Exam 5: Lecture 6 Flashcards
1
Q
RNAi Pathway
A
- process by which mRNA transcripts are either degraded or prevented from being translated
- most basic form, segment of double stranded RNA cleaved into relatively small fragments by Dicer enzyme
- resulting pieces are called short interfering RNAs (siRNAs)
- appears to be present in all metazoans and is used to regulate gene expression
2
Q
siRNAs
A
- bound by complex of proteins called RISC
- this complex unwinds the two strands
- one strand discarded while other is retained
3
Q
RISC-RNA complex
A
- searches for complementary sequences within messenger RNAs and forms localized regions of double stranded RNA
- formation of these complexes triggers destruction of mRNA transcript which then prevents translation
- some circumstances mRNA transcript not degraded but just prevented from being translated
4
Q
microRNAs
A
- miRNAs
- type of siRNAs involved in silencing of gene expression
- most cases gene expression is down-regulated and not eliminated therefore miRNAs are thought to function in the “fine-tuning” of gene expression
- most metazoan genomes predicted to encode 100’s to 1000’s of miRNA genes
- these genes transcribed by RNA Pol II
5
Q
pri-miRNA
A
- initial transcript
- around 500 bases
- folded into single stem loop with trailing tails
- then cleaved into smaller (~70nt) stem loop pre-miRNA fragment by two nuclear enzymes called Pasha and Drosha
6
Q
pre-miRNA
A
- exported into cytoplasm by Exportin-5
- once in cytoplasm, Dicer enzyme cleaves loop section leaving 21-24nt double-stranded RNA fragment (miRNA)
- this fragment bound by RISC
- important component of this complex is RNA binding protein Argonaute (Ago)
7
Q
Within RISC
A
- two miRNA strands separated (one degraded, one retained)
- RISC-miRNA complex then searches for complementary sequences within messenger RNA transcripts present within cell
8
Q
Region of Complementarity
A
- if this region between miRNA mRNA transcript is perfect, then transcript is degraded by RISC
- only partial homology between two fragments then RISC will prevent translation without degrading transcript
9
Q
miRNAs Binding
A
- most common position of miRNAs to bind within mRNA transcripts is 3’ UTR
- have been shown to bind to other positions as well
- binding of miRNA to complementary sequences within 3’ UTR can lead to degradation or blocks on translation
10
Q
Most Important Determinant of Degredation
A
- whether or not first 7-8 bases within miRNA have perfect or imperfect homology with mRNA transcript
- if perfect transcript degraded
- less than perfect transcript survives and translation is blocked
- degree of homology between remaining 13-14 bases of miRNA and mRNA transcript is not a factor in determining whether or not transcript will be degraded
11
Q
Binding of mRNA transcript
A
- can be bound by multiple miRNAs
- many mRNAs will contain multiple binding sites for a single type of miRNA
- several different types of miRNAs can simultaneously bind to a single mRNA transcript
- regulation of single transcript dependent upon combinatorial code of miRNAs that bind to 3’ UTR
12
Q
Lengthy of mRNA 3’ UTR
A
- factor in how many different miRNAs can bind to transcipt
- mRNAs with long 3’ UTR segments can be bound by more miRNAs than mRNAs with very short 3’ UTR segments
- a single type of miRNA can bind to multiple different mRNA transcripts thereby regulating expression of multiple genes
13
Q
Inhibiting translation Initiation
A
- PABP binds to 3’ end of transcript interacts with several eukaryotic IF’s which bind to 5’ end making circular RNA species thus improving stability and allows higher levels of translation
- as miRNAs bind to 3’ end of transcript, accompaning Ago protein displaces IF’s and instead binds to 5’ end
- without IF’s ribosome can’t initate translation
14
Q
Post-initiation Inhibition
A
- recruitment of miRNA-Ago complex to mRNA transcript allows for Ago protein to displace ribosome from transcript
- shortened proteins are then degraded
15
Q
Nucleases Inhibition
A
- degradation of transcript can occur when Ago protein recruits RNA specific nucleases
- enzymes digest messenger RNA transcript
