small RNAs

Question 1

dsRNA and interference

Answer 1

dsRNA = double stranded RNA
antisense RNA bind strongly to mRNA
make dsRNA
dicer binds
dicer is Rnase III like endonuclease - digests dsRNA into 21-25bp fragments - leave 1 strand in tact
siRNA-guided the endonuclease removes one of the siRNA strands - passenger strand - requires Argonaute-Piwi proteins
RISC complex formed (multiprotein RNA-induced silencing complexes) - recognise mRNA, bind and cleave target mRNA molecules which are complementary to siRNA guide - inactivate it

Question 2

Complexity of genome and gene regulation in eukaryotes

Answer 2

number of genes doesn't correspond to the complexity of the organism 
protein coding RNA 
non-coding (nc) functional RNA - tRNA
nc structural RNA - alter mRNA 
exons not continuous  
10-15% of genome is used for protein

Question 3

what is the affect if dicer is missing

Answer 3

lethality in embryo

cells unable to differentiate - depletion of multipotent stem cells

Question 4

describe the action of long ncRNA

Answer 4

X chromosome inactivation
Xist - random, every cell, same DNA as male, happen in lineage - clone all cells same
methylation of x chromosome - heterochromatin - transcriptionally inactive
formation of Barr body

Question 5

describe the action of miRNA

Answer 5

controls translation
lin-14 is a TF
null mutation of lin-14 make faster development
prolonged expression of lin-14 make retarded development
null mutation of lin-4 make retarded development
lin-4 make 2 RNA products - 61 and 21 nucleotides
21 nucleotide - inhibits lin-14 - binds to target site in lin-14 mRNA UTR

Question 6

describe miRNA

Answer 6

typically 18-26 nucleotides 
evolutionarily conserved 
uniquely expressed in metazoan cells 
>700 in humans 
regulate expression >30% of protein coding genes

Question 7

miRNA transcription and processing

Answer 7

transcribed by rna pol II
into long pri-miRNA with stem and loop structure
RNase III endonuclease in complex with DiGeorge syndrome critical region 8 (DGCR8) - precursor miRNA
DGCR8 - interact with pri-miRNA through ssRNA regions - stem helps endonuclease to cleave
precursor miRNA transported to cytoplasm by Exportin-5 using Ran-GTP

Question 8

miRNA binding sites

Answer 8

inhibit target mRNA through base pairing
seed region - near 5’ end
mismatches in centre form bulge then less stringent binding

Question 9

diseases associated with ncRNA or miRNA expression

Answer 9

cancer
fat metabolism 
blood cells 
stem cells
apoptosis
biomarkers

Question 10

what is piRNA important for

Answer 10

germ cell production