L26 Regulatory RNA

Question 1

Discovering regulatory RNA

Answer 1

Genetics - mutations in genes lacking ORFs

RNA-sequencing of purified RNA (small)

• deep sequencing - rare transcripts
• depletion of ribosomes-associated RNAs (mRNA)

Immunoprecipitation and RNA-sequencing
- UV cross-linking of protein to RNA (CLIP) => led to discovery of Piwi RNA

Question 2

Early approach for targeted gene activation

Answer 2

See onenote diagram

• bacteria utilize antisense inhibition to regulate gene activity
• homologous base-pairing between antisense and sense RNA causes sequence specific gene silencing

Question 3

Gene silencing in a nematode

Answer 3

see onenote slides

- antisense mechanism cannot account for gene silencing in the worm

Question 4

Unc 22 gene and key findings

Answer 4

See onenote slide
- twitching phenotype w

Key findings
- dsRNA 100x more effective than ssRNA in causing silencing
- very small quantities of dsRNA cause silencing - catalytic
- gene silencing is sequence specific
- gene silencing results in the loss of target mRNA
- gene silencing occurs post-transcriptionally (no silencing with dsRNA targeting promoters and introns
=> concluded = RNA interference (RNAi)

Question 5

RNAi

Answer 5

• RNAi is a ubiquitous phenomenon in eukryotes

Fire and Mello concluded:
Eukaryotic cells perceive dsRNA as a sequence specific signal to inhibit expression of the corresponding mRNA

Question 6

RNAi also observed in plants

Answer 6

See onenote slide

co-suppression/post-transcriptional gene silencing (PTGS)

Question 7

Possible explanation for PTGS

Answer 7

See onenote slide

Question 8

RNAi is associated with small RNAs

Answer 8

See onenote slide

• small RNAs associated with PTGS first discovered in plants

Question 9

Summary of RNAi

Answer 9

1. dsRNA - must have homology to target transcript
2. homology to exons not introns (processed transcript)
3. effectors are small single-stranded RNA - called siRNA
4. RNAi involves transcript degradation
5. small amounts of dsRNA have a big effect (non-stochiometric)

Question 10

miRNA - lin mutants

Answer 10

See onenote slides
microRNA

• heterochronic mutants = developmental timing altered
• focus on defects to vulva development
• lin-4 = development progression delayed
• lin-14 = developmental progression occurs earlier than normal

Question 11

lin-14

Answer 11

see onenote slide

• lin-14 mutants do not produce the pattern of cell divisions and differentiation normally seen at L1 stage of development (infer than lin-14 is active at the L1 stage)
• Lin14 required for patterned cell division
• Lin14 required for L1 stage of development
• Active specifically in the first larval stage as lin14 mutant effects all developmental stage
• Lin14 protein initially abundant then reduced but lin14 mRNA high across all four stages, something blocking lin14mRNA translation

Question 12

lin-4 regulates lin-14

Answer 12

See onenote slide

• lin-4 mutants repeat the L1 pattern of cell division and differentiation at later stages of development => infer that lin-14 remains active in all developmental stages of lin-4 mutants (hence L1 stage of development is repeated)
• lin-4 promotes developmental progression by reducing lin-14 protein accumulation in later larval stage (L2-L4)

Question 13

lin-4 encodes a small RNA

Answer 13

see onenote slides

• lin4 produces a ~60nt and 22nt transcript - no obvious ORF
• 22nt molecule derived from the 60nt transcript (stem-loop)
• lin4 regulates lin14 post-transcriptionally and in trans, regulation involves inhibition of protein synthesis

Question 14

microRNAs

Answer 14

• majority of small RNAs are not temporally regulated - called microRNAs
• miRNAs present in all multicellular eukaryotes
• miRNAs regulate more than 70% of each organisms protein-coding genes
• miRNAs are conserved
• animal miRNA not found in plants and vice versa

Question 15

miRNA biogenesis - transcription

Answer 15

see onenote slide

Primary miRNA (pri-miRNA)

• ssRNA (>70nt) generated by RNA pol 2 and has 5’ CAP and poly-A tail
• 1 or more ~70-80nt foldback region with an imperfect hairpin stem-loop structure
• foldback arises from intramolecular interactions due to reverse sequence complementarity
• pri-miRNAs transcript has one, but sometimes have multiple foldbacks (polycistronic)
• most prim-miRNA transcripts do not have an ORF
• miRNA can arise from splicing e.g. within intron of a protein-coding RNA but not common

Question 16

miRNA biogenesis - nuclear processing

Answer 16

see onenote slides

Question 17

siRNA biogenesis

Answer 17

See onenote slide

• Multiple siRNA duplexes arise from a single template, precursor DS RNA
• But for miRNA, only one miRNA per transcript assuming only one stem and loop structure per transcript
• Drosha not needed for processing
• Dicer processes long dsRNA in cytoplasm

Question 18

RNA-induced silencing complex (RISC)

Answer 18

see onenote slide

• one strand of the ds duplex is incorporated into a cytoplasmic multi-component ribonucleoprotein complex - RISC
• RISC always contains a 100kDa Agonaute (AGO) protein => guide RNA strand base-pair with complementary mRNA

Question 19

Summary - miRNA/siRNA

Answer 19

1. miRNA/siRNA are 21-25 nt ss RNA molecules that are derived from ds intermediate
2. miRNA/siRNA always associated with an AGO protein in RISC
3. work in trans to silence gene activity post-transcriptionally
4. interactions between the guide RNA in RISC and target transcript have different outcomes:
   - siRNA = target transcript degraaded
   - miRNA = translational target transcript is blocked (worms)