Protocol for an in vitro assay to study HIV-1 Tat methylation.

A critical virus-encoded regulator of HIV-1 transcription is the Tat protein, which is required to potently activate transcription. Tat is regulated by a wide variety of post-translational modifications. This protocol describes an in vitro assay to study Tat methylation.

The HIV-1 Tat Protein Is Monomethylated at Lysine 71 by the Lysine Methyltransferase KMT7.

The HIV-1 transactivator protein Tat is a critical regulator of HIV transcription primarily enabling efficient elongation of viral transcripts. Its interactions with RNA and various host factors are regulated by ordered, transient post-translational modifications. Here, we report a novel Tat modification, monomethylation at lysine 71 (K71).

Activation of HIV transcription by the viral Tat protein requires a demethylation step mediated by lysine-specific demethylase 1 (LSD1/KDM1).

The essential transactivator function of the HIV Tat protein is regulated by multiple posttranslational modifications. Although individual modifications are well characterized, their crosstalk and dynamics of occurrence during the HIV transcription cycle remain unclear.We examine interactions between two critical modifications within the RNA-binding domain of Tat: monomethylation of lysine 51 (K51) mediated by Set7/9/KMT7, an early event in the Tat transactivation cycle that strengthens the interaction of Tat with TAR RNA, and acetylation of lysine 50 (K50) mediated by p300/KAT3B, a later process that dissociates the complex formed by Tat, TAR RNA and the cyclin T1 subunit of the positive transcription elongation factor b (P-TEFb).

Acetylation of cyclin T1 regulates the equilibrium between active and inactive P-TEFb in cells.

The elongation competence of the RNA polymerase II complex is critically dependent on the positive transcription elongation factor b (P-TEFb). P-TEFb exists in two forms in cells, an active form composed of cyclin T1 and CDK9 and an inactive form, in which cyclin T1/CDK9 is sequestered by Hexim1 and 7SK snRNA. Here, we report that partitioning of active and inactive P-TEFb is regulated by acetylation of cyclin T1.

Formation of iminium ions by fragmentation of a2 ions.

Tandem mass spectrometric experiments have been carried out on the protonated amides H-Gly-Ala-NH2, H-Ala-Gly-NH2, H-Ala-Val-NH2, H-Val-Ala-pNA, H-Leu-Phe-NH2, H-Phe-Leu-NH2, H-Phe-Tyr-NH2 and H-Tyr-Phe-NH2 with particular emphasis on the fragmentation of the isomeric a2 ions derived therefrom. Primary fragmentation reactions of the protonated amides involve formation of the y1" and b2 ions with further fragmentation of the b2 ion to form the a2 ion which fragments to form iminium ions. Collision-induced dissociation studies of the mass-selected a2 ions were carried out.