Hello. What do you mean by polysome fractions before preparing the total RNA. Did you perform a  grandient to analyse the profile of the ribosomes from sub-units to polysomes??? And did you only take the ribosomes (single, polysomes & polyribosomes) for your RNA preparation???

This is a very common problem encountered in RNC-mRNA extraction. Since this process is very tricky and you'd better not to add RNase inhibitor in most of the steps, the RNA degradation is very likely to happen. RNC-mRNA is very fragile and extremely difficult to obtain in intact and full-length form. That's why you see mostly the high-abundance mRNAs like actin. Extreme care must be taken during the whole experimental process.

40S subunit attaches the mRNA during the translation initiation. This complex moves slowly in sucrose gradient. Therefore you should detect it not in the polysome fraction. Meanwhile, during the ultracentrifugation, 40S-mRNA complex is easy to disassemble if the process lasts long. Crosslinking might be a useful way to keep the 40S-mRNA complex.

Ok. Seems like my question has not been interpreted correctly. Let me rephrase it. I have performed polysome analysis on growing Drosophila larvae, and fractionated the contents into 12 equal fractions, with fractions 1-4 containing 40S, 60S and 80S peaks and the remaining fractions containing the polysomes. I performed qRT-PCR analysis to calculate the percentage of an mRNA of interest in the 12 fractions. I have data for  actin, Myc and many other mRNAs , all of which indicate that most mRNAs have a high ribosome occupancy (% of mRNA associated with 1 or more ribosomes ) of greater than 90-95%. I have enough RNAse inhibitors in the form of Heparin and Ribolock (Fermentas). I have no problems with RNA degradation, as the above data has been replicated convincingly multiple times. I do detect mRNAs in the sub-polysome fractions (fractions 1-4), but as mentioned previously they represent only 5-10% of the total mRNA content. So my question comes down to this - does this low percentage binding to sub-polysomes, represent degradation of non-polysomal mRNAs, or is there some other reason for low-levels of sub-polysomal mRNA association in-vivo (in Drosophila)?

OK, understood. For actin, the translation is usually very active, especially in the proliferating cells and tissues. Therefore, 90-95% ribosome occupancy (as you defined) can be true.

This is in response to the point raised by Dr. Zhang. I don't think I am getting disassembly of 40S-bound mRNAs. The reason is pretty simple. If I have a higher percentage of mRNAs bound to the 40S mRNP, even after disassembly, the disassembled mRNAS should be found as free mRNAs or as some other smaller complex in P-bodies or stress granules. This doesn't seem to be the case, I have very less percentage of mRNA in fraction 1 which should contain free mRNAs. Since, I have lots of Heparin and RNAse inhibitor present, degradation can also be discounted, and the reproducible nature of this experiment confirms that degradation can be ruled out. Also, I do get sub-polysomal association for a few mRNAs, suggesting that 40S binding should be intact for these mRNAs. Most other mRNAs behave differently. I do have mRNA in all of the sub-polysomal fractions for all mRNAs, however, the percentage of mRNAS bound to ribosomes turns out be much much greater, amounting to over 90% of mRNAs being to ribosomes. Overall, my data suggests that most mRNAs have a preferential association with polysomes. 

If the translation is very efficient in your Drosophila (I am not an expert in the drosophila model)  and actin mRNA I guess is rather represented within the mRNA total population, the actin mRNA when taken in the tertiary complex, the scanning starts to produce the first  ribosome. It is followed rapidly by a succession of new tertiary complexes taken up by the same thread of mRNA and so.  Therefore,  a  number of ribosomes accumulate  on the tread of mRNA  given rise to polysomes (n2-nx). That is why the presence of this specific mRNA is low in sub-ribosome fractions and since  highly translatable is recovered in your polysome fractions. 

Actin was just an example. Almost all mRNAs that I tested using qPCR - Myc, 4EBP etc had 90-95% ribosomal occupancy. a few ribosomal proteins had lower occupancy values of  60-80%. The only ribosomal occupancy data that I have access to is in Drosophila embryos and perhaps adult flies. My polysome profiles were done on Drosophila larvae, for which I have not seen data anywhere else. Which is why I put forth the question, in order to confirm my findings.

Drosophila larvae, undergo rapid growth - 200 fold increase in mass and 1000 fold increase in volume in 5 days of larval development. It is my guess that such rapid growth requires high translation rates which is why I see such high ribosomal occupancies for most mRNAs.

The other possibility is that the non-ribosomal mRNAs might be degraded. Is there any way by which I can test if this might be the case?

Yes the marked mass  increase is due to translation enhancemen,t I guess connected to increase in transciription. Did you look at transcription because  translation of  the transcription factor mRNA  is probably increased as well  leading to transcription increase. Translation increase might occur before transcription increase.

To study the degradation maybe you could at least verifiy that the mRNAs of interest are still present in the ribosomal and sub-ribosomal preparation. And control that it is still active for translation in an vitro cell lysate.   Study mRNA degradation is quite tricky and mRNP preparation!!!!. 

Thank you Catherine.

Comparing translation and transcription in Drosophila right from embryogenesis is tricky, as there is no transcriptional activity during early embryogenesis. Almost all the cellular activities, including axis specification occurs purely via translational control. Hence, I have not compared the translation and transcription between embryos and larvae. 

About the degradation, yeah I was wondering about degradation in-vivo, and so posed the question. I do see mRNA (all the transcripts) in the fractions containing 40S subunit, but as I mentioned previously compared to the percentage of mRNA bound to ribosome  the 40S bound mRNA is only 5-10%.

If I understood well the question you ask :  are the mRNAs that are not engaged in 40S,  80S and polysomes degraded?

For the poor % of mRNA found in the 40S I think that I answered,already: as soon as the mRNA is  engaged on the 40S, if the translation is very efficient "good mRNA thas is with a nice relaxed  5' UTR structure ", new ternary complexes  are rapidly engaged  driving  topolysomes (according to  the scanning model of translation). 

Ok. Thank you for your explanation Catherine.