W7L1 introduction to small non-coding DNA

Question 1

RNA type percentage

Answer 1

-Only 1-2% are coding RNA, the rest are none coding
-the non-coding include rRNA, tRNA,… in regulatory fuction, siRNA, miRNA, piRNA and lncRNA

Question 2

Modern approaches to RNA characterisation

Answer 2

RNA sequencing innovations:
v High throughput steady-state RNA-seq→ cell-specific transcripts and rare transcripts e.g. lncRNAs
v Nascent RNA-seq (GRO-seq, PRO-seq)→ rare transcripts e.g. lncRNAs, eRNAs, promoter upstream transcripts and upstream antisense RNAs
v Modified RNA-seq (PANDORA-seq) →Modified RNAs e.g. transfer and ribosomal RNA-derived small RNAs (tsRNAs/rsRNAs)
Improvements to the isolation of small RNAs:
v Crosslinking RNA/protein –immunoprecipitation (CLIP) → siRNAs, miRNAs, piRNAs
v Depletion of ribosome-associated RNAs → siRNAs, miRNAs

Question 3

Targeted gene inactivation (gene silencing)

Answer 3

§ Bacteria utilize antisense inhibition to regulate gene activity
§ Homologous base-pairing between antisense and sense RNA causes sequence-specific gene silencing
-RNA pairing lead to duplex formation which is untranslatable and unstable

Question 4

ssDNA Gene silencing experiment in nematodes

Answer 4

-Use antisense inhibition to silence a nematode gene - inject single-stranded antisense RNA. Very effective gene silencing
- in the control which used sense transgene, the same phenotype as the antisense transgene was observered
# bacterial antisense mechanism cannot account for gene silencing in the worm

Question 5

Discovery of dsRNA as a trigger for gene silencing in nematodes

Answer 5

Compared effectiveness of single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) to induce gene silencing. Inject into the worm. Antisense rnA ss does not have any effect
1. dsRNA 100x more effective than ssRNA in causing silencing of uncoordinated-22 (unc-22)
2. Very small quantities of dsRNA cause silencing – catalytic activity suggested

Question 6

Furthered experiment on nematodes

Answer 6

More experiment showed that gene silencing is sequence specific
-gene silencing result in the loss of target mRNA
-gene silencing occur post-transcriptionally, no silencing with.dsRNA targeting promoters and intron

Question 7

The discovery of gene silencing in plants

Answer 7

Introduce a chalcone synthase (CHS) transgene into Petunia plants
WT petinia have a purple colour
Small proportion of CHS transgenic plants displayed flowers with white sectors - example of gene silencing
Called co-suppression as sense transgene suppresses activity of endogenous gene

Question 8

Small RNAs are associated with co-suppression in plants

Answer 8

Introduction of ACO transgene into tomato plants results in a small number displaying co-suppression
-small RNA blot identify a population of small 22nt sense and antisense RNA in co-suppression lines
Small RNAs also associated with RNAi in worms, insects and protozoa - called short interfering RNAs (siRNAs)

Question 9

Key feature of RNAi

Answer 9

• Double-stranded RNA is the trigger for RNAi
• dsRNA must have homology to target transcript
• siRNAs are 21-24 nt ssRNA molecules
• Work in trans to silence gene activity post transcriptionally
• RNAi involves transcript degradation
• Small amounts of dsRNA have a big effect (non-stoichiometric)

Question 10

Isolation of nematode lineage mutants

Answer 10

Isolated lineage (lin) mutants - affect pattern/timing of cell divisions and differentiation
Lin4 mutant lack many adult body structures (include vulva)
Lin14: Develop adult body structure at larval stage- small poorly formed adult
-both classes of mutant display vulva defects and inability to lay eggs-bag of worm phenotype

Question 11

Vulva defects of lin-4 and lin-14 mutants

Answer 11

-Vulva formed from characteristic cell divisions in all four larval stages
lin4 mutants – L1 cell divisions repeated at later larval stages (delay developmental progression )
lin14 mutants – L1- cell divisions skipped, causing developmental progression occurs earlier than normal

Question 12

what are heterochronic mutant

Answer 12

Altering in developmental timming

Question 13

Characterisation of lin-14 and Lin 4 WT

Answer 13

Developmental progression associated with post-transcriptional regulation of lin14. Lin 14 protein abundant in L1 stage, start to drop in L2 stage.
Post-transcriptional regulation of lin14 involves lin-4

Question 14

Characteristic of Lin 4 mutant

Answer 14

Lin protein level always high

Question 15

Characteristic of lin 14 mutant

Answer 15

Lin 14 protein alway low

Question 16

Characterisation of lin-14 gain-of-function mutants

Answer 16

-same phenotype as lin4 mutant, repeated L1 pattern
gain-of-function lin-14 mutation does not display post-transcriptional regulation

Question 17

lin-4 encodes a small non-coding RNA

Answer 17

lin-4 produces an ~60nt and 22nt transcript - no obvious ORF
22nt molecule derived from the 60nt transcript (stem-of hairpin loop)
22nt lin-4 transcript partially complementary to multiple sites in the lin-14 3’ UTR
-lin14 protein level inversely porpotional to lin4 mRNA level
Lin-4 function in trans to block synthesis of Lin-14 protein

Question 18

Other small temporal RNAs in the worm

Answer 18

lin-4 sequence ONLY found in worms, only example of small RNA
let-7 orthologs found in other metazoans – small RNAs are common

Question 19

Transcription of miRNAs

Answer 19

Came from primary mRNA
§ Transcript generated by RNA pol II, - 5’ m7G cap, poly-A tail and spliced
§ 1 or more ~70-80nt foldback region with an imperfect(bludge due to lack of complimentry) hairpin stem-loop structure
§ Foldback arises from intramolecular interactions due to reverse sequence complementarity pri-miRNAs transcript has one, but sometimes multiple foldbacks (polycistronic) in insect
Most pri-miRNA transcripts do not have an open reading frame
Some miRNAs located in protein coding mRNAs (intron)

Question 20

Processing of miRNA transcripts

Answer 20

Type III RNA endonuclease Drosha in nucleus cleaves stem-loop at the base of primary miRNA transcript to form 60-70nt precursor miRNA (pre-miRNA)
2. pre-miRNA actively transported into cytoplasm
Type III RNA endonuclease Dicer removes loop and trims stem of pre-miRNA to form miR:miR* duplex

Question 21

siRNA biogenesis

Answer 21

1. Arise from long perfectly paired dsRNA precursor
2. Dicer produces multiple duplexes
   from a single dsRNA precursor 3. 3’ overhangs generated