RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases
- Max Planck Institute for Biophysical Chemistry, Germany
- Center for Innovative Medicine and Science for Life Laboratory, Novum, Karolinska Institutet, Sweden
Figures
Figure 1 with 1 supplement
Many elongation factors (EFs) bind RNA in vivo.
PAR-CLIP signal strength for EFs varies. The barplots show log2 fold-enrichments of transcript-averaged PAR-CLIP signals over the averaged PAR-CLIP signal for initiation factor TFIIB, which shows …
Figure 1—figure supplement 1
Confirmatory information on PAR-CLIP experiments.
(A) Smoothed, raw PAR-CLIP signals (as measured by the number of PAR-CLIP U-to-C transitions per U site) over a set of 2532 selected mRNAs were aligned at their 5′-end (TSS), scaled to a common …
Figure 2 with 1 supplement
Normalization of PAR-CLIP data shown for two representative EFs, Ctk2 (top) and Spt5 (bottom).
(A) Smoothed, raw RNA-binding strength as measured by the number of PAR-CLIP U-to-C transitions per U site for all mRNAs sorted by length and aligned at their RNA 5′-end (transcription start site, …
Figure 2—figure supplement 1
Normalization of PAR-CLIP data shown for two representative EFs, Ctk2 (top) and Spt5 (bottom), at mRNAs (left) versus CUTs (right).
Smoothed, raw and normalized PAR-CLIP signals as shown in Figure 2D but averaged over mRNAs (left) and CUTs (right). After normalization, average mRNA and CUT profiles were rescaled together, …
Figure 3 with 1 supplement
mRNA-binding profiles of EFs.
(A) Smoothed, transcript-averaged Pol II normalized PAR-CLIP occupancy profiles of EFs centered around the transcript 5′-end (transcription start site, TSS) [−150 nt to +400 nt] and pA site [−400 nt …
Figure 3—figure supplement 1
Non-averaged elongation factor RNA occupancies over mRNAs and introns.
(A) Transcript-wise Pol II normalized elongation factor PAR-CLIP occupancies. Smoothed occupancy profiles derived from PAR-CLIP data for a set of 2532 selected mRNAs. Transcripts were sorted by …
Figure 4 with 1 supplement
Asymmetric distribution of EFs at coding and non-coding transcripts.
(A) PAR-CLIP occupancies over mRNAs (left) and non-coding CUTs (right). Smoothed, averaged Pol II normalized RNA occupancy profiles were aligned at the RNA 5′-end (transcription start site, TSS) and …
Figure 4—figure supplement 1
Asymmetric distribution of EFs at coding and non-coding transcripts of similar length.
(A) PAR-CLIP occupancy heat plot similar to that shown in Figure 4A, but with mRNAs and CUTs selected to be of similar lengths, 350–700 nt. (B) and (C) PAR-CLIP occupancy profiles for elongation …
Figure 5
Chromatin association of EFs depends on RNA.
Western blot analysis (top) and quantitative densitometry (bottom) of exemplary EFs bound to chromatin before and after treatment with RNase A/T1 mix. H3 was used as loading control. Densitometry …
Figure 6 with 1 supplement
Recombinant CTDK-I complex is active and binds RNA in vitro.
(A) The three-subunit CTDK-I complex from S. cerevisiae was recombinantly expressed in insect cells and purified to homogeneity. The purified complex was run on a 4–12% gradient sodium dodecyl …
Figure 6—figure supplement 1
Recombinant and active CTDK-I complex binds preferentially U-rich ssRNA in vitro.
Binding of CTDK-I to U- and A-rich ssRNA sequences with 24% GC. Increasing concentrations (0–5.8 µM) of the full-length CTDK-I kinase complex were incubated with 8 nM of a U-rich ssRNA (blue line; Kd…
Figure 7 with 1 supplement
Comparison of PAR-CLIP and ChIP-Seq occupancy profiles.
Averaged ChIP-Seq (red) and PAR-CLIP (blue) occupancy profiles of EFs and ChIP-Seq of the histone marks H3K4me3, H3K79me3 and H3K36me3 (yellow) centered around TSSs [−150 bp to +600 bp] and pA sites …
Figure 7—figure supplement 1
Comparison of replicate measurements for ChIP-Seq.
Comparison of ChIP-Seq IP replicate measurements for elongation factors Bur1, Ctk1 and Rtf1 and histone methylation mark H3K36me3. The scatterplots compare average log2 read counts of all …
Figure 8
Deletion of Set1 RRMs impairs its recruitment to genes.
(A) Western blot analysis of Set1-TAP (top) and Rpb3 (bottom) in a Set1-TAP strain (left), a strain lacking the first 579 amino acids of Set1 (ΔRRM-Set1-TAP; middle) and a ΔPaf1 Set1-TAP strain …
Tables
Table 1
PAR-CLIP analysis of elongation factors (EFs).
| EF | Complex* | Number of crosslink sites† |
|---|---|---|
| Bur1 | BUR kinase complex | 77931 |
| Bur2 | 46293 | |
| Ctk1 | CTDK-I | 129352 |
| Ctk2 | 98993 | |
| Cdc73 | Paf1C | 57603 |
| Ctr9 | 55807 | |
| Leo1 | 27665 | |
| Paf1 | 20742 | |
| Rtf1 | 60068 | |
| Set1 | COMPASS | 189723 |
| Set2 | 68875 | |
| Dot1 | 42848 | |
| Spt5‡ | DSIF | 517568 |
| Spt6 | 93902 | |
| TFIIB§ | 16686 |
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*DSIF, DRB sensitivity inducing factor; CTDK, C-terminal domain kinase; Paf1C, Paf1 complex; COMPASS, Complex Proteins Associated with Set1.
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†Average number of crosslink sites with p-values<0.005.
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§Initiation factor, used to determine the level of RNA background crosslinking
Additional files
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Supplementary file 1
Sequences of primer pairs used for ChIP-qPCR.
YER: Heterochromatic region on chromosome V. Sequences of primer pairs used for strain generation.
- https://doi.org/10.7554/eLife.25637.017
