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. 2013 Oct 9;425(19):3625-38.
doi: 10.1016/j.jmb.2013.01.035. Epub 2013 Feb 8.

The non-coding B2 RNA binds to the DNA cleft and active-site region of RNA polymerase II

Steven L Ponicsan  1 , Stephane Houel, William M Old, Natalie G Ahn, James A Goodrich, Jennifer F Kugel
Affiliations

The non-coding B2 RNA binds to the DNA cleft and active-site region of RNA polymerase II

Steven L Ponicsan et al. J Mol Biol. .

Abstract

The B2 family of short interspersed elements is transcribed into non-coding RNA by RNA polymerase III. The ~180-nt B2 RNA has been shown to potently repress mRNA transcription by binding tightly to RNA polymerase II (Pol II) and assembling with it into complexes on promoter DNA, where it keeps the polymerase from properly engaging the promoter DNA. Mammalian Pol II is an ~500-kDa complex that contains 12 different protein subunits, providing many possible surfaces for interaction with B2 RNA. We found that the carboxy-terminal domain of the largest Pol II subunit was not required for B2 RNA to bind Pol II and repress transcription in vitro. To identify the surface on Pol II to which the minimal functional region of B2 RNA binds, we coupled multi-step affinity purification, reversible formaldehyde cross-linking, peptide sequencing by mass spectrometry, and analysis of peptide enrichment. The Pol II peptides most highly recovered after cross-linking to B2 RNA mapped to the DNA binding cleft and active-site region of Pol II. These studies determine the location of a defined nucleic acid binding site on a large, native, multi-subunit complex and provide insight into the mechanism of transcriptional repression by B2 RNA.

Keywords: C-terminal domain of RPB1; CTD; EM; EMSA; LC; MS; MS/MS; Pol II; RNA polymerase II; RPB; TATA binding protein; TBP; TFII; XIC; electron microscopy; electrophoretic mobility shift assay; extracted ion chromatogram; liquid chromatography; mass spectrometry; ncRNA; non-coding RNA; protein–RNA cross-linking; subunit of RNA polymerase II; transcription; transcription factor of RNA polymerase II.

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Figures

Figure 1
Figure 1
The loss of the Pol II CTD does not alter the ability of B2 RNA to bind Pol II and repress transcription in vitro. (A) The Pol II CTD was digested by limited proteolysis with chymotrypsin. Shown is a Western blot of the RPB1 subunit of Pol II after incubation with increasing amounts of chymotrypsin. Molecular weight markers (kDa) are labeled on the left. (B) CTD-less Pol II binds B2 RNA as well as untreated Pol II. ncRNA/Pol II complexes were resolved by EMSAs. (C) Loss of the Pol II CTD does affect the ability of B2 RNA to repress transcription in vitro. Shown are the transcripts produced from in vitro reactions containing CTD-less Pol II and untreated Pol II in the absence and presence of 2.5 nM B2 RNA, or 2.5 nM B1 RNA as a negative control.
Figure 2
Figure 2
Biotinylation of B2 RNA(81-130) does not affect its ability to potently repress transcription in vitro. B2 RNA(81-130), 5’-biotin-B2 RNA(81-130), or 5’-biotin-B2 RNA(81-115) were individually added to transcription reactions where indicated. 390 nt transcript was monitored. The values indicate the amount of transcript produced normalized to the reaction that did not contain ncRNA.
Figure 3
Figure 3
Schematic overview of the ncRNA-Pol II formaldehyde crosslinking protocol. (1) Pol II was immunoprecipitated from nuclear extracts using bead-immobilized antibody. (2) 5'-biotinylated RNA was bound to Pol II, followed by washes with a nonspecific competitor DNA and higher salt to reduce nonspecific binding. (3) Complexes were treated with formaldehyde to covalently crosslink ncRNA to Pol II. (4) The entire solution containing the RNA and bead-bound Pol II was digested with trypsin. (5) After centrifugation, the supernatant was mixed with streptavidin beads to purify the ncRNA and enrich for RNA-crosslinked peptides. (6) After centrifugation the supernatant was heated to reverse formaldehyde crosslinks. (7) Peptides were then re-digested with trypsin. (8) Recovered peptides were subjected to LC MS/MS sequencing and identified using Mascot. (9) The intensities of peptides recovered from the crosslinked samples were compared to those from the control samples as described in the text.
Figure 4
Figure 4
Properties of Pol II peptides isolated using the approach shown in Figure 3 are highly reproducible. (A) The elution times for peptides identified in the two crosslinking experiments were highly reproducible. The times at which peptides eluted from the column prior to ionization for the two B2 RNA(81-130) crosslinked samples were plotted against one another. (B) Comparison of the total XIC values of the peptides crosslinked to B2 RNA(81-130) from two separate experiments. The total XIC value for each peptide was normalized by the sum of all XIC values from that sample, and the log of these numbers were plotted. (C) Comparison of the total XIC values of the peptides from two separate trypsin treated Pol II control samples. The total XIC value for each peptide was normalized by the sum of all XIC values from that sample, and the log of these numbers were plotted.
Figure 5
Figure 5
Nine peptides in the crosslinked samples are significantly enriched compared to the control samples. (A) Comparison of the average XIC values of peptides crosslinked to B2 RNA(81-130) to the average XIC values from the control samples. For each peptide, the normalized total XIC values from the two crosslinked samples were averaged, and normalized total XIC values for the two control samples were averaged. The log of the average total XIC values for the crosslinked samples were plotted versus those for the control samples. The dashed line represents the theoretical line along which the peptides would fall if they were recovered at the same intensity in the crosslinked and control samples. The blue squares represent peptides found to be significantly enriched in the crosslinked samples using the statistical analysis in Figure 5B, whereas the grey circles represent peptides that were neither over- nor under-represented in the crosslinked samples versus the control samples. (B) Statistical analysis to determine which peptides were significantly enriched in the crosslinked samples. The residuals of each point from the theoretical line in panel A were plotted versus the peptide name. The solid black line represents the mean of the residuals, the grey dashed lines are one standard deviation from the mean, and the black dashed lines are two standard deviations. The bottom panel displays the points remaining after omitting points removed by the statistical analysis explained in detail in the Results section. The solid line represents the average of the remaining residuals and the dashed lines are two standard deviations.
Figure 6
Figure 6
The majority of the Pol II peptides significantly enriched in the crosslinked samples map to the surface of Pol II containing the DNA cleft and active site region. The top left panel shows a front view of Pol II, and the bottom panel shows a top view. The significantly enriched peptides are shown in blue, with crosslinkable amino acids highlighted in light blue. RPB5-2 and RPB1-8, which were not found in the control Pol II samples, are shown in green, with crosslinkable amino acids in light green. For the smaller image on the top right, the image on the top left was flipped horizontally to show the lack of peptides that map to the back side of Pol II. The images were generated using Pymol and PDB structure 3FKI.,
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