Basic question about siRNA, shRNA..etc - What's the difference? (Nov/03/2006 )

I am quite confused about these names.. si RNA, shRNA, RNAi... miRNA(?).. I am doing an "siRNA" experiment by transfecting oligo to the cells, but why it is called siRNA?...

Can I get some answers or some link to the basic knowledge of this topic?

I am quite confused about these names.. si RNA, shRNA, RNAi... miRNA(?).. I am doing an "siRNA" experiment by transfecting oligo to the cells, but why it is called siRNA?...

Can I get some answers or some link to the basic knowledge of this topic?

I hope I hear some feedback about these attempts at definitions.

siRNA - short interfering RNA
These are short synthetic oligos, duplexes of RNA about 21 bases in length with overhanging ends. siRNA is introduced into cells where one strand of the siRNA will complex with proteins (including argonaute) to form RISC (RNA induced silencing complex). The oligo then cleaves target sites in single-stranded RNA that have at least partial complementarity with the siRNA sequence, where complementarity is especially critical with a ~7 base region of the siRNA called the seed sequence. When an siRNA interats with a target sequence with slightly less complementarity, the translation of the target sequence is suppressed. This can lead to widespread gene modulation by a single siRNA sequence. (see: Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Scacheri PC, Rozenblatt-Rosen O, Caplen NJ, Wolfsberg TG, Umayam L, Lee JC, Hughes CM, Shanmugam KS, Bhattacharjee A, Meyerson M, Collins FS. Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):1892-7. Epub 2004 Feb 09.)


shRNA - short interfering hairpin RNA
The precursors to natural miRNA are regions within an RNA strand that form hairpins. When these hairpins are cleaved from the RNA strand by the drosha enzyme they form free stem-loops of RNA with an overhanging end. Cleaved again by dicer, the loop is excised and a duplexes of RNA about 21 bases in length with overhanging ends is released. shRNA with a structure similar to the intermediate hairpin can be manufactured and introduced into cells as oligos. Once cleaved by dicer, their behavior is as an siRNA or miRNA, cleaving RNA or suppressing its translation depending on the degree of homology between oligo and target.


RNAi - RNA interference
Long double strands of RNA with sections complementary to mRNA, when introduced into cells, can be cleaved by Diver to release siRNA. Use of long strands of duplex RNA to trigger suppression is termed RNAi.


miRNA - micro RNA
These are RNAs expressed naturally in cells. They start as regions within an RNA strand that form hairpins. When these hairpins are cleaved by the drosha enzyme they fform free stem-loops of RNA with an overhanging end. Cleaved again by dicer, the loop is excised and a duplexes of RNA about 21 bases in length with overhanging ends is released. These usually suppress translation of RNA strands bearing partially complementary targets in their 3'-UTRs.

Regards,

- Jon

cool! I am reading and figuring out what's what...

Thank u both!

shRNA is "small hairpin" RNA and expressed from a plasmide transfected into the nucleus. By that it is generated like any RNA but the promotor is important (U6 for example, as it has a clearly defined transcription start point). Drosha cuts probably the hairpin and the resulting siRNA (!) is than exported by Exportin 5 into the cytosol where it forms a RISC with Argonaute and other proteins like any siRNA that is directly transfected into the cytosol.
Overexpression of shRNA seems to be toxic in vivo at least as the Exportin-5 pathway might be saturated and so the export of natural miRNAs ("micro RNAs") out of the nucleus is blocked.

Because siRNA's are chemically synthesized they are way more expensive than having a plasmide that codes for a shRNA. But siRNA's seem not to block the processing of endogenous miRNA's. The in silico design of siRNAs is already pretty advanced while the in silico prediction of effective shRNA's still has a way to go (and no, it is not enough to just copy the siRNA with the common loop into a plasmide, probably the Drosha processing is what is not completelly understood and the resulting knock down efficiency is very different compared to the siRNA efficency than sometimes).
Cloning of shRNA plasmids is not trivial either. The common strategy is to have primer who span the complete shRNA (sense, loop, antisense) and just anneal those followed by ligation. But because of the internal complementarity of the sequences, the primer tend to anneal with themself and you will get many wrong clones. Sequencing of clones is essential here - but needs some experience too because of the secondary structures.
In other words: what you should decide for is a matter of what you want to achieve, what system you want to work in and what you have available moneywise.

Edit: Just to prevent confusion concerning Dincer: Dicer only processes the shRNA if that one reaches the cytoplasm and is still long enough. As Dicer cuts depending on length while Drosha seems to cut depending on structure. But as I said, Drosha is still not really understood.

Thanks Wincel, that clarified shRNA for me. As you wrote, they are not introduced as oligos but are expressed as longer transcripts in cells.

I've found two commonly-used expansions of the acronym now, short hairpin RNA and small hairpin RNA.

I'll watch for developments of the drosha-recognition story; that's an interesting unsolved problem.

Best,

- Jon