The function of proteins that interact with mRNA

Specific proteins are associated with mRNA in the cytoplasm of eukaryotic cells. The complement of associated proteins depends upon whether the mRNA is an integral component of the polysomal complex being translated, or, alternatively, whether it is part of the non-translated free mRNP fraction. By subjecting cells to ultraviolet irradiation in vivo to cross-link proteins to mRNA, mRNP proteins have been shown to be associated with specific regions of the mRNA molecule. Examination of mRNP complexes containing a unique mRNA has suggested that not all mRNA contain the same family of associated RNA binding proteins. The function of mRNA associated proteins may include a role in providing stability for mRNA, and/or in modulating translation. With the recent demonstrations that both free and polysomal mRNPs are associated with the cytoskeletal framework, specific mRNP proteins may play a role in determining the subcellular localization of specific mRNPs.     

“In situ” translation: Use of the cytoskeletal framework to direct cell-free protein synthesis

Summary We have developed a novel, “in situ” translation system derived from cultured cells that are subject to mild detergent extraction. By using a low concentration of nonionic detergent to gently permeabilize cells while they remain adherent to a substrate, cytoskeletal frameworks are obtained that are devoid of membraneous barriers yet retain much the same topological arrangement of mRNA, ribosomes and cytostructure that exists “in vivo”. Data indicate that when these cytoskeletal frameworks are supported by a ribosome-depleted, nuclease-treated, reticulocyte lysate supernatant, they are capable of resuming translation of their attached polysomes for at least 40 minutes. Emulsion autoradiography of ongoing protein synthesis demonstrates that protein synthetic activity is ubiquitous throughout the population of extracted cells, and not confined to a less well-extracted subset. Computer-assisted, two-dimensional gel analysis reveals that the pattern of proteins produced by such extracted cells is approximately 70% coincident with that produced by unextracted cells, including proteins of molecular weight as great as 200 kilodaltons. Furthermore, a continued increase in intensity of almost all proteins during the first 40 minutes of translation suggests that translational re-initiation, in addition to polysome run-off, is also taking place. Collectively, these findings indicate that much of the translational machinery remains both intact and competant in this cytoskeletal-based translation system. As such, this system should prove extremely useful in identifying molecular factors operant during certain types of translation control and in further examining the role played by the cytoskeleton in regulating gene expression. This work was supported by grants from the American Cancer Society (#NP-683) and from the University of Connecticut Health Center Research Foundation.     

Changes in distribution of actin mRNA in different polysome fractions following stimulation of MPC-11 cells

Individual mRNA species have been shown to differ both with respect to localization in the cell, and in their distribution upon stimulation of cells with different signals. In this study we have examined the distribution of actin mRNA in the free, cytoskeletal-bound, and membrane-bound RNA fractions, both in starved cells, and in response to stimulation by feeding. These results were then compared with mRNAs for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and histone H4. The results we obtained showed that actin mRNA was located in the free RNA fraction in starved cells, while upon stimulation it was located both in the free, and in the cytoskeletal fraction; no redistribution of GAPDH mRNA occurred between the three RNA fractions, while H4 mRNA showed a different localization upon stimulation. Incubation with the drugs actinomycin-D and cycloheximide showed that an altered localization of actin mRNA from free in starved cells to free and cytoskeletal mRNA fractions following stimulation, was dependent on RNA synthesis, and not on protein synthesis.     

Free,cytoskeletal-bound and membrane-bound polysomes isolated from MPC-11 and Krebs II ascites cells differ in their complement of poly(A) binding proteins

A three-step detergent/salt extraction procedure (Vedeleret al., Mol Cell Biochem 100: 183–193, 1991) was used to isolate free polysomes (FP), cytoskeletal-bound polysomes (CBP) and membrane-bound polysomes (MBP) from MPC-11 and Krebs II ascites cells. Polysomes were pelleted, washed with high salt buffer and re-pelleted. Proteins in the dialysed high-salt extracts were subjected to poly(A) Sepharose chromatography and poly(A) binding and non-binding proteins were separated by SDS-PAGE. In MPC-11 cells the FP fraction contains thirteen poly(A) binding proteins and four non-poly(A) binding proteins while the corresponding fraction in Krebs II ascites cells has four poly(A) binding proteins and six proteins which do not bind poly(A). The CBP fraction isolated from MPC-11 cells has a complement of ten poly(A) binding proteins, four which are non-poly(A) binding, and a protein of 105 kDa which has both poly(A) binding and non-poly(A) binding properties. In the CBP fraction prepared from Krebs II ascites cells a protein band at 32 kDa exhibits both poly(A) binding and non-poly(A) binding properties. In this fraction there are six poly(A) binding proteins and an additional eight which do not bind poly(A). Of the total number of proteins eight of these have a molecular weight below 40 kDa. The MBP fraction in MPC-11 cells contains three poly(A) binding proteins and eleven with non-poly(A) binding properties. In contrast this fraction in Krebs II ascites cells has a complement of thirteen poly(A) binding and ten non-poly(A) binding proteins. The results show differences in the poly(A) binding properties of the proteins in the three polysome fractions and that the complements of polysome-associated proteins are different in the two cell lines. This may be related to the differences in the growth characteristics of MPC-11 and Krebs II ascites cells.     

Identification of RNase-resistant RNAs in Saccharomyces cerevisiae extracts: Separation from chromosomal DNA by selective precipitation

High-quality chromosomal DNA is a requirement for many biochemical and molecular biological techniques. To isolate cellular DNA, standard protocols typically lyse cells and separate nucleic acids from other biological molecules using a combination of chemical and physical methods. After a standard chemical-based protocol to isolate chromosomal DNA from Saccharomyces cerevisiae and then treatment with RNase A to degrade RNA, two RNase-resistant bands persisted when analyzed using gel electrophoresis. Interestingly, such resistant bands did not appear in preparations of Escherichia coli bacterial DNA after RNase treatment. Several enzymatic, chemical, and physical methods were employed in an effort to remove the resistant RNAs, including use of multiple RNases and alcohol precipitation, base hydrolysis, and chromatographic methods. These experiments resulted in the development of a new method for isolation of S. cerevisiae chromosomal DNA. This method utilizes selective precipitation of DNA in the presence of a potassium acetate/isopropanol mixture and produces high yields of chromosomal DNA without detectable contaminating RNAs.