RNA interference and microRNAs

Question 1

siRNA pathway process

Answer 1

retrovirus inserts double stranded RNA genome into host genome. Dicer cuts up viral genome into siRNAs which enter RISC. One strand ejected from the complex, guide strand retained. Complex scans cell for sequence complementarity to siRNA. base pairing triggers enzymatic activity of complex>degrades target mRNA. viral infection prevented

Question 2

siRNA pathway evolution

Answer 2

viral defence mechanism as early as in plants

Question 3

dicer action

Answer 3

creates overhangs at the 3’ ends of both strands of the siRNA
3’ end bound to dicer

Question 4

RNase 3 domains of dicer

Answer 4

cleave RNA
precise cutting
not aligned>make incisions 2 nucleotides apart to generate overhangs

Question 5

ruler helix domain of dicer

Answer 5

measures length of RNA to ensure accurate cleavage into fragments of ~22 nucleotides

Question 6

RISC

Answer 6

RNA induced silencing complex
recognises specific molecular configuration of the siRNAs processed by dicer

Question 7

argonuate component of RISC

Answer 7

core protein
recognises and binds guide strand
has domains:PIWI,MID,PAZ,N terminal

Question 8

PIWI domain of argonuate

Answer 8

endonuclease/RNAse activity
degrades target when complementary RNA found

Question 9

PAZ domain of argonuate

Answer 9

binds 3’ end of guide strand

Question 10

MID domain of argonuate

Answer 10

binds 5’ end of siRNA to ensure correct configuration

Question 11

N terminal of argonuate

Answer 11

helicase activity
unwinds double stranded siRNA

Question 12

miRNA genes

Answer 12

encode larger precursors that are processed into shorter mature miRNAs
used in translational control

Question 13

miRNA processing

Answer 13

pri-miRNA transcribed from the miRNA gene. has several hairpin structures. drosha complex recognises desired section and dicer cleaves hairpin and one strand is retained
recognised by RISC
complex searches cytoplasm for sequence complementarity

Question 14

seed sequence

Answer 14

miRNAs have specificity to target mRNA
perfect base pairing at 5’ end at seed sequence
rest is mismatched

Question 15

inhibition of ribosome elongation

Answer 15

RISC interacts with mRNA, the complex triggers inhibition of translation
not degradation due to imperfect base pairing

Question 16

miRNA degrades mRNA

Answer 16

when miRNA has perfect complementarity to the target mRNA

Question 17

piRNAs

Answer 17

piwi-interacting RNAs
expressed in germline cells

Question 18

retrotransposons

Answer 18

highly active in mammalian germline cells
piRNAs suppress their activity

Question 19

how piRNAs work

Answer 19

have complementary sequence to transposon
transposon transcribed into line RNA
piRNA transcribed into piRNA precursor
piwi proteins recognise precursor and cleave it into small fragments that can silence line RNA

Question 20

how do piRNA-piwi complex cause packing of DNA

Answer 20

recruits DNMts, HMts and HP1 proteins