Non-coding RNAs & their Applications (11) Flashcards
how do you determine the function of a newly discovered gene?
create a loss-of-function mutant & look for a change in the phenotype
antisense RNA
RNA complementary to the mRNA
when binding to mRNA will prevent ribosome from binding
why does the cell signal DS RNA as an invasion?
some viruses have DS RNA genomes
RNAi
silencing of a gene by destroying the mRNA from a dsRNA
RNAi steps
- dsRNA enters cell
- Dicer cuts dsRNA into siRNAs
- RISC binds to siRNAs, unwinds siRNAs & keeps one strand
- RISC & SS of siRNA scans mRNA in the cell & looks for any mRNA that match the siRNA
- Once a match is found, the splicer enzyme in RISC splices the mRNA. mRNA pieces are then attacked by exonucleases & destroyed
What are some exs of short-term gene silencing?
- Make dsRNA in vitro (in a test tube, will polymerize DS RNA)
- Liposomes (lipid bubbles) – transfect cells
o Micelle, DS RNA gets packaged up, micelle fuses with membrane dumping DS RNA into the cell - Inject
- Shoot
- Feed –> ants eating DS RNA
o Enables DS RNA to spread
What are some exs of long-term gene silencing?
genetic transformation
applications for gene silencing in animals & humans?
turn off disease-causing cancer genes & restore cells to its original type
turn off genes of invading pathogens & boost immune sys
applications for gene silencing in pests?
turn off genes in reproduction
turn off genes that allow mosquitos to spread disease
what is a new anticancer therapy in liver tumour of mice? not on midterm
siRNAs injected into mice
dsRNA targeted the BCI-1 gene mutant & wild-type remained
liver tumour size reduced & normal livers remained
how does siRNA effect HIV? not on midterm
cells treated with siRNA specific for the CD4 gene prevented HIV infection
how can we make transgenic indivs?
place a promoter in front of the gene
combine a gene with a certain promoter
transposon (not on midterm) & what is its major use?
DNA fragments capable of squeezing themselves into c’somes
parasitic molecule
enable the addition of a promoter to genetically modify an organism
how do we introduce DNA into a new host organism?
o Injection o Gene gun o Electric shock o Lipid capsules o Viruses
how do we detect transgenic indivs?
use a reported gene (ex: green fluorescent PRO)
gain of function & what type of genotype would display the trait? & when does this method work best
mutants add gene & ask what is the change in phenotype
homozygous recessive
looking at dominant genes
loss of function & what causes the complete loss?
mutants remove or turn off the gene & ask what is the change in the phenotype
homozygous recessive
RNA interference technology makes it much easier to create ____ -of-function mutants:
loss
natural functions of RNAi
o Defense mechanism against dsRNA virus
o Regulate development
drosha
nuclear enzyme that processes pre-miRNAs
also processes long DS RNA
processing of miRNA
Drosha processes pre-miRNAs & dicer processes pre-miRNAs into miRNAs
where do miRNAs bind?
3’ UTR (non-coding sequence)
RISC scans for what type of sequence?
exon sequences
miRNA effect in plants
Match to the exon sequences of genes –> RNA will make miRNAs typically bind to 3’UTR & be degraded in plants
miRNA effect in animals
miRNAs don’t bind to their target with 100% complementarity
Translation is blocked ( mRNA isn’t degraded)
what is the diff in miRNA among plants & animals?
plants –> binds perfectly to 3’ UTR
animals –> binds imperfectly to 3’ UTR
plants –> mRNA degraded
animals –> blocks translation
what enables miRNA to bind to more than one gene?
imperfect matches of the 3’ UTR
multiple layers of regulation of a gene is caused b/c of
more than one miRNA can target a single gene
what helps determine tissue type
miRNAs expressed in a tissue specific manner
have miRNAs to a specific tissue type
gene expression can differ in tissues due to:
diffs in transcription factors
tissue-specific alternative splicing
diffs in miRNAs
in a recent study, one human’s c’some was predicted to have 700 genes but had how many transcripts?
7000 diff transcripts
many were non-coding –> miRNAs? or other functions?
what are the dsRNAs in the body?
siRNA & miRNA
siRNA vs. miRNA
o miRNA attacks 3’ UTR & blocks translation
o siRNA is used by RISC to find a mRNA match
o Both: processed by dicer
o miRNA can bind to multiple genes
o siRNA is specific to an mRNA
o siRNA come from outside the cell & miRNA is made within the cell
in animals, miRNA binds imperfectly & siRNA binds perfectly to their
both: involved in gene silencing
