Structural studies on the RNA-recognition motif of NELF E, a cellular negative transcription elongation factor involved in the regulation of HIV transcription

Abstract

The elongation of transcription of HIV RNA at the TAR (transactivation-response element) is highly regulated by positive and negative factors. The cellular negative transcription elongation factor NELF (negative elongation factor) was suggested to be involved in transcriptional regulation of HIV-1 (HIV type 1) by binding to the stem of the viral TAR RNA which is synthesized by cellular RNA polymerase II at the viral long terminal repeat. NELF is a heterotetrameric protein consisting of NELF A, B, C or the splice variant D, and E. In the present study, we determined the solution structure of the RRM (RNA-recognition motif) of the RNA-binding subunit NELF E and studied its interaction with the viral TAR RNA. Our results show that the separately expressed recombinant NELF E RRM has alpha-helical and beta-strand elements adopting a betaalphabetabetaalphabeta fold and is able to bind to TAR RNA. Fluorescence equilibrium titrations with fluorescently labelled double- and single-stranded oligoribonucleotides representing the TAR RNA stem imply that NELF E RRM binds to the single-stranded TAR RNAs with K(d) values in the low-micromolar range.

NELF and GAGA Factor Are Linked to Promoter-Proximal Pausing at Many Genes in Drosophila

“…NELF was previously shown to associate with a variety of RNAs, including some with substantial secondary structure and some without it (63). Our conclusion contrasts with studies of the human immunodeficiency virus (HIV) provirus, for which sequence-specific association of NELF-E with the TAR element has been detected and is thought to control elongation (15,46). Recent analysis of an HIV provirus, however, indicates that NELF associates with the elongation complex and causes Pol II to pause at position ϩ45, well before the TAR element has been transcribed and well within the range where we find Pol II pausing on Drosophila genes (67).…”

Enhancer RNA Facilitates NELF Release from Immediate Early Genes

“…The NELF-E subunit contains an RNA recognition motif (RRM) that mediates direct interactions with various RNA sequences with little or no apparent sequence or structural constraint, which is suitable for binding to nascent RNAs derived from many genes (Rao et al, 2006; Yamaguchi et al, 2002). The RRM of NELF-E was also shown to be critical for the RNAPII pausing activity of NELF in an in vitro transcription assay (Yamaguchi et al, 2002).…”

The Receptor Tyrosine Kinase RON Represses HIV-1 Transcription by Targeting RNA Polymerase II Processivity

“…Both DSIF and NELF contribute to Tat-dependent activation of HIV-1 transcription and are phosphorylated by P-TEFb (39,51). Furthermore, the E subunit of NELF associates with the TAR element (39,52), and ectopic expression of this NELF subunit inhibits transient basal transcription from the HIV LTR (39). Depleting NELF in the context of U1 cells, which harbor latent HIV-1 provirus, released the Pol II paused at ϳϩ42 and increased processive HIV transcription (28).…”