miRNA and siRNA Flashcards
what is coding RNA
RNA that codes for a protein; mRNA
what is noncoding RNA
RNA that doesn’t code for a protein; everything other than mRNA
which type of RNA, coding or noncoding, regulates gene expression
noncoding
what is the role of noncoding RNA
regulation of gene expression
what is another term for regulation of gene expression
RNA interference aka RNAi
what is RNAi
RNA interference (regulation of gene expression)
what is miRNA
microRNA
what is miRNA responsible for
regulating gene expression by inhibition
does miRNA activate or inhibit gene expression
inhibit
what does miRNA regulate specifically
the translation of specific mRNAs and the stability of specific mRNAs
what transcribes the gene for miRNA
RNA pol II
right after transcription of miRNA, what happens to the transcript
it folds into a hairpin structure
how many nucleotides is the hairpin structure of miRNA
70
what is the name of the miRNA gene once it’s transcribed
pri-miRNA
describe the structure of pri-miRNA
the hairpin structure is 70 nt long and has imperfect base pairing in the stem
how does pri-miRNA become pre-miRNA
hairpin is cleaved = pre-miRNA
what cleaves the pri-miRNA hairpin
Drosha protein with DGCR8
how many strands in the pri-miRNA
two; it’s dsRNA
what type of molecule is Drosha
an RNase
once the hairpin pri-miRNA is cleaved, what is the molecule called
pre-miRNA
what happens to pre-miRNA now that the hairpin has been cleaved by Drosha + DGCR8
it’s recognized by a nuclear export factor (Exportin5) and it’s transported out of the nucleus
what transports pre-miRNA out of the nucleus
Exportin5
what happens to the pre-miRNA once it’s in the cytoplasm
Dicer and TRBP cleaves it and processes it into miRNA
what cleaves the pre-miRNA
Dicer and TRBP
describe the structure of miRNA that was produced by Dicer and TRBP
double stranded RNA, 21-23 nucleotides long
now that we have miRNA fully processed and it’s double stranded, what happens to it
one strand is selected and assembles into a RISC
what is a RISC
RNA-induced silencing complex
what is the RISC made up of
ss miRNA, an Argonaute protein, and other proteins
what happens once RISC has one strand of the miRNA attached to it
miRNA guides the RISC to mRNA molecules with complementary sequences in the 3’ UTR
when the miRNA strand is in the RISC, what two things bind once mRNA is around
5’ end of miRNA with the 3’ UTR of the mRNA
once miRNA is bound to the 3’ UTR of mRNA, what happens
two possibilities, both including mRNA degradation
what two types of pairing can occur between the 5’ miRNA and the 3’ UTR of mRNA
extensive and less extensive
describe what occurs when miRNA is extensively bound to mRNA
Arg protein slices through the mRNA = rapid mRNA degradation
extensive matches between miRNA and mRNA are common in ____
plants
describe what occurs when miRNA is less extensively bound to mRNA
Arg doesn’t slice through the mRNA, leading to rapid translational repression and eventual degradation of mRNA (repression occurs via proteins that inhibit translation, and the mRNA may be moved to P bodies for degradation)
less extensive matches between miRNA and mRNA are common in ____
humans
T or F: in mammals, base pairing between miRNA and mRNA needs to be perfect in order to get translation inhibition
false; does not need to be perfect
how many bases need to be matched between miRNA and mRNA to get translation inhibition
2-7
what are the implications of not needing perfect base pairing between miRNA and mRNA
it allows one miRNA to regulate many different mRNAs
what is another function of Dicer and Argonaute proteins other than controlling gene expression
they play a role in cellular defense against viruses and transposons by cleaving dsRNA
describe the mechanism of Dicer and Argonaute defending against viruses
virus puts dsRNA into cytoplasm of the host cell, and this is recognized by TRBP and Dicer, which produce small fragments of siRNA. Argonaute cleaves siRNA so one strand is in a RISC, this complex then binds to virus RNA, and RNA is cleaved by Argonaute = rapid destruction
what is siRNA
short interfering RNA
what is the siRNA knockdown method
used to study the function of any gene by inactivating it with siRNA
what is the role of siRNA in the siRNA knockdown method
siRNA will inactivate a gene of interest, allowing us to determine the function of that gene
what is the first step of siRNA knockdown method called
in vitro production of dsRNA
describe the in vitro production of dsRNA in the siRNA knockdown method
you take the gene of interest and clone it into a plasmid, making a sense and an antisense transcript. Hybridizing the two transcripts together = dsRNA, and now these can be put into cultured cells or whole organisms
once we have dsRNA for siRNA knockdown, how does it get processed into siRNA
dsRNA is processed into siRNA by Dicer
in siRNA knockdown, what occurs once Dicer forms the siRNA from the dsRNA
siRNA forms a RISC, and it directs the RISC to bind to any mRNA that has complementary sequences (note: the mRNA it will bind to is from an organism of interest when we want to determine the funciton of some of its genes)
in siRNA knockdown, what occurs once siRNA and RISC binds to the mRNA with genes of interest
binding = mRNA is destroyed, so no protein is produced. Then we can observe the phenotype to determine the function of the destroyed gene
in siRNA knockdown, how will we determine the function of a gene once we finish the procedure?
siRNA binding to mRNA destroyed it so no protein is produced, so we will observe the phenotype
