Lecture 17 Flashcards
Coding RNA:
- RNAs that encode some type of information
- A conduite between the RNA and the protein
- messenger RNAs
Functional RNA:
- Ribosomal RNA
- transfer RNAs
- Decoding the information within the mRNA
- short nuclear RNAs
- part of processing RNA, splicing introns out of transcripts in eukaryotes
Regulatory (small) RNAs:
- siRNAs (discovered through silencing RNAs)
- miRNAs (discovered developmental mutants)
- piRNAs
- circRNAs
Regulatory (long) RNAs:
- IncRNAs
- Involved with X inactivation in placental mammals
Gene silencing:
- Trans-acting antisense RNA in E.coli silence their targets by forming dsRNA duplexes
- 100 nt in size, complimentary sequences to mRNA
- Selectively inactivate a gene/nucleotide sequence within a genome
Antisense inibition:
Paring between antisense and sense RNA, inhibiting gene expression as the duplex cannot be translated
Kemphues group and gene silencing experiment:
- Anti-sense inhibition for silencing Par-1
- Antisense mechanism cannot account for sense inhibition
- Phenotype observed in half of the worms injected
- Injection is easy, but the silencing effect is not easy to replicate
What does partition-1 (par-1) gene do?
- It promotes early cell division in the nematode Caenorhabditis embryo
Can antisense/sense suppression be induced by aberrant RNA?
- Gene silencing is mediated/induced by dsRNA
- 100x more effective than ssRNA
What does the unc22 gene code for?
- Uncordinated-22
- Codes for a muscle microfilament
- Mutations in this gene result in twitching worms
Is gene silencing sequence specific?
Yes
What gene is unc24?
- Paralysis
Fem-1 gene mutant:
- No sperm, so feminises the worm
hlh-1 gene mutant:
- Lumpy-dumpy phenotype
dsRNA causes
- Loss of target mRNA, either because
- dsRNA is interfering with transcription
- dsRNA is causing degradation of the transcript, post-transcriptionally
Does gene silencing occur post-transcriptionally?
- Yes!
- Injecting dsRNAs homologous to promoters and introns does not cause silencing
RNA interference:
- RNAi
- Eukaryotic cells perceive dsRNA as a sequence specific signal to inhibit expression of the corresponding mRNA
Co-suppresion/post-transcriptional gnee silencing (PTGS)
- Manipulate petal colour by introducing a transgene into plants
WT flowers and transgenic flowers are produce - mRNA of transgene/endogenous gene are degraded
Virus-induced gene silencing (VIGS)
Induce virus resistance by introducing viral transgene into plants
- Expression of untranslatable viral transgene in plants, these plants are immune to virus as viral RNA/transgene mRNA is degraded
- Non-transgenic plants exhibit disease symptoms as viral RNA accumulates
Virus induced gene silencing:
- Natural defence against viruses exists in some plants
- Virus produce dsRNA either via secondary structures in the RNA or as a duplication event
- dsRNA is recognised as being foreign, triggering RNAi
RNAi is associated with small RNAs:
- Antisense RNAs
- Short interfering RNAs (siRNAs)
- Target the genes/genetic elements from which they are derived
- Work in trans and complementary to target
- Induces mRNA degrading, post-transcription and sequence specific
Discovery of endogenous small RNAs occured via:
- Study of the lin mutant with altered patterns of cell lineage in vulva development
Lin-4 mutant:
- Bag of worms phenotype
- Vulva doesn’t form correctly, so eggs cannot come out of the worm
Heterochronic mutants affect developmental timing (slowing it down or speeding it up):
- Four stages of larval development
- In lin-4 and lin-14(gf) there is a delay in developmental progression
- Lin-14 and lin-28 loss of function shows developmental process earlier than normal
Lin-14 mutation:
- Required for the cell type of the first larval stage
- The protein is only required for the first larval stage
- But lin-14 mutants do not produce any lin-14 transcript so larval stage 1 is skipped
Lin-4 mutation:
- Stuck with L1 cell types in all four larval stages
- Limits lin-14 activity to the l1 larval stage
- Effects protein abundance
Lin-14(gf) mutants with a loss of the 3’ UTR region show
- 7 partially complementary sequences
- that the 3’ UTR region is essential for the degradation of the lin-4 protein in the 2nd, 3rd and 4th larval stages
let-7
- regualtes lin-41
- found in all metozoans
- works in larval development, regulating lin-41
Primary miRNA is generated by:
- RNA pol II and has a 5’ CAP and poly-A tail
Primary miRNAs can form:
- an imperfect hairpin stem-loop strucutre throuh the intramolecular interactions
Drosher, a type III endonuclease:
- Found in the nucleus
- Recognises the hairpin loop, and binds to primary micro RNA transcript
- precursor miRNA is exported from the nucleus
- Produces a single duplex
Dicer, another type III endonuclease:
- In the cytoplasm
- Type III endonuclease
- Dices the precursor RNA, removes the loop from the stem
- Produces multiple duplexes
- 5’ phosphate and a 2nt 3’ overhang
- Needs to be processed still further
siRNA biogenesis
- long perfectly pair dsRNA
- it is processed in the cytoplasm by dicer
- produces multiple duplexes
- 5’ phosphate and a 2nt 3’ overhang
RNA-induced silencing complex (RISC)
- A complex that modifies dsRNA further
- Argonaut degrades the target transcript leading to gene silencing
- The end that enters RISC is determined by the sampling of thermodynamic stability of the 5’ end
- The weakest end of the duplex enters
