Polysome analysis

Noll H (1969) Polysomes Analysis of structure and function. In Campbell PN, Sargent JR (eds) Techniques in protein biosynthesis, vol II. Academic Press, London New York, pp 101-179... 

Genome-Wide Analysis of mRNA Polysomal Profiles with... 

Arava, Y. (2003). Isolation of polysomal RNA for microarray analysis. Methods Mol. Biol. 224, 79-87. 

Figure 10.1 Experimental schemes for microarray analysis. All experimental schemes start with a separation step of the cell lysate by velocity sedimentation in a sucrose gradient (top scheme). Collection of the desired fractions is assisted by a continuous ultraviolet (UV) reading of the gradient (an example of such UV reading is shown in each section). This allows determination of the sedimentation position of the 40S, 60S, 80S, and polyribosomal complexes (2,3, and more).Three general ways for fraction collection and analysis are presented (sections A, B, and C) (A) Collection of two fractions (free and polysomes) and direct comparison between them, with the free mRNA fraction labeled with green dye and the polysome fraction labeled with red dye. (B) Collection of two fractions and indirect comparison between them by utilizing an unfractionated reference RNA. (C) Collection of multiple fractions (four in this case), where each fraction is compared to an unfractionated reference sample. The blue arrows indicate the addition of spike-in RNA to each fraction and to the reference RNA.

There are numerous protocols for polysomal gradients preparations that differ mainly at the step for harvesting the cells, and the gradient composition and separation times. The protocol presented later was optimized for isolation of polysomal mRNA from the yeast Saccharomyces cerevisiae, yet many steps will be similar to other eukaryotes and the procedure can easily be modified for other organisms. We will use this protocol as a template on which we will indicate and highlight points that are critical for the microarray analysis. Generally, the RNA isolated by this protocol can be used for analysis by DNA microarray, Northern blot, or RT-PCR. 

We utilize three independent methods to assess a compound s effect in vivo on translation—metabolic 35S-methionine/cysteine labeling, polysome profile analysis, and effects on transfected reporter gene activity. 

It is likely that protein binding and other macromolecular interactions also play a role in stabilizing the rRNA structures. A recent analysis12 reveals that the binding of specific ribosomal protein to rRNA in vitro results in changes within the rRNA modification patterns. Because the experiments detailed herein examine a population of rRNA within the cell, and thus may be analyzing a variety of RNA conformations, it is remarkable that very consistent results are observed. However, the consequence of polysome assembly and translation on the structure of the rRNA and how these structures might be distinct are not addressed in this analysis. In vitro DMS modification of soybean RNA has revealed some differences in base reactivity relative to that observed on RNA modified in vivo as described herein.36 ... 

In weak composite acrylamide-agarose gels Dahlberg et al. (1969) reported that not only ribosomes but polysomes could be made to migrate, and this provides indeed an elegant and convenient procedure for the analysis of polysome populations (Fig. 10.15). Addition of the antibiotic streptomycin which is known to bind to ribosomes, causes... 

Figure 5 Starting from natural mRNA, a cDNA library (A blue) is produced and like ribosomal display, the cDNA is transcribed into mRNA (B) with no stop codons. The 3 -end of each mRNA molecule is ligated to a short synthetic DNA linker (C) and sometimes a polyethyleneglycol spacer, which terminates with a puramycin molecule (small red sphere). The ligation is stabilized by the addition of psoralen (green clamp), which is photoactivated to covalently join both strands. Addition of crude polysomes or purified ribosomes (D) results in translation of the mRNA into protein, but the ribosome stalls at the mRNA-DNA junction. Since there are no stop codons, release factors cannot function and instead the puromycin enters the A-site of the ribosome (A). Because puramycin is an analog of tyrosyl-tRNA, the peptidyl transferase subunit catalyzes amide bond formation between the puromycin amine and the peptide carboxyl terminus, but is unable to hydrolyze the amide link (which should be an ester in tyrosyl-tRNA) to release the dimethyladenosine. The ribosome is dissociated to release the mRNA-protein fusion (E), which is protected with complementary cDNA using RT-PCR (F). The mRNA library can then be selected against an immobilized natural product probe (G), nonbinding library members washed away and the bound mRNA (H) released with SDS. PCR amplification of the cDNA provides a sublibrary (A) for another round of selection or for analysis/ sequencing.

There is a detailed review of the effect of ions and detergents on the release of subcellular mRNAs during lysis,20 indicating that the inclusion of sodium deoxycholate is crucial for efficient release of membrane-bound polysomes. However, based on Northern analysis of the debris following lysis in 140 mM KC1,1.5 mM MgCl2... 

The method has been used for the separation of enzymes, hormones, RNA-DNA hybrids, ribosomal subunits, subcellular organelles, for the analysis of distribution of samples of polysomes and for lipoprotein fractionation. 

Fig. 14. Polysomal mRNP from rat liver. A. Sucrose gradient analysis of the polyribosomes preparation treated with EDTA. Labeling period, 40 minutes. Centrifugation was at 21.000 rpm for 16 hours. —A A Cts/min -------, absorbance. B. CsCI equilibrium gradient analysis of the poly-...

A very important question concerns the nature of the proteins of polysomal mRNP. This question has not yet been answered because of the difficulties in the isolation of polysomal mRNPs. Since reticulocyte mRNPs can now be obtained in a pure state, the question may be answered in the near future. In another approach, Schweiger and Hannig (1970) tried to find proteins typical of nuclear D-RNP by direct analysis of polysomes. They separated the total polysomal protein into several fractions using free flow electrophoresis and then compared the fractions with nuclear D-RNPs obtained by means of polyacrylamide gel electrophoresis. In free polysomes and in membrane-bound polysomes, proteins similar to proteins of the nuclear D-RNP were found. If the membrane-bound polysomes are separated from the membranes by treatment with pure deoxycholate, these proteins are dissociated from polysomes and thus resemble the proteins of nuclear D-RNP in their sensitivity to deoxycholate. 

This means that the artificial interaction of RNA with polysomal proteins proceeds at the moment of dissociation of the polysomes. After dissociation these proteins combine irreversibly with mRNA or with ribosomal subunits. These results complicate the question of whether mRNPs are native structures. However, if the mRNA in polysomes is free, then it shoidd be possible to obtain it in an almost free state after the dissociation of polysomes in the presence of a large excess of D-RNA. Such experiments have been done, but it was found that even in the presence of 2 mg of free D-RNA per 1 mg of RNA of polysomes the endogenous polysomal-mRNA is recovered in heterogeneous complexes with a buoyant density of 1.40 to 1.60 g/cm. This figure may correspond to the protein content that seems to be the lower limit of the protein content in polysomal mRNA-containing complexes. In any case the experiments by Leytin et al. (1970) indicate that it is necessary to be very cautious in the analysis of the composition of polysomal mRNP since many artifacts may be created during their isolation. 

Another approach to the analysis of mRNA transfer to the ribosomes is the study of cytoplasmic particles containing newly formed cytoplasmic mRNA. Leytin et al. (1971) have found that the material containing newly formed mRNA is somewhat less dense in the CsCl gradient than polysomes formed earlier. For example, while the bulk of polysomes have a buoyant density of 1.55 to 1.56 g/cm, the newly formed material possesses a density of 1.47 to 1.53 g/cm. Newly formed particles comprise only a small fraction of the total material since they cannot be detected by routine UV absorption methods. With more detailed analysis two newly formed components may be observed component A with p = 1.49 g/cm and component B with p = 1.52 g/cm. After long-term incubation, or in the presence of actinomycin D (chase experiment), almost all radioactive material is transferred from A to B peaks to the main peak of the polysomes. Inhibition of protein synthesis by cycloheximide interferes with these polysome transformations, and the label representing newly synthesized material accumulates in component A (p = 1.49 g/cm ). Thus one can suggest that component A converts to B and the latter is transformed into mature polysomes. It was found further than... 

Fig. 4. Quantification of LHC2 message levels in the total mRNA fraction (a) and in the mRNA fraction associated with polysomes ( ). Quantification was by Northern blot analysis.

Alkaline hydrolysates of mRNA from vaccinia virus (Kates, 1970) and mouse sarcoma 180 cells (Mendecki et al, 1972) show that the poly (A) tracts, 100-200 nucleotides in length, are located at the 3 -ter-minal end of the RNA molecules. Analysis of the reaction products of highly purified exoribonuclease specific for 3 -OH termini also indicates that most (and possibly all) of the poly (A) sequences are at the 3 -terminal end of the mRNA s (Molloy et al, 1972). This is supported by the fact that the time course of poly( A) labeling in polysomes indicates that this sequence is assembled after the rest of the RNA molecule has been completed (Mendecki et al, 1972). Poly (A) synthesis is sensitive to actinomycin D but to an extent less than that of the rest of the RNA molecule which further argues that the poly (A) segment is added after transcription is completed (Darnell et al, 1971b Mendecki et al, 1972). 

A search for these polypeptides in other cell fractions gave inconclusive results. Electrophoretic analysis of polysome-associated proteins,for example, showed a very complex pattern with many, poorly resolved bands. At the present time, it seems however possible that these polypeptides may be associated with other components of infected cells, namely with the viral ENA. 

Recent evidence points to the presence of protease activity-associated with polysomes and ribosomes when extracts of uninfected cells are assayed (refs. 27 32, Figure j). Characteristic of infection of cells by poliovirus is drastic, rapid inhibition of protein synthesis. Poliovirus infection also depresses the ribosomal protease activity (27, 29, 55) Ribosomes from uninfected cells have been reported to possess an autoproteolytic activity (31, 32), and this has been confiimed by two-dimensional gel analysis (Figure 4) Poliovirus infection of HeLa cells reduces the autoproteolysis of isolated 808 ribosomes markedly (not shown). The inhibition of HeLa cell ribosomal protease activity requires protein synthesis, but proceeds in the presence of guanidine (55) ... 

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