non coding RNA

Question 1

what is sanger sequencing?

Answer 1

old method to determine DNA seq

1. dideoxynucleotide triphosphates (ddNTPs) are added to growing chain -> NTPs cannot be added, chain terminates
2. size separation by gel electrophoresis
3. gel analysis to determine sequence of DNA

Question 2

what is new generation sequencing?

Answer 2

to determine DNA sequence

1. sample prep
2. cluster generation
3. sequencing
   - 1st read: when fluorescent tagged NT added to growing chain, excited by a light source -> fluorescent signal emitted -> wavelength and signal intensity determines the base
   - 2nd read: for the reverse strand
4. data analysis

Question 3

what is a non coding RNA and its functions?

Answer 3

a functional RNA molecule transcribed from DNA but not necessarily transcribed into proteins
regulates gene expression at transcriptional and post-transcriptional level

Question 4

what are the two types of RNAi and what do they do?

Answer 4

miRNA (micro)
siRNA (small interfering)
they silence their target genes

Question 5

what is miRNA derived from?

Answer 5

rnas that are transcribed in the nucleus, which then fold and are processed, then exported into cytoplasm as ds precursor mirnas
- RNA transcripts that fold back on themselves to form short hairpins

Question 6

what is siRNA derived from?

Answer 6

longer dsRNA produced in cell itself or delivered into cell experimentally

Question 7

how are miRNA produced?

Answer 7

• ds precursors of mi/siRNAs bind to dicer
  dicer: an endoribonuclease protein, which cuts the dsRNA into short segments (~21nts long)
• short dsRNA binds to argonaut protein (guide strand)
• the other strand is degraded
• ssRNA + argonaut + other proteins = RISC (RNA-induced silencing complex)

Question 8

what does siRNA do? (what are its targets?)

Answer 8

Directs RISC to bind to specific mRNAs
- precise targetting
- base pairing between siRNA and target mRNA -> perfect complementarity to target sites
- argonaut catalyses cleavage (degradation) of mRNA

Question 9

what does miRNA do? (what are its targets?)

Answer 9

Directs RISC to mRNAs
- only a part of miRNA (seed) pairs with mRNA
- perfect seed binding to mRNA leads to degradation
- imperfect seed binding to mRNA leads to inhibition of translation
- imperfect binding allows miRNAs to target hundreds of endogenous mRNAs
- typically binds to 3’UTR of target genes
- results in degradation, destabilisation of mRNA, translational repression

Question 10

what are mirtrons?

Answer 10

a type of miRNA located in the introns of mRNA-encoding genes

Question 11

what is pri-miRNA (primary)?

Answer 11

miRNAs firstly transcribed as pri-miRNAs -> pre-miRNA -> mature miRNA

Question 12

what is a gene knock down?

Answer 12

temporarily stops or decrease the expression of one or more targeted genes
- uses RNAi (siRNA)
- used to determine a function of a gene

Question 13

what is an application of miRNA?

Answer 13

can be used as a biomarker for cancer

Question 14

what do lncRNAs act as?

Answer 14

1. signal molecules
2. decoy molecules (blocks proteins from regulating DNA and mRNA molecules)
3. guide molecules
4. scaffold molecules (assemble the target area)

Question 15

what is XIST?

Answer 15

• a large lncRNA
• required for transcriptional silencing of one X chr in females (dosage equivalence)
• expressed on the inactive X chr: XIST binds to chr -> recruits histone modification enzymes (methylases) (H3K27me3) -> X-inactivation