miRNA* - miRNA star forms - (May/18/2008 )
maybe someone can help me to grasp this..
What exactly are these miRNA*?
They derive also from the same hairpin structure like the "normal" miRNA. So if the "normal" miRNA is then later called the "mature miRNA" or "guided strand", the miRNA* is the passenger strand. To what extent are these miRNA* complementary to the "normal" miRNA? Possible, that the miRNA star forms can act as antimiRs?
And why have only few star forms been discovered or predicted????
Thanks a lot for any answer!
Somebody else correct me if I'm wrong, but I think it's predominantly about cloning percentages:
The first cloning strategies only had a few clones for each miRNA discovered. These were named with no * - i.e. the mature, guide miRNA strand (miR-xx). But then later on, and more recently with the high throughput sequencing, people started to also clone/sequence the opposite strand. The if the opposite strand is found at low frequency (less than 15% of the guide strand) it is named miR-xx*. If, however, the two "arms" of the pre-miRNA are more equal in their distribution then they are not named *, but instead miR-xx-3p and miR-xx-5p, depending based upon their location to either 5' or 3' of the miRNA molecule. Both species could potentially have a biological effect.
As to your second question, why there are few miRNA*, well it has been hypothesized that the strand with the highest stability at the 5' end is degraded. I guess if they are rapidly degraded then they would not be frequently cloned. But you could still predict them, but I think miRbase only puts them in their database if there is evidence for their existence??
The star strand is mostly complementary to the guide strand, but there are usually single-stranded overhangs on each end, there is usually one or a few mispairs and there are sometimes extra or missing bases causing single-stranded "bubbles". Looking over the files in miRBase is a good way to get a feel for the degree of complementarity of guide and star strands.
The passenger strand of an siRNA is analogous to the star strand of a miRNA.
Synthetic oligos that are intended to interfere with maturation or activity of miRNAs, such as antimiRs or Morpholinos, are designed to be completely complementary to their targets. Since a star strand is not completely complementary to a guide strand, an inhibiting oligo would not have a sequence identical to the star strand even if they bound to the same guide strand sequence; the artificial oligo would be perfectly bound, while the star strand would (at least) contain mispairs.
Thanks for your replies..
To my next problem: We tried to overexpress miRNAs by transfecting the miRNAs that are encoded in an expression vector (either cloned by ourself or ordered) into human endothelial cells. For quantification of the miRNA level we use AB Taqman Real time kits. As results we have higher miRNA level in the cells transfected with the vector ctrl (empty vector) than in the cells transfected with the vector enconding the miRNA.
So can you imagine that it is possible that after transfection the star form/passenger strand is incorporated into RISC complex instead of the guided strand? And by that, the endogenous miRNA level is inhibited? If yes, to what extent? 50% "knockdown"..less..more? And what about the frequency? I haven't read about this problem in any papers..
We have this problem with at least 4 miRNAs..but we CAN overexpress them when transfecting them as precursor miRNAs (Ambion).
Thanks a lot!!
Exactly how did you clone into your plasmid? PCR or oligo? Most people PCR amplify the mature miRNA +/- ~100bp 5' and 3' so that you clone the precursor miRNA, which gets processed by drosha and dicer...