Defining NELF-E RNA Binding in HIV-1 and Promoter-Proximal Pause Regions

Abstract

The four-subunit Negative Elongation Factor (NELF) is a major regulator of RNA Polymerase II (Pol II) pausing. The subunit NELF-E contains a conserved RNA Recognition Motif (RRM) and is proposed to facilitate Poll II pausing through its association with nascent transcribed RNA. However, conflicting ideas have emerged for the function of its RNA binding activity. Here, we use in vitro selection strategies and quantitative biochemistry to identify and characterize the consensus NELF-E binding element (NBE) that is required for sequence specific RNA recognition (NBE: CUGAGGA(U) for Drosophila). An NBE-like element is present within the loop region of the transactivation-response element (TAR) of HIV-1 RNA, a known regulatory target of human NELF-E. The NBE is required for high affinity binding, as opposed to the lower stem of TAR, as previously claimed. We also identify a non-conserved region within the RRM that contributes to the RNA recognition of Drosophila NELF-E. To understand the broader functional relevance of NBEs, we analyzed promoter-proximal regions genome-wide in Drosophila and show that the NBE is enriched +20 to +30 nucleotides downstream of the transcription start site. Consistent with the role of NELF in pausing, we observe a significant increase in NBEs among paused genes compared to non-paused genes. In addition to these observations, SELEX with nuclear run-on RNA enrich for NBE-like sequences. Together, these results describe the RNA binding behavior of NELF-E and supports a biological role for NELF-E in promoter-proximal pausing of both HIV-1 and cellular genes.

Author Summary

RNA polymerase II (Pol II) is a molecular machine that is responsible for transcribing all protein coding genes in the eukaryotic genome. Transcription by Pol II is a highly regulated process consisting of several rate-limiting steps. During transcription elongation, a number of transcription factors are essential to modulate Pol II activity. One of these factors is the Negative Elongation Factor (NELF), and it plays a major role in promoter-proximal pausing, a widespread phenomenon during early transcription elongation. NELF-E, a protein subunit of the NELF complex contains a conserved RNA binding domain that is thought to regulate transcription through its interaction with newly transcribed RNA made by Pol II. However, the function of the RNA binding activity of NELF-E remains unresolved due to prior conflicting studies. Here, we clarify the RNA binding properties of NELF-E and provide insight into how this protein might facilitate promoter-proximal pausing of Pol II in transcription. Moreover, we identify the precise region of NELF-E binding in one of its known regulatory targets, HIV-1. Taken together, the results presented indicate a dynamic interplay between NELF and specific RNA sequences around the promoter pause region to modulate early transcription elongation.