Eliott Spencer and Dr. Roger Kaspar, Chemistry

Poly(A)-binding protein or PABP is found in high concentrations in all eukaryotic cells. Cell biologists have speculated for a long time that PABP plays an important role in stabilizing the fragile MRNA molecule so that it can be more effectively translated into its final gene product. PABP does this by binding to the Poly(A) tail at the 3′ end of the MRNA. 1,2

The PABP gene is believed to be controlled at the translational or MRNA level.4 One evidence of this is the large amount of PABP MRNA found in the cytoplasm with one or fewer ribosomes attached. Fewer ribosomes indicate a lower level of translation. As a preliminary part of the experiment we lysed human lymphocytes and centrifuged the cell parts in a sucrose gradient (a tube of sucrose of varying density along its length) then fractionated and blotted the fractions onto nitrocellulose paper. A binding of radioactive probe (in this case radioactive pieces of the PABP gene) showed that most of the PABP MRNA was in the less dense region of the sucrose, or the layer in the sucrose gradient that would indicate fewer ribosomes binding to it. This is a contrast to what we saw when we lysed cells expressing the human growth hormone gene and probed the extracts with radioactive human growth hormone (hGH) DNA. This distribution showed the majority of the hGH mRNAs in the more dense layers of the sucrose gradient (more ribosomes attached). These results are typical for most mRNAs and indicate that hGH is actively translated.6 This shows that the PABP MRNA is different from hGH and most mRNAs because it is present but not being efficiently translated into its final gene product This anomaly is strong evidence of a translational repression mechanism.

The 5′ untranslated region (UTR) of the PABP MRNA has short Poly(A) tracts that are unique to the PABP MRNA.3 PABP may be binding to these tracts and inhibiting translation.5 By attaching this unique 5′-UTR to other genes that do not exhibit translational repression we were able to observe a distribution of hGH MRNA similar to that of the translationally repressed PABP MRNA. The first blots are promising but need to be improved before the data can be taken seriously. With further repetition of the experiment we expect to show that PABP’s translational repression can be attributed entirely to the sequence of the 5′-UTR of the PABP MRNA.

Two specially designed DNA plasmids were used. Each contained genes that gave the cells that contained them resistance to a killing drug as well as the genes we used for studying the mechanism of the PABP translational repression mechanism. These plasmids and two types of human cells were used to create three unique and stable cell lines. T– lymphocytes were used to create a cell line that expressed the unaltered hGH gene and another cell line that expressed the hGH gene with the PABP 5′-UTR attached in place of the hGH 5′-UTR. A third cell line was made by introducing the modified hGH plasmid into a B-lymphocyte cell line to show that the translational control mechanism was not unique to a certain cell type. The plasmids were put into the cells using chemical vectors, then the cells with the plasmid were selected for by introducing a drug that killed all cells that had not taken up the plasmid. These cell lines were lysed and spun in a sucrose gradient, then fractionated and probed with radioactive fragments of hGH exactly like the cells mentioned above.

The results are not ready for publication yet. The experiment needs to be repeated and better blots need to be made. All of the cell lines have been frozen in liquid nitrogen so the difficult and time consuming task of making stable cell lines will not be necessary again.

References

1. Blobel, G. (1973). A protein of molecular weight 78,000 bound to the polyadenylate region of eukaryotic messenger RNAS. PNAS, 1
   70:924.
2. Gorlach, M., C. G. Burd, and G. Dreyfuss, 1994. The MRNA poly(A)-binding protein: localization, abundance, and RNA-binding 2
   specificity. Experimental Cell Research, 211:400.
3. Grange, T., C. Martins de Sa, J. Oddos, and R. Pictet, 1987. Human mRNA polyadenylate binding protein: evolutionary conservation 3
   of a nucleic acid binding motif. Nuc. Acids Res., 15:477.
4. Berger, L. C., J. Bag, and B. H. Sells, 1992. Translation of poly(A)-binding protein MRNA is regulated by growth conditions. Biochem. Cell Biology, 70:770.
5. Nomura, M., R. Gourse, and B. G, 1984. Regulation of the synthesis of ribosomes and ribosomal components. Annual Review of 5
   Biochemistry, 53:75.
6. Hill, J. R. and D. R. Morris, 1992. Cell-specific translation of S-adenosylmethionine decarboxylase MRNA. Regulation by the 5′ 6
   transcript leader. J Biol Chem, 267:21886.