Small RNAs and control of gene expression

Micro-RNAs and RNA interference

-          a phenomenon in which RNA stability or translation is controlled by short RNAs (micro-RNAs)

-          micro-RNAs made like other RNAPII transcripts initially (Fig. 18.17a)

o       capped, polyA

o       no ORF, so ignored for a long time

o       all have at least one stem-loop structure (and that is the key)

§         stem-loop not perfectly basepaired

-          these initial transcripts are processed (Fig. 18.18a) by two nucleases

o       Drosha crops the RNA so there is just the stem-loop left

o       Dicer cuts off the loop and maybe a little more processing

o       This imperfect doublestranded molecule is incorporated into a protein complex called RISC

§         If the RNA carried by RISC complex basepairs incompletely with mRNA, generally get translational silencing

§         If the RNA carried by RISC basepairs completely with mRNA, generally get mRNA destruction

·         Operates catalytically, so one RISC complex destroys many RNAs

§         The general term for both these mechanisms is RNA interference (RNAi)

How much does this happen?

-          now have found lots of miRNAs in lots of different plants and animals

o       2% of plant genes may be regulated this way

o       1/3 of human genes may be regulated this way

o       These numbers are very fluid, this is a very new field

o       Doggone bunch of this, for sure

siRNAs

-          Found by accident

-          injected long double strand RNAs, found that all mRNAs containing that sequence were destroyed, not only in cells where injected, but all over the organism and through several generations

-          found that long dsRNA gets cut up into pieces, short interfering RNAs (siRNAs)

-          induces destruction of mRNA of matching gene

-          turns out that the siRNAs are processed by the same machinery the processes micro-RNAs in cells

-          just stumbled onto this in that way

-          Nobel prize 2006

Knocking out expression of a gene

Use siRNA to eliminate mRNA of your choice

-          all you need to do is make a

o       dsRNA of 21 to 24nt or so with 3’ overhang

o       hairpin

-          that has perfect complementarity to the mRNA you want to destroy

-          get this RNA into cells in culture or into an entire animal

o       transfection into cells

§         check mRNA levels to see if the mRNA you targeted is gone

§         see what effect that has on cells

o       in animals

§         inject lipid vesicles containing siRNAs into animal

§         look for repression of mRNA in the animal

§         see what effect it has