-induced silencing complex, that cuts mRNA complementary to the guide strand using nuclease called slicer
4. if complementarity is imperfect, repression of translation results followed sometimes by mRNA degradation

Question 7

How does RNAi pathway regulate viral gene expression?

Answer 7

Incorporating viral derived siRNA into cells RISC machinery results in specific targeting of viral RNA for degradation

Question 8

What is the counter measure of the RISC machinery?

Answer 8

protein: p19, binding siRNA preventing it from associating into RISC

Question 9

What are micro RNAs (miRNAs)

Answer 9

18-25 nt RNAs produced naturally in plant and animal cells, cleavage from 75 nt stem-loop precursor RNA

Question 10

What is the pathway for miRNA?

Answer 10

1. Dicer RNase cleaves ds stem part of precursor to yield miRNA in ds form
2. Strands of miRNA are separated and a guide strand compelxes with RISC control gene expression by base-pairing to their mRNAs
3. single stranded miRNA can direct the degradation of complimentary mRNA or blocks translation

Question 11

What is the Dorsha pathway of miRNA maturation in humans?

Answer 11

1. Primary RNA transcribed by RNA polymerase II and forms a stem loop
2. Cleavage at the stem results from processing by a complex of proteins microprocessor that includes ribonuclease Dorsha
3. Export into nucleus
4. Cleavage of the loop by Dicer
5. Separation of strands and assembly of RISC including one miRNA strand to direct translation inhibition/mRNA degradation

Question 12

What does Mitron stand for?

Answer 12

miRNA intron

Question 13

What is the Mitron pathway of miRNA formation?

Answer 13

1. Transcription of pre-mRNA
2. Intron excision
3. Debrancing of lariat
4. Pri-miRNA formation
5. Further processing by Dicer to form miRNA and formation of RISC from complex of proteins and one of the miRNA strands

Question 14

What is the pathway to gene silencing by miRNAs in animals?

Answer 14

miRNA base pair imperfectly to 3’ UTR of their target mRNA. this leads to inhibition of protein product accumulation from miRNA targeted mRNA

Question 15

What is the pathway to gene silencing by miRNA in plants?

Answer 15

miRNAs tend ot basepair perfectly or near-perfectly with target mRNA and cause mRNA cleavage

Question 16

How foes RNAi provide a useful tool for molecular biologists?

Answer 16

silencing of genes by RNAi can be used to study gene function, a vector can deliver RNA to cell, processing to siRNA results in targeting of specific gene transcripts, silencing them and preventing them from being transcribed