MRNA polysome

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

The rate of protein synthesis is about 6 peptide bonds per minute, thus, it takes, about 1 to 2 minutes to synthesize an average sized protein. Because mRNA is often several thousand nucleotides in length, the same mRNA molecules can be simultaneously bound by many ribosomes. An mRNA that is bound by multiple ribosomes is called a polysome. Polysomes provide a mechanism for many copies of a protein to be translated from a single mRNA. Polysomes in the cytosol synthesize most of the proteins and enzymes required by the body for intracellular processes such as metabolism. 

The bacterial cell also contains tRNA (cytoplasm), rRNA (ribosomes) and mRNA (polysomes). 

Changes in the spectrum of ribonucleic acids during morphogenesis and differentiation have also been studied intensively in recent times. Work in this field has been concerned with determination of changes in the composition of ribosomal RNA (Nemer and Infante, 1967), mRNA-polysome complexes (Infante and Nemer, 1967, 1968) in early sea urchin embryos, the characteristics of different types of RNA in the embryonic organs of various animals (Bresnick et al., 1967 Wicks, 1967), and other matters. 

The rapid repression of pre-existing protein synthesis caused by anaerobic treatment is correlated with a near complete dissociation of polysomes in primary roots of soybeans (Lin Key, 1967) and maize (E.S. Dennis and A.J. Pryor, personal communication). This does not result from degradation of aerobic mRNAs, because the mRNAs encoding the pre-existing proteins remain translatable in an in vitro system at least five hours after anaerobic treatment is initiated (Sachs et al., 1980). This is in agreement... 

A ribosome is a cytoplasmic nucleoprotein stmcture that acts as the machinery for the synthesis of proteins from the mRNA templates. On the ribosomes, the mRNA and tRNA molecules interact to translate into a specific protein molecule information transcribed from the gene. In active protein synthesis, many ribosomes are associated with an mRNA molecule in an assembly called the polysome. 

Many ribosomes can translate the same mRNA molecule simultaneously. Because of their relatively large size, the ribosome particles cannot attach to an mRNA any closer than 35 nucleotides apart. Multiple ribosomes on the same mRNA molecule form a polyribosome, or polysome. In an unrestricted system, the number of ribosomes attached to an mRNA (and thus the size of polyribosomes) correlates positively with the length of the mRNA molecule. The mass of the mRNA molecule is, of course, quite small compared with the mass of even a single ribosome. 

Bagni, C., Mannucci, L., Dotti, C. G., and Amaldi, F. (2000). Chemical stimulation of synaptosomes modulates alpha —Ca2+/calmodulin-dependent protein kinase II mRNA association to polysomes. J. Neurosci. 20, RC76. 

Collecting the correct fraction is greatly facilitated by the use of a continuous ultraviolet (UV) detector, such as the ISCO UA-6 system. The OD254 reading allows accurate determination of the sedimentation of various complexes (e.g., 40S, 80S, and various polysomal complexes Fig. 9.1, step 3a). This information can be used to correct for small variations in sedimentation. Importantly, in many cases, it enables the determination of the number of ribosomes on the mRNA or on the resulting fragments, thereby allowing more accurate conclusions. 

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. 

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