MRNA-protein complex

Bernstein DS, Buter N, Smmpf C, Wickens M. Analyzing mRNA-protein complexes using a yeast three-hybrid system. Methods 2002 26 123-141. 

The first success was demonstration in 1994, with the report of a large, diverse library of decapeptides displayed and selected while associated with E. coli S30 polysomes and RNA.262 The key to Dower s success was the application of natural product antibiotics that were known to interfere with protein synthesis by stabilizing the ribosome-mRNA-protein complex. Thus, rifampicin and chloramphenicol (for prokaryotic system) or cycloheximide (for eukaryotic system) were used.2 3 Because these antibiotics halt the translation at random locations, the ensuing libraries were composed of mostly truncated peptides and thus not really suitable for the generation of cDNA libraries. Later, removal of the stop codon from mRNA was used to stall the translation at the end of the mRNA.264,265 Several improvements have been made more recently to stabilize the... 

In 1964 Spirin et al. discovered in embryonic cells cytoplasmic ribonucleoprotein particles containing rapidly labeled RNA with many properties of mRNA. The particles contain RNA and protein in a ratio of about 1 3 or 1 4. These particles, which are free cytoplasmic mRNA-protein complexes not combined with ribosomes, were named informosomes. Under ultracentrifugation they form a number of discrete peaks with sedimentation coefficients equal to 20, 30, 40, 50, 55, 65, and 75S (Spirin et al., 1964). 

A third type of mRNA-protein complex has been derived from cytoplasmic polysomes (Perry and Kelley, 1968 Henshaw, 1968). It was shown that after dissociation of polysomes into subunits by EDTA treatment, mRNA is liberated in the form of ribonucleoprotein with an RNA to protein ratio equal to 1 2 or 1 3. 

Bag J (1984) Cytoplasmic mRNA-protein complexes of chicken muscle cells and their role in protein synthesis. Eur J Biochem 141 247-254... 

Formation of a site-specific mRNA-protein complex is involved in the translational control of the biosynthesis of ferritin, an iron storage protein, which is stimulated in response to the presence of iron. In this instance, a cytoplasmic repressor protein of 85 kDa binds to a highly conserved 28-nucleotide stem-loop structure in the 5 untranslated region of ferritin mRNAs in the absence of iron. In the presence of iron, the protein dissociates from the mRNA, which is then available for translation. A similar loop motif occurs in the 3 untranslated region of transferrin receptor mRNA, which is also subject to translational control by an iron-responsive repressor. 

Splicing is a processing step of the pre-mRNA to become a mature transcript. This involves the excision of intervening noncoding sequences (introns) from coding sequences (exons) by a multiple protein complex, the spliceosome. After splicing the mRNA molecule is ready for translation, since it contains a continuous sequence that encode an entire protein. 

As pDNA and mRNA transfection differ in both the timing of mRNA expression and the gross amount of mRNA delivered to the cell, it is important to identify a suitable time point to measure miR-mediated repression. We observe that at any time point after transfection, pDNA transfections have higher measurable levels of miR-mediated repression compared to mRNA transfections (Fig. 6.2C). This difference may, in part, reflect a time lag of active miR-protein-complex formation relative to the onset of translation of the transfected Renilla luciferase mRNA. For single time point experiments, we decided to measure miR-mediated repression in mRNA and DNA transfections at 16 and 24 h, respectively. 

Izaurralde, E., Lewis, J., McGuigan, C., Jankowska, M., Darzynkiewicz, E., and Mattaj, I. W. (1994). A nuclear cap binding protein complex involved in pre-mRNA splicing. Cell 78, 657-668. 

Spliceosome a protein complex that catalyzes the splicing out of introns in pre-mRNA. 

Weng Y, Czaplinski K, Peltz SW (1996) Identification and characterization of mutations in the UPFl gene that affect nonsense suppression and the formation of the Upf protein complex but not mRNA turnover. Mol Cell Biol 16 5491-5506... 

FIGURE 27-22 Protein complexes in the formation of a eukaryotic initiation complex. The 3 and 5 ends of eukaryotic mRNAs are linked by a complex of proteins that includes several initiation factors and the poly(A) binding protein (PAB). The factors elF4E and elF4G are part of a larger complex called elF4F. This complex binds to the 40S ribosomal subunit. 

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