miRNA single or double stranded? - (Jan/10/2007 )

I was told today by a coworker that miRNA does not exist as a double stranded RNA molecule EVER!

I am trying to confirm if anyone in the forum has heard of this concept. I always tought that the product of DICER cleavage is a double stranded molecule.
Then, somehow, this molecule is separated into two single strands and the "mature" single stranded RNA goes into the RISC.

He claims that the product of DICER is directly a single stranded miRNA that goes into the RISC. He also told me that what I extract using a mirvana miRNA isolation kit are all single stranded miRNAs.

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DICER is a ribonuclease, so it's product is single-stranded not double..






he is correct..
miRNA exists as double only before maturation (looped) and at this time it is called pre-miRNA
double-stranded pre-miRNA>bind to DICER>mature single-stranded miRNA>RISC

click here

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For your first statement:

DICER is a RNAse III enzyme. It cuts double stranded RNA. Its product should be double stranded RNA.
This is an assumption that several people have made (including myself).

Here is a quote from a publication in Science (Lund, 2003):

"First, nuclear Drosha cleaves long primary transcripts releasing 60- to 70-nt pre-miRNAs...Then, cytoplasmic Dicer processes pre-miRNAs into 20- to 22-nt duplexes bearing two nucleotide single-stranded 3' extensions. Generally, only one strand of the duplex serves as the mature miRNA."


For your second statement (and the ambion slideshow):

you cannot assume such a simple process as: pre-miRNA>bind to DICER>mature single-stranded miRNA>RISC

I am certain that at no point in time you will have single stranded miRNA floating in the cytoplasm.


Here is what another collegue has suggested (the man is an RNAi expert so I am inclined to believe him):

The product of DICER must be a double stranded RNA product. What happens next after DICER cutting is that the product
is (somehow) separated into its two strands. The strand that does not go into the RISC is degraded quickly.
Thus, it is hard to find this small duplexes in cellular rna extractions.


BTW, I appreciate your response. I just want to mention that this thread was created for the sole purpose of advancing my (and anyone else who reads this thread) knowledge on the miRNA pathway.

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I remember there is a kit based on the concept of dsRNA form of miRNA
have any idea for it?

http://www.systembio.com/microRNA_Discovery.htm

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I feel most of miRNA pruifed from tissue are in the form of single strand.
While there should still a portion miRNA existed in double strand form, think about the concentration of miRNA*, where are them come from?

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well, thanx for your correction
i have no deep interest in miRNA, what i mentioned is just in general
and of course, experts can dig into it better than me

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I'll give a shot at summarizing my current understanding of the steps in miRNA maturation.

An interesting recent paper addresses the timing of the double strand -> single strand conversion:
http://www.rnajournal.org/cgi/content/abstract/rna.283207v1

The authors show that the double strand binds to the Scr2-R2D2 complex and after association of other factors to form a pre-RISC complex (including Ago2), the Argonaute protein employs the same catalytic activity it uses to cleave complementary mRNA to cleave the passenger strand of the small dsRNA, leaving the guide strand intact and active on the now-mature holo-RISC complex.

So, Drosha cleaves the stem-loop from the pri-miRNA complex in the nucleus producing a pre-miRNA, Dicer cleaves the loop from the stem producing the short dsRNA, the pre-RISC complex forms around the short dsRNA and cleaves one strand which then dissociates, leaving a single RNA strand in the holo-RISC complex.

Good discussion, I hope this thread lasts.

- Jon

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Hi
I found more information about this subject in following paper.
Check page 3 under the topic MicroRNA Biogenesis.

MicroRNAs and their regulatory roles in animals and plants

Hi

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Jon Moulton

http://www.rnajournal.org/cgi/content/abstract/rna.283207v1

Mici

MicroRNAs and their regulatory roles in animals and plants

above two paper` IF are very poor! so,I prefer to don`t believe it.

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Hi,

This confused me for a while also but after reading the papers that were available on the subject this is what I understand happens:

After Drosha processing the pre-miRNA is transported to the cytoplasm by exportin 5, at which stage it is a duplex i.e. double stranded. Action by the enzyme Dicer then cleaves these duplexes to 22nt.

When this duplex is then taken up by the RISC complex one strand of this short-lived duplex (the star strand) disappears[lost or degraded], whereas the other strand remains as a mature miRNA. Therefore the miRNA associated with RISC is single stranded.

This is in part due to the protein Ago 2 which is involved in to the association of the miRNA with RISC. The structure of the Ago protein, in particular its PAZ domain, is such that it contains a cleft which can base pair with the 3' overhang of the miRNA thus anchorring one of the strands of the duplex to the RISC. During RISC assembly, the cleavage products (approximately 22-nt miRNA duplexes) are rapidly converted into single strands. The strand with relatively unstable base pairs at the 5’ end typically remains in siRISC [Schwarz, D.S. et al., 2003, Khvorova, A. et al., 2003] and aligns with the mRNA while the other strand is released into the cytoplasm.

These star strands are shortlived, ususally degrading, but it is important to note that sometimes the star strands can be detected during cloning or northern blots.

Hope this is some help!

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