Role of the SKIP complex in HIV-1 Tat transactivation
Zsolt Toth, Salk Institute for Biological Studies
Human immunodeficiency virus (HIV) that belongs to the genus of lentiviruses is the etiological agent of Acquired Immune Deficiency Syndrome (AIDS). One of the characteristic features of this virus is that although it has a single stranded RNA genome, its replication and gene expression require double stranded DNA integrated into the host chromosomes and the RNA polymerase II transcription machinery of the infected host cell.
It is known that the replication rate of HIV-1 depends on the level of viral transcription, which is tightly regulated at the elongation step. In this process the HIV-1 protein Tat has an essential role. In the absence of Tat the synthesis of full-length viral mRNAs is blocked resulting in short transcripts and consequently abrogated viral replication. Tat induces the production of full length viral transcripts by directly binding to the cis-acting RNA element TAR found at the 5' end of each viral mRNAs and recruiting the cellular kinase P-TEFb and its associated factors to the TAR RNA element. Subsequently P-TEFb phosphorylates the C terminal domain of RNA polymerase II stalled downstream close to the HIV-1 promoter LTR, which relieves the transcription block and leads to the synthesis of full-length viral mRNAs.
It was shown that in the absence of P-TEFb the replication of HIV-1 is severely impaired presumably due to inefficient viral transcription. This finding suggests that inhibition of P-TEFb and its associated factors might represent novel targets in AIDS therapeutics. For this, however, we need to know in details how P-TEFb and its associated factors can regulate Tat-mediated viral transcription. My research proposal will address this question in which I am going to study the cellular splicing factor SKIP in the regulation of Tat-mediated HIV-1 transcription. The host laboratory has shown that SKIP is a P-TEFb associated cellular protein, which is required for the Tat-mediated transcription elongation but its mechanism is still unknown.
My preliminary data from nuclear extract fractionation indicates that SKIP is present in distinct splicing and transcription complexes in vivo. Thus, my first aim is to purify the native SKIP-transcription elongation complex separately from the SKIP-splicing complex, and identify the SKIP elongation complex subunits. I will test the role of the new elongation subunits in HIV-1 Tat:P-TEFb-regulated transcription in vivo by siRNA-mediated knockdown as well as using in vitro transcription assays . I will examine whether the noncoding SRA RNA, which was recently reported to regulate SKIP activity, is part of the SKIP elongation complex, and whether P-TEFb-dependent RNAPII elongation is controlled by this regulatory RNA. Finally, I will investigate whether SKIP-associated factors and SRA RNA are required for methylation of histone H3K4, which is a chromatin modification that occurs during the RNAPII elongation checkpoint present near to the HIV-1 promoter. This histone methylation mark is the sign for a transcriptional active chromatin, which is required for the expression of the HIV-1 genome integrated into the cellular chromosome.
P-TEFb and its associated factors are also involved in regulation of gene expression of other viruses in addition to HIV-1, which affect their replication in the host cells. Therefore, understanding of the mechanism of how P-TEFb and its associated factors such as SKIP control the transcription may advance our knowledge of viral pathogenesis.