Tuesday, July 31, 2007

learning note about ncRNA

  • siRNA and miRNA
Both are kinds of ncRNA(non-coding RNA);
Both are small, and short, ~20nt in length;
Both are involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene;
Difference in structure:
siRNA is a short (usually 21-nt) double-strand of RNA (dsRNA) with 2-nt 3' overhangs on either end.
while, miRNA is single-stranded RNA molecules of about 21-23 nucleotides in length with step-loop secondary structure.

Another essential differcence I guess is siRNA is exogenous, while miRNA is endogenous. So, when people use this techonology(I think siRNA is just kind of technology, not RNA in cell naturally like miRNA) to do RNAi, two issues (innate immunity, and off-targeting) are chanllenging us.
Direct transfection of an exogenous siRNA can be problematic, since the gene knockdown effect is only transient, particularly in rapidly dividing cells. One way of overcoming this challenge is to modify the siRNA in such a way as to allow it to be expressed by an appropriate vector, e.g. a plasmid. This is done by the introduction of a loop between the two strands (like a miRNA in form), thus producing a single transcript, which can be processed into a functional siRNA. People also use this way to avoid the non-specific effects by siRNA; converting siRNA into a miRNA. MicroRNAs occur naturally, and by harnessing this endogenous pathway it should be possible to achieve similar gene knockdown at comparatively low concentrations of resulting siRNAs. This should minimize non-specific effects.
  • about miRNA
miRNAs are first transcribed as part of a primary microRNA (pri-miRNA). This is then processed by the Drosha with the help of Pasha/DGCR8 (=Microprocessor complex) into pre-miRNAs. The ~75nt pre-miRNA is then exported to the cytoplasm by exportin-5, where it is then diced into 21-23nt siRNA-like molecules by Dicer. In some cases, multiple miRNAs can be found on the pri-miRNA.
It's hard to know how long and what's the original form of pri-miRNA on genome, since it's not stable after transcribed. Could be very long, ~several kb, connected with many stem-loop structure. (Is it possible to contain intron, either? I guess it's possible. And also alternative transcription, why not?)
pri-miRNA then is Drosha-proceeded into pre-miRNA. pre-miRNA is ~60-100nt long, hairpin structure (only one loop?) . Then it's exported outside to cytoplasm, where they are digested into a sequence-specific single-strand mature miRNA by Dicer.
The mature miRNA then binds to a complex called RNA-Induced Silencing Complex(RISC). The RISC-bound miRNA then binds to specific mRNA by significantly but not completely complementary to the mRNA.
The ways that miRNA inhibits gene expression are different in plant and animal. In plant, the formation of the double-stranded RNA(dsRNA) through the binding of the miRNA triggers the degradation of the mRNA transcript through a process similar to RNA interference (RNAi); while in animal, it prevents translation without causing the mRNA to be degraded. Animal miRNAs are usually (partially) complementary to a site in the 3' UTR whereas plant miRNAs are usually (highly) complementary to coding regions (also found in 5' UTR and 3' UTR) of mRNAs.

2 comments:

Anonymous said...

Good day Dong Xianjun,

You might be interested in joining the miRNA group just started on Nature Networks:
http://network.nature.com/group/mirna

Regards,

- Jon

Dong Xianjun said...

Thanks for visiting, Jon

I will have a look later.