Exam 4 part 1

Question 1

RNAi

Answer 1

the pathway where specific genes are inhibited

short RNA oligonucleotides in a protein complex regulate gene expression

Question 2

RNA oligonucleotide

Answer 2

targets protein complex to the desired RNA

Question 3

protein complex

Answer 3

inhibits gene expression

Question 4

RNA component of RNAi pathway

Answer 4

a short single strand of RNA that provides specificity

Question 5

protein component of RNAi pathway

Answer 5

made up of a collection of nucleic acid binding proteins, nucleases, and other enzymes that interact with RNA to regulate gene activity

Question 6

exogenous source of RNAi

Answer 6

RNA viruses or experiments

siRNA- short-interfering RNA

Question 7

siRNA

Answer 7

1. mostly exogenous
2. dsRNA precursors
3. may be target specific
4. degrade the target RNA

Question 8

endogenous source of RNAi

Answer 8

miRNA

Question 9

miRNA

Answer 9

1. encoded by endogenous genes
2. hairpin precursors
3. recognize multiple targets
4. can trigger transcriptional inhibition or mRNA cleavage

Question 10

RNAi two phase process

Answer 10

1. initiation- generation of mature siRNA or miRNA

2. execution- silencing of target gene, degradation or inhibition of translation

Question 11

initiation

Answer 11

1. cropping
2. dicing
3. activation

Question 12

cropping

Answer 12

1. occurs in nucleus
2. made of Pasha and Drosha “crop” pri-miRNA to form pre-miRNA
3. exported to cytoplasm
4. pre-miRNA has a 5’ phosphate and a two-nucleotide 3’ overhang

Question 13

Pasha

Answer 13

dsRNA binding protein

Question 14

Drosha

Answer 14

RNase III (cleaves dsRNA)

Question 15

dicing

Answer 15

1. occurs in cytoplasm
2. Dicer binds and dices pre-miRNA to form short dsRNAs
3. dicer has a “ruler” built in, cuts dsRNAs the same length
4. dicer cleaves ~22 nucleotide RNA duplex with 2bp 3’ overhang

Question 16

activation

Answer 16

1. process of combining the short miRNA with effector proteins to form complex
2. generates mature RNA-induced silencing complexes (RISCs)

Question 17

Key members of RISCs

Answer 17

argonautes
humans have 4 argonauts proteins
C. elegans have 29

Question 18

RISC formation

Answer 18

1. dsRNA generated by dicer is bound by RISC-loading protein
2. once loaded Argonaute (Ago2) selects one strand of dsRNA
3. this is guide strand, other “passenger” strand is either degraded or removed

Question 19

guide strand

Answer 19

retained in holo-RISC

Question 20

RNAi targeting

Answer 20

miRNA guide strand has a seed sequence at the 5’ end that is complementary to the target mRNA

Question 21

inhibition

Answer 21

if miRNA is complementary, Argonaute cleaves the mRNA

incomplete complementarity, translation is inhibited

Question 22

RNAi useful

Answer 22

can silence a gene without creating a mutant

Question 23

HCV infection

Answer 23

can only infect cells that express miR-122

silencing miR-122 represses viral infection

Question 24

CRISPRs

Answer 24

Clustered Regularly Interspaced Short Palindromic Repeats

Question 25

CRISPR adaptation

Answer 25

short pieces of viral RNA get integrated into the bacterial CRISPR loci

Question 26

CRISPR crRNA biogenesis

Answer 26

CRISPR RNA is now synthesized (tracr RNA and guide RNA)

Question 27

CRISPR interference

Answer 27

interact with the protein (Cas9) and cleave the viral DNA

Question 28

tracr RNA

Answer 28

helps to activate Cas9

Question 29

gRNA (guide RNA)

Answer 29

helps to silence the target viral DNA