Defining NELF-E RNA binding in HIV-1 and promoter-proximal pause regions

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

RNA synthesis precision is regulated by preinitiation complex turnover

TATA-binding protein (TBP) nucleates the assembly of the transcription preinitiation complex (PIC), and although TBP can bind promoters with high stability in vitro, recent results establish that virtually the entire TBP population is highly dynamic in yeast nuclei in vivo. This dynamic behavior is surprising in light of models that posit that a stable TBP-containing scaffold facilitates transcription reinitiation at active promoters. The dynamic behavior of TBP is a consequence of the enzymatic activity of the essential Snf2/Swi2 ATPase Mot1, suggesting that ensuring a highly mobile TBP population is critical for transcriptional regulation on a global scale. Here high-resolution tiling arrays were used to define how perturbed TBP dynamics impact the precision of RNA synthesis in Saccharomyces cerevisiae. We find that Mot1 plays a broad role in establishing the precision and efficiency of RNA synthesis: In mot1-42 cells, RNA length changes were observed for 713 genes, about twice the number observed in set2Δ cells, which display a previously reported propensity for spurious initiation within open reading frames. Loss of Mot1 led to both aberrant transcription initiation and termination, with prematurely terminated transcripts representing the largest class of events. Genetic and genomic analyses support the conclusion that these effects on RNA length are mechanistically tied to dynamic TBP occupancies at certain types of promoters. These results suggest a new model whereby dynamic disassembly of the PIC can influence productive RNA synthesis

Regulation of rRNA Synthesis by TATA-Binding Protein-Associated Factor Mot1▿

Mot1 is an essential, conserved, TATA-binding protein (TBP)-associated factor in Saccharomyces cerevisiae with well-established roles in the global control of RNA polymerase II (Pol II) transcription. Previous results have suggested that Mot1 functions exclusively in Pol II transcription, but here we report a novel role for Mot1 in regulating transcription by RNA polymerase I (Pol I). In vivo, Mot1 is associated with the ribosomal DNA, and loss of Mot1 results in decreased rRNA synthesis. Consistent with a direct role for Mot1 in Pol I transcription, Mot1 also associates with the Pol I promoter in vitro in a reaction that depends on components of the Pol I general transcription machinery. Remarkably, in addition to Mot1's role in initiation, rRNA processing is delayed in mot1 cells. Taken together, these results support a model in which Mot1 affects the rate and efficiency of rRNA synthesis by both direct and indirect mechanisms, with resulting effects on transcription activation and the coupling of rRNA synthesis to processing

The NBE is necessary and sufficient for dNELF-E binding.

<p>(a) A representative F-EMSA of full length dNELF-E binding to Napt1NBEmut RNA, Napt1+hairpin, or Napt1-Δstem. Below each gel is a visual representation of each sequence tested. Mutations made in the NBE are colored red. (b) A normalized plot of fraction bound for each RNA sequence tested in (a). The data and fit are annotated in the graph to indicate measured K_d and fit error. For comparison, the fit of dNELF-E binding to Napt1min is shown as a dashed line.</p

Relative enrichment of the NBE in <i>Drosophila</i> genomic regions near transcription start sites (TSSs).

<p>(a) Heat map of DNA sequence similarity to NBE in active <i>Drosophila</i> genes (n = 5471). Each row in the heat map represents a <i>Drosophila</i> gene from −50 to +150 base pairs from the TSS, and colors indicate the p-value of the sequence similarity index calculated from permutated 7-mer sequence scores. The asterisk indicates the position of NBE enrichment relative to the TSS. A heat map comparison of DNA sequence similarity for NBEs between paused (n = 3225) and non-paused (n = 2246) genes is shown to the right. Genes in each group are ordered by the strength of NBE similarity for comparison. (b) The average profile of the NBE similarity index in active genes. (c) A sequence logo representation of NBE-like sequences in active genes between +0 and +50 base pairs from the TSS for all genes. (d) The average profile of the NBE similarity index in paused and non-paused genes (p-value<7.2×10⁻⁷ by a Kolmogorov-Smirnov test or p-value<1.3×10⁻⁵ by a two-sample unequal variance t-test).</p

NELF-E binding affinity for RNA targets.

<p>Values given are the average K_d ± s.d. for <i>n</i> independent replicates. K_d values determined by FP and EMSA were statistically different (p<0.01) only for HIV-1 TAR+A RNA.</p

A humanized dNELF-E reveals an amino acid region that contributes to dNELF-E RNA recognition.

<p>(a) A sequence alignment of the RRM domain from a family of NELF-E proteins. Shaded in blue are the highly conserved ribonucleoprotein motifs RNP2 and RNP1. The boxed residues contain the seven amino acids that are mutated in the experiment shown in (b). Amino acids in red are the glutamate/aspartate residues that shift four positions toward the C-terminus in <i>Drosophila</i> and several other organisms. Asterisks represent positions that are thought to make RNA contacts <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004090#pgen.1004090-Rao2" target="_blank">[25]</a>. Below the alignment is a secondary structure prediction obtained from jnetpred and a normalized quality alignment <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004090#pgen.1004090-Cole1" target="_blank">[55]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004090#pgen.1004090-Henikoff1" target="_blank">[57]</a>. The brown arrows are beta sheets and the red tubes are alpha helices. (b) A summary of the mutagenesis performed on dNELF-E. The seven amino acid region boxed in (a) was humanized as illustrated in the domain structures. The grey region denotes the human RRM, while red signifies <i>Drosophila</i>. (c) The ΔΔG° for each NELF-E variant binding to either Napt1min and TAR RNA or TAR+A and TAR RNA. The K_d of each protein construct to its target was used to calculate the free energy (ΔG = −RT(lnK_d)) from which the ΔΔG° values are derived. All experiments used full length protein constructs. Error bars represent the propagation of error derived from the standard deviations for indicated binding experiments. A ΔΔG° of 0.5 kcal mol⁻¹ is shown by the dotted line.</p

Identification of the NELF-E Binding Element within high affinity aptamers.

<p>(a) MEME analysis of the top 3,000 unique clustered sequencing reads from a SELEX experiment of dNELF-E or its RRM domain. The sequence logo derived is shown for both proteins. (b) Secondary structure of Napt1min RNA aptamer. An additional GC base pair was added to the end of the aptamer (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004090#s4" target="_blank">Materials and Methods</a>). The consensus sequence is highlighted with coloring that corresponds to the sequence logo. (c) Full length dNELF-E binds to Napt1min with high affinity. Shown is a representative fluorescence electrophoretic mobility shift assay (F-EMSA) with increasing concentrations of dNELF-E protein from 1.4 nM up to 2 µM and a fixed concentration of fluorescently labeled aptamer. (d) A plot of the fraction of bound Napt1min against protein concentration is presented for the gel in panel (c) and fit to the Hill equation. The equilibrium dissociation constant (K_d) is shown in the graph and the error represents the standard deviation of the uncertainty of the fit. (e) A plot of fluorescence polarization of the same binding experiment and its measured K_d and fit error are presented. Raw polarization values are given in units of milipolarization (mP).</p

Human and <i>Drosophila</i> NELF-E bind specifically to HIV-1 TAR RNA.

<p>(a) A secondary structure of HIV-1 TAR RNA. The predicted NBE is colored according to the sequence logo shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004090#pgen-1004090-g001" target="_blank">Figure 1a</a>. (b) A representative F-EMSA of full length dNELF-E binding to TAR and TAR+A is shown. Below each gel is a visual representation of the RNAs tested. Mutations are indicated in red. (c) As described in (b), a representative F-EMSA of full length hNELF-E to TAR, TAR+A, or TAR-ΔhNBE are shown. (d) A normalized plot and fit of fraction bound RNA for experiments shown in (b) and (c). The binding constant and fit standard error for each experiment is included next to its label.</p

Function and Structural Organization of Mot1 Bound to a Natural Target Promoter*S⃞

Mot1 is an essential, conserved TATA-binding protein (TBP)-associated factor in Saccharomyces cerevisiae and a member of the Snf2/Swi2 ATPase family. Mot1 uses ATP hydrolysis to displace TBP from DNA, an activity that can be readily reconciled with its global role in gene repression. Less well understood is how Mot1 directly activates gene expression. It has been suggested that Mot1-mediated activation can occur by displacement of inactive TBP-containing complexes from promoters, thereby permitting assembly of functional transcription complexes. Mot1 may also activate transcription by other mechanisms that have not yet been defined. A gap in our understanding has been the absence of biochemical information related to the activity of Mot1 on natural target genes. Using URA1 as a model Mot1-activated promoter, we show striking differences in the way that both TBP and Mot1 interact with DNA compared with other model DNA substrates analyzed previously. These differences are due at least in part to the propensity of TBP alone to bind to the URA1 promoter in the wrong orientation to direct appropriate assembly of the URA1 preinitiation complex. The results suggest that Mot1-mediated activation of URA1 transcription involves at least two steps, one of which is the removal of TBP bound to the promoter in the opposite orientation required for URA1 transcription