Genetic analysis of the structure and function of 7SK small nuclear ribonucleoprotein (snRNP) in cells

J Biol Chem. 2014 Jul 25;289(30):21181-90. doi: 10.1074/jbc.M114.557751.

Abstract

The positive transcription elongation factor b (P-TEFb), comprised of cyclin-dependent kinase 9 (CDK9) and cyclins T1 (CycT1) or T2 (CycT2), activates eukaryotic transcription elongation. In growing cells, P-TEFb exists in active and inactive forms. In the latter, it is incorporated into the 7SK small nuclear ribonucleoprotein, which contains hexamethylene bisacetamide-induced proteins (HEXIM) 1 or 2, La-related protein 7 (LaRP7), methyl phosphate capping enzyme, and 7SK small nuclear RNA (7SK). HEXIM1 inhibits the kinase activity of CDK9 via interactions between 7SK, HEXIM1, and CycT1. LaRP7 and methyl phosphate capping enzyme interact with 7SK independently of HEXIM1 and P-TEFb. To analyze genetic interactions between HEXIM1 and/or LaRP7 and 7SK using a cell-based system, we established artificial heterologous RNA tethering assays in which reporter gene expression depended on interactions between selected regions of 7SK and its cognate binding partners fused to a strong activator. This system enabled us to map the HEXIM1- and LaRP7- binding regions of 7SK. Assays with various mutant 7SK plasmid targets revealed that the 5'U-Ubulge and central loop of stem-loop I or RNA motif 3 of 7SK are required for transactivation, suggesting that HEXIM1 and CycT1 form a combinatorial binding surface for 7SK. Moreover, a region in HEXIM1 C-terminal to its previously mapped RNA-binding motif was also required for interactions between HEXIM1 and 7SK. Finally, a tyrosine-to-alanine mutation in HEXIM1, which is critical for its inhibitory effect on CDK9, changed HEXIM1 into an activator. These cell-based assays elucidate this important aspect of transcription elongation in vivo.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Cyclin T / genetics
  • Cyclin T / metabolism
  • Cyclin-Dependent Kinase 9 / genetics
  • Cyclin-Dependent Kinase 9 / metabolism
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Nucleic Acid Conformation
  • Protein Structure, Tertiary
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • Ribonucleoproteins / genetics
  • Ribonucleoproteins / metabolism
  • Ribonucleoproteins, Small Nuclear / genetics*
  • Ribonucleoproteins, Small Nuclear / metabolism*
  • Structure-Activity Relationship
  • Transcriptional Activation / genetics

Substances

  • CCNT1 protein, human
  • Cyclin T
  • HEXIM1 protein, human
  • Larp7 protein, human
  • RNA-Binding Proteins
  • Ribonucleoproteins
  • Ribonucleoproteins, Small Nuclear
  • CDK9 protein, human
  • Cyclin-Dependent Kinase 9