Higher Cell Gene Expression Research Articles

For Want of a Template

For Want of a Template

Cellular mRNA Activates Transcription Elongation by Displacing 7SK RNA

The positive transcription elongation factor P-TEFb is a pivotal regulator of gene expression in higher cells. Originally identified in Drosophila, attention was drawn to human P-TEFb by the discovery of its role as an essential cofactor for HIV-1 transcription. It is recruited to HIV transcription complexes by the viral transactivator Tat, and to cellular transcription complexes by a plethora of transcription factors. P-TEFb activity is negatively regulated by sequestration in a complex with the HEXIM proteins and 7SK RNA. The mechanism of P-TEFb release from the inhibitory complex is not known. We report that P-TEFb-dependent transcription from the HIV promoter can be stimulated by the mRNA encoding HIC, the human I-mfa domain-containing protein. The 3′-untranslated region of HIC mRNA is necessary and sufficient for this action. It forms complexes with P-TEFb and displaces 7SK RNA from the inhibitory complex in cells and cell extracts. A 314-nucleotide sequence near the 3′ end of HIC mRNA has full activity and contains a predicted structure resembling the 3′-terminal hairpin of 7SK that is critical for P-TEFb binding. This represents the first example of a cellular mRNA that can regulate transcription via P-TEFb. Our findings offer a rationale for 7SK being an RNA transcriptional regulator and suggest a practical means for enhancing gene expression.

Synthesis of DNA from vaccinia messenger RNA templates.

POLYADENYLIC acid sequences have been demonstrated in most messenger RNA molecules isolated from eukaryotic cells and animal viruses1–7. These sequences, ranging in size from 50 to 200 nucleotides, have been shown to occur on the 3-prime end of mRNA molecules of vaccinia virus and of HeLa cells (ref. 2 and unpublished results of W. Maloy and R. Perry and R. S. Sheldon, J. Kates and R. Perry). Aside from its intrinsic interest, the discovery of poly A sequences attached to messenger RNAs helps the study of gene expression in higher cells. For example, the ability of poly A containing mRNAs to bind quantitatively to immobilized poly U or poly dT is the basis of an extremely powerful method for the purification of most of the mRNA present in the polyribosomal fraction of cells2,6. Another potentially important application of the poly A sequence in mRNA, which will be the subject of this report, is its ability greatly to facilitate the synthesis of DNA copies of mRNA molecules using the RNA dependent DNA polymerase purified from oncornaviruses such as avian myeloblastosis virus (AMV) and Rous sarcoma virus (RSV)8–10. The simple principle involved depends on the fact that the reverse transcriptase enzyme functions very efficiently when an RNA template such as poly A is coupled with a small complementary DNA primer such as oligo dT11–13. The DNA product of this reaction is found covalently attached to the 3-prime end of the primer oligomer (David Baltimore, personal communication). We report here the success of the latter experimental approach in the efficient production of DNA complements of vaccinia virus messenger RNAs. Vaccinia virus mRNA was chosen purely as a model system in view of the fact that it can readily be synthesized in vitro in large quantities using the endogenous viral transcriptase15,16 and because about 90% of the RNA contains 3-prime poly A sequences.