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.
Highlights
RNA polymerase II (Pol II) is a molecular machine responsible for transcribing all protein coding genes in the eukaryotic genome in a highly regulated multistep process
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
The results presented indicate a dynamic interplay between NELF and specific RNA sequences around the promoter pause region to modulate early transcription elongation
Summary
RNA polymerase II (Pol II) is a molecular machine responsible for transcribing all protein coding genes in the eukaryotic genome in a highly regulated multistep process. Three protein complexes have a major role in Pol II pausing. NELF (Negative elongation factor) and DSIF [DRB (5,6-dichloro-1-b-D-ribofuranosylbenzimidazole) sensitivity inducing factor], form a stable complex with Pol II and inhibit its elongation shortly after initiation. P-TEFb (Positive transcription elongation factor b), a complex of CDK9 kinase and CyclinT, overcomes the influence of these factors and promotes the release of Pol II into productive elongation [6,7,8,9]. Experimental evidence indicates that P-TEFb phosphorylates NELF, DSIF, and the C-terminal domain (CTD) of Pol II and that one or more of these modifications alleviate the pause [10,11,12]
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