Exam 5: Lecture 6 Flashcards Preview

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RNAi Pathway

-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



-bound by complex of proteins called RISC
-this complex unwinds the two strands
-one strand discarded while other is retained


RISC-RNA complex

-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



-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



-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



-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)


Within RISC

-two miRNA strands separated (one degraded, one retained)
-RISC-miRNA complex then searches for complementary sequences within messenger RNA transcripts present within cell


Region of Complementarity

-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


miRNAs Binding

-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


Most Important Determinant of Degredation

-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


Binding of mRNA transcript

-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


Lengthy of mRNA 3' UTR

-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


Inhibiting translation Initiation

-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


Post-initiation Inhibition

-recruitment of miRNA-Ago complex to mRNA transcript allows for Ago protein to displace ribosome from transcript
-shortened proteins are then degraded


Nucleases Inhibition

-degradation of transcript can occur when Ago protein recruits RNA specific nucleases
-enzymes digest messenger RNA transcript


pb Gene

-pb expressed in anterior segments of embryo that will eventually give rise to parts of proboscis
-mutations that eliminate pb activity lead to transformation of parts of pb into two legs


Analysis of Milkweed Bug Genome

-indicates that all eight Hox genes in fruit fly are also present in milkweed bug
-pb loss-of-function mutants do not yet exist in milkweed bug


How can you determine if the pb gene plays a similar role in establishing the identity of the proboscis of the milkweed bug?

-inject short double stranded RNAs that have sequences complementary to the pb mRNA into developing milkweed bug embryo
-RNAi pathway processes the dsRNA and will target pb mRNA
-levels of pb protein reduced due to degradation of transcript
-reductions of pb in milkweed bug lead to similar proboscis-leg transformations
-indicates role of pb has been conserved between Drosophila and milkweed bug