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One reason that microscopy is such an important tool for biological research is that biological entities function in part through their organization. Just take a look at any micrograph and you will see order, organization and compartmentalization.

i-630809d72bf3e99744e4cf7796f96995-cell_131_1.gifBut much of the organization that produces biological activity is yet to be uncovered. To highlight this I'll just point out a recent paper on mRNA distribution within a developing embryo.

In this study (published in a recent issue of Cell, see cover left), researchers embarked on a large scale analysis of mRNA localization in developing Drosophila oocytes. In total, they localized endogenous transcripts from 25% of the fly genome. Of course many transcripts are localized to distinct parts of the fly embryo. Some famously localized transcripts, which drive the differentiation of flies, are oscar, bicoid and nanos to name a few. But what about the distribution of mRNA within cells*? Here is what they found:

Of the 2314 mRNAs expressed, a remarkable 71% are subcellularly localized.

Some types of localization patterns seen were:
- Nuclear associated (seen with 3.5% of the transcripts)
- Perinuclear associated (3.5%)
- Excluded from the apical region (49.5% !!!)
- Excluded from the basal region (11.1%)
- Membrane associated (0.3%)
- RNA "islands" (1.9%)

Some categories are quite intriguing such as:
- Apical foci (1.0%)
- Apical clusters (1.2%)
- Microtubule associated (0.4%)
- Centrosomal associated (0.3%)
- Cell Junction associated (0.5%)
- Chromatin associated (0.6%)
- Spindle mid-zone (0.4%)

A full list can be seen in Table 1.

Below is a nice collage of FISH fluorescence from embryos used in the study.

i-bd2851265b7bffd4f70a0f188ab084eb-droppedImage.jpg

How is this localization acheived? From the paper:

Gene ontology (GO) term enrichment analysis reveals that maternal transcripts are enriched for genes involved in RNA metabolism (Figures 1D, S3, S4A, and Table S1), including, for example, many components of the spliceosome (crn, prp8, SmD3, snRNP69D, snRNP70K, U2af50). This finding is consistent with the large and diverse maternal mRNA contingent, and the need to organize aspects of their processing, translation, stability and localization. Interestingly, these terms are more strongly enriched among the more stable maternal mRNA subsets that continue to be detected through stages 6-9 (Figure S4A and Table S1), in agreement with recent observations (Tadros et al., 2007a).

So many mRNAs that are pumped into the embryo from the mother (i.e. maternal transcripts) encode RNA binding proteins. During the course of developement, these maternal transcripts are the earliest mRNAs to be translated.

Other small items:

A big surprise is that distinct endoplasmic reticulum (ER) targeting of transcripts were not widely seen despite the fact that 20-40% of the transcripts code for proteins whose synthesis occurs at the ER. This may be due to the fact that the ER was not well preserved in their samples or that protein synthesis at the ER is low during development.

Another intreaguing finding is that much of the localization of the mRNA matched the distribution of the transcript's encoded protein. This indicates that compartmentalization of proteins within subcellular loci may be in part dictated by where the protein was synthesized. I must point out some studies in budding yeast. In those studies although the mRNA was localized to the daughter's ER and the protein was confined to the daughter cell, disruption of mRNA localization did not lead to a disruption of protein segregation. Thus it may be that several redundant factors help to compartmentalize most proteins and that mRNA location plays only a small part.

The entire collection of images is available in a public resource: Fly-FISH Home, University of Toronto

So this goes to show you that there is a tremendous amount of organization in biological organisms, even at the mRNA level.

Ref:
Eric Lécuyer, Hideki Yoshida, Neela Parthasarathy, Christina Alm, Tomas Babak, Tanja Cerovina, Timothy R. Hughes, Pavel Tomancak and Henry M. Krause
Global Analysis of mRNA Localization Reveals a Prominent Role in Organizing Cellular Architecture and Function
Cell (07) 131:174-187 doi:10.1016/j.cell.2007.08.003

Shepard KA, Gerber AP, Jambhekar A, Takizawa PA, Brown PO, Herschlag D, DeRisi JL, Vale RD
Widespread cytoplasmic mRNA transport in yeast: identification of 22 bud-localized transcripts using DNA microarray analysis.
PNAS (03) 100:11429-11434 doi:10.1073/pnas.2033246100

*As PhysioProf notes, the early Drosophila embryo is a large multi-nucleated syncytia, which is technically one cell. Most of the classic asymmetrically localized transcripts are distributed asymmetrically across the syncytia as a whole, but not much was known about the distribution of transcripts at the local level.

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I recently saw Pat Brown talk about just this, and he mentioned this paper in his talk. He says that his "bold hypothesis" is that subcellular compartmentalization is a significantly more active process than we appreciate. I think that hypothesis is becoming less bold as more data piles up to support it :)

I am kind of curious, however...the textbook explanation we get in biology classes is that diffusion of these proteins occurs very rapidly. So how is the compartmentalization achieved? I can understand localizing things to the nucleus, the mitochondria, or tethering it to the cell membrane. Is there a lot more tethering to other substructures and less diffusion than we assume?

I know what you mean. A colleague of mine once told me of an experiment that tracked single GFP monomers in a fission yeast cell - on average it took 5 sec for a molecule to diffuse from one end to the other (~10um).

For quite a while Rob Singer's group has had data that actin mRNA is localized to the leading edge, however many groups have shown that inhibiting translation does not affect cell migration. Where local translation does seem to mater is in the post-synapse. Translation inhibition down regulates LTP, memory and a whole plethora of phenotypes that rely on synaptic remodelling.

It could be that these mRNA localization phenomena may have more to do with feedback mechanisms where local concentrations of protein, or local protein modification, may feedback on local protein synthesis. In this way there is a global regulation but it requires the mRNA to be concentrated where the protein is.

"Of course many transcripts are localized to distinct parts of the fly embryo. Some famously localized transcripts, which drive the differentiation of flies, are oscar, bicoid and nanos to name a few. But what about the distribution of mRNA within cells?"

This is just a semantic quibble, and I'm sure you know this, but at the stage of development when many of the "famous" transcripts are localized, the whole fly embryo is a single multi-nucleate cell.

Damn syncytia! (Moreover most of the classic mRNAs I mentioned are asymmetrically localized in the oocytes). I'll clean it up thanks.