Tuesday, July 24, 2007

Somatodendritic microRNAs   posted by amnestic @ 7/24/2007 10:54:00 PM

Kosik and colleagues used laser capture microdissection to get RNA populations from dendrites or cell bodies of cultured rat neurons. They optimized their technique so that mRNAs known to be enriched in dendrites, such as CaMKII and MAP2, showed about equal levels from soma and dendrite. They then performed multiplex PCR for several mRNAs and 187 miRNAs. The distribution of mRNAs and miRNAs is similar with a large somatic population and a gradient going out into the dendrites. Some small proportion of miRNAs have a little bit of dendritic enrichment. One point the authors are trying to get across is that there is no such thing as a 'dendritic RNA' because even the mRNAs and miRNAs that show some dendritic localization usually show just as much in the cell body.

Two nice things are that this paper validates a couple miRNA target prediction programs (Pictar and Targetscan) and that they provide a quantitative view of the miRNA copy number per cell. Both of these prediction programs suggested that miR-26a would target MAP2. This is convenient since both showed a somatodendritic distribution, meaning they hang out together even out at the farthest dendritic reaches. Inhibition of miR-26a with a synthetic oligonucleotide resulted in increases in MAP2 protein expresion, as one would expect from the classic miRNA-target relationship. As far as I am aware this brings the total of known dendrtici miRNA target pairs up to three, the other two being mir-268 and CaMKII (in drosophila) and miR-134 and LIM-Kinase. Quantification was achieved using PCR with known copy number standards. They knew how many cell bodies they captured, so they could get a copy number per cell estimate (probably a minor undershoot since even if they are awesome they probably couldn't save allllll the RNA from degradation). Anyway, they found... well I'll let them explain it:

rno-miR-124a is among the most abundant miRNAs in neurons and fell in the range of 10^4 copies per neuronal cell body. Despite its abundance, rno-miR-124a is enriched in cell bodies. rno-miR-26a and rno-miR-16 are less abundant miRNAs and fell in the range of 10^3 copies per neuronal cell body (Table 6). Because (delta)Ct of 2.61 +/- 0.39 describes the distribution of most miRNAs between the cell body and neurite, the number of copies of many miRNAs distributed along this gradient may be as low as in the hundreds of copies in the dendritic compartment. Even a one-order-of-magnitude error in this number is far below the number of synapses on the dendritic tree, and, therefore, the copy numbers of many miRNAs are likely to fall below one per synapse.

Delta Ct refers to the number of PCR cycles (i.e. doublings) it takes for the dendritic levels to reach the somatic levels. For instance, a delta Ct of 2.61 means that there are 2^2.61 (~6.1) times more somatic copies of the miRNA than there are dendritic copies.

I was particularly intrigued by this last sentence even though I have no idea what it means:

Stochasticity derived from the effects of miRNAs will contribute to the activation barrier for coherent responses, to the utilization of information provided by translational bursting, and to the flexibility needed by dendrites to sample alternative states (Kaern et al. 2005).

Guess I'll have to read Kaern et al. real quick.

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