Transcriptome maps of general eukaryotic RNA degradation factors
- Max-Planck-Institute for Biophysical Chemistry, Germany
Figures
Figure 1 with 2 supplements
Overview of PAR-CLIP experiments performed in this study.
(A) Overview of degradation pathways studied. (B) Number of high-confidence PAR-CLIP cross-link sites obtained for each factor after merging data from replicates.
Figure 1—figure supplement 1
Biological replicate PAR-CLIP experiments have high correlation.
(A) Total transcript occupancy of factors in replicate experiments are plotted (in log2 space) and Spearman correlation values are shown for each pair. Each dot corresponds to a transcript. The …
Figure 1—figure supplement 2
Western Blot analysis for all degradation factors analyzed in this study show IP efficiency.
IP using the TAP-tag is detected by Western Blot analysis with tag specific antibody to show the IP quality of the different PAR-CLIP experiments representative for one replicate per factor. The …
Figure 2 with 2 supplements
Distribution of degradation factor cross-link sites over the yeast transcriptome.
(A) Fractions of high confidence PAR-CLIP sequencing reads of 30 yeast degradation factors fall into various transcript classes. Depicted classes are the following: messenger RNA (mRNA) in turquoise …
Figure 2—figure supplement 1
Metagene profiles for subunits of the TRAMP complexes on snoRNA genes.
Transcript averaged PAR-CLIP occupancy profiles are shown for Air1, Trf5, Mtr4, Air2, and Trf4. snoRNA genes are aligned either at their 5´ end or at their 3´ end (n = 77). Occupancy profiles are …
Figure 2—figure supplement 2
Different transcript classes have comparable U-content.
Fraction of U over all bases in transcript classes studied in Figure 2 (untranslated region (UTR); intron; coding sequence (CDS), ribosomal RNA (rRNA), transfer RNA (tRNA), small nucleolar RNA …
Figure 3 with 3 supplements
Metagene analysis of degradation factor binding on mRNAs.
Averaged occupancy profiles of degradation factors over mRNAs aligned around their transcription start site (TSS) (n = 3,193, left) and around their poly-adenylation (pA) site (n = 3,193, right) in …
Figure 3—figure supplement 1
Metagene profiles of yeast RNA degradation factors centered on translation start and stop sites in comparison to TIF-annotated TSS and pA sites.
Transcript-averaged PAR-CLIP occupancy profiles are shown for RNA degradation factors involved in (A) deadenylation, (B) decapping, (C) 5´→3´ exonuclease Xrn1, (D) exosome, (E) TRAMP, (F) Ski, and (G…
Figure 3—figure supplement 2
Comparison of binding profiles on genes containing annotated upstream sense NUTs with all mRNAs.
(A) Binding enrichment of degradation factors around the TSS of genes with an upstream sense NUT. Enrichment is defined as the ratio of the average occupancy in the interval [±300 nt] of the TSS on …
Figure 3—figure supplement 3
Metagene analysis of degradation factor binding on mRNAs after removing signals from known NUTs and CUTs.
Cross-link sites were filtered to exclude regions that were previously annotated as NUTs and CUTs (Neil et al., 2009; Schulz et al., 2013). Averaged occupancy profiles of degradation factors are …
Figure 4 with 2 supplements
Surveillance of aberrant nuclear antisense RNAs by the exosome and the TRAMP4 complex.
Averaged occupancy profiles of degradation factors binding to transcripts antisense of mRNAs aligned around transcription start site (TSS) (n = 3,076, left) and around their poly-adenylation (pA) …
Figure 4—figure supplement 1
Motif enrichment analysis shows enrichment of Nrd1/Nab3 motifs for the TRAMP4 and the exosome complex.
Motif analysis was performed for all degradation factors in this study. Nrd1 and Nab3 are included for comparison (Schulz et al., 2013). Occurrences of Nrd1 motif (GTAG) and Nab3 motif (CTTG) are …
Figure 4—figure supplement 2
The aberrant nuclear ncRNAs bound by components of the exosome and the TRAMP4 complex are primarily NUTs and CUTs.
Cross-link sites were filtered to exclude regions that were previously annotated as NUTs and CUTs (Neil et al., 2009; Schulz et al., 2013). Averaged occupancy profiles of degradation factors are …
Figure 5 with 3 supplements
Global co-occupancy and co-localization analysis reveals unexpected cooperation between factors from different complexes and pathways.
(A) Matrix of pairwise correlation coefficients of factor occupancies evaluated over all transcripts. (B) Matrix of co-localization based on the enrichment of factor x binding within 40 nt of the …
Figure 5—figure supplement 1
Co-occupancy for 74 RNA processing factors.
Matrix of pairwise correlation coefficients of factor occupancies evaluated over all transcripts. Analysis for 74 RNA processing factors, including 30 factors from this study, and 44 factors from …
Figure 5—figure supplement 2
Co-localization coefficients for all 74 RNA processing factors.
Pairwise correlation between normalized co-localization profiles of factors in a window of 40 nt centered at PAR-CLIP cross-link sites. Analysis for 74 RNA processing factors, including 30 factors …
Figure 5—figure supplement 3
Two-dimensional embedding of co-localization between 74 RNA processing factors.
tSNE plot visualizes similarities in co-localization profiles between RNA processing factors (factors are color-coded based on their function). Analysis for 74 RNA processing factors, including 30 …
Figure 6 with 9 supplements
Binding preferences reveal a link between decapping-mediated degradation and translation.
(A) Total occupancy per mRNA (according to TIF-seq annotation) for six factors as a function of the average mRNA codon optimality (transcript optimality). The occupancy of factors from the 5´→3´ …
Figure 6—figure supplement 1
Occupancies of deadenylation factors (Ccr4, Pop2, Not1, Caf40, Pan2, and Pan3) compared to transcript length, optimality, expression level, and half-life.
(A) To understand binding specificity of deadenylation factors, the total occupancy of each factor on a transcript is plotted against various transcript features (Gray shading: 95% confidence …
Figure 6—figure supplement 2
Occupancies of decapping factors (Dcp2, Dcp1, Edc2, Edc3, and Dhh1) compared to transcript length, optimality, expression level, and half-life.
(A) To understand binding specificity of decapping factors, the total occupancy of each factor on a transcript is plotted against various transcript features (Gray shading: 95% confidence intervals …
Figure 6—figure supplement 3
Occupancy of Xrn1 compared to transcript length, optimality, expression level, and half-life.
(A) To understand binding specificity of Xrn1 on various mRNAs, the total occupancy of Xrn1 on a transcript is plotted against various transcript features (Gray shading: 95% confidence intervals …
Figure 6—figure supplement 4
Occupancies of exosome components (Rrp6, Csl4, Rrp40, Rrp4, and Rrp44) compared to transcript length, optimality, expression level, and half-life.
(A) To understand binding specificity of exosome components, the total occupancy of each factor on a transcript is plotted against various transcript features (Gray shading: 95% confidence intervals …
Figure 6—figure supplement 5
Occupancies for components of the TRAMP complex (Air1, Trf5, Mtr4, Air2, and Trf4) compared to transcript length, optimality, expression level, and half-life.
(A) To understand binding specificity of TRAMP components, the total occupancy of each factor on a transcript is plotted against various transcript features (Gray shading: 95% confidence intervals …
Figure 6—figure supplement 6
Occupancies for components of the Ski complex (Ski2, Ski3, Ski7, and Ski8) compared to transcript length, optimality, expression level, and half-life.
(A) To understand binding specificity of factors in the Ski complex, the total occupancy of each factor on a transcript is plotted against various transcript features (Gray shading: 95% confidence …
Figure 6—figure supplement 7
Occupancies for components of the NMD pathway (Upf1, Upf2, Upf3, and Nmd4) compared to transcript length, optimality, expression level, and half-life.
(A) To understand binding specificity of factors in the NMD pathway, the total occupancy of each factor on a transcript is plotted against various transcript features (Gray shading: 95% confidence …
Figure 6—figure supplement 8
Distributions of transcript length, half-life, expression level and transcript optimality for yeast mRNAs.
Histograms on the diagonal show distributions of length, half-life (Materials and methods), expression level (Baejen et al., 2017) and transcript optimality (Pechmann and Frydman, 2013). Pairwise …
Figure 6—figure supplement 9
Correlation between binding to degradation factors and transcript length, codon-optimality, expression, and half-life.
Pearson correlation values between the binding strength of degradation factors (total occupancy over each transcript) and transcript length, transcript optimality (Pechmann and Frydman, 2013), …
Figure 7
Location and recruitment of the decapping complex Dcp1/Dcp2 and decapping enhancers Edc3, Dhh1, and Edc2.
(A) Smoothed, transcript-averaged PAR-CLIP occupancy profiles aligned at TSS and pA sites [±750 nt] of unstable and stable transcripts (first and fourth quantile of half-life distribution, …
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (S. cerevisiae, BY4741) | Ccr4_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000000019 | |
| Strain, strain background (S. cerevisiae, BY4741) | Pop2_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000005335 | |
| Strain, strain background (S. cerevisiae, BY4741) | Not1_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000000689 | |
| Strain, strain background (S. cerevisiae, BY4741) | Caf40_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000005232 | |
| Strain, strain background (S. cerevisiae, BY4741) | Pan2_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000003062 | |
| Strain, strain background (S. cerevisiae, BY4741) | Pan3_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000001508 | |
| Strain, strain background (S. cerevisiae, BY4741) | Dcp1_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000005509 | |
| Strain, strain background (S. cerevisiae, BY4741) | Dcp2_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000005062 | |
| Strain, strain background (S. cerevisiae, BY4741) | Edc2_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000000837 | |
| Strain, strain background (S. cerevisiae, BY4741) | Edc3_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000000741 | |
| Strain, strain background (S. cerevisiae, BY4741) | Dhh1_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000002319 | |
| Strain, strain background (S. cerevisiae, BY4741) | Xrn1_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000003141 | |
| Strain, strain background (S. cerevisiae, BY4741) | Rrp6_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000005527 | |
| Strain, strain background (S. cerevisiae, BY4741) | Csl4_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000005176 | |
| Strain, strain background (S. cerevisiae, BY4741) | Rrp40-TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000005502 | |
| Strain, strain background (S. cerevisiae, BY4741) | Rrp4_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000001111 | |
| Strain, strain background (S. cerevisiae, BY4741) | Rrp44_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000005381 | |
| Strain, strain background (S. cerevisiae, BY4741) | Air1_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000001341 | |
| Strain, strain background (S. cerevisiae, BY4741) | Trf5_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000005243 | |
| Strain, strain background (S. cerevisiae, BY4741) | Mtr4_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000003586 | |
| Strain, strain background (S. cerevisiae, BY4741) | Air2_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000002334 | |
| Strain, strain background (S. cerevisiae, BY4741) | Trf4_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000005475 | |
| Strain, strain background (S. cerevisiae, BY4741) | Ski2_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000004390 | |
| Strain, strain background (S. cerevisiae, BY4741) | Ski3_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000006393 | |
| Strain, strain background (S. cerevisiae, BY4741) | Ski7_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000005602 | |
| Strain, strain background (S. cerevisiae, BY4741) | Ski8_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000003181 | |
| Strain, strain background (S. cerevisiae, BY4741) | Upf1_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000004685 | |
| Strain, strain background (S. cerevisiae, BY4741) | Upf2_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000001119 | |
| Strain, strain background (S. cerevisiae, BY4741) | Upf3_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000003304 | |
| Strain, strain background (S. cerevisiae, BY4741) | Nmd4_TAP | C-terminally tagged gene (Open Biosystems, Germany). | SGD: S000004355 | |
| Antibody | IgG | Sigma-Aldrich | Cat#: I5006, RRID:AB_1163659 | IP: 0.1 mg per IP |
| Antibody | PAP anti-TAP | Sigma Aldrich | Cat#: P1291, RRID:AB_1079562 | WB (1:2000) |
| Commercial assay or kit | Dynabeads Protein G | Invitrogen | Cat#: 10003D | 330 µl per IP |
| Commercial assay or kit | RNase T1 | Thermo Fisher Scientific | Cat#: EN0541 | |
| Commercial assay or kit | Antarctic Phosphatase | NEB | Cat#: M0289S | |
| Commercial assay or kit | RNase OUT | Invitrogen | Cat#: 10777019 | |
| Commercial assay or kit | T4 Polynucleotide Kinase | Invitrogen | Cat#: EK0032 | |
| Commercial assay or kit | T4 RNA ligase 2, truncated KQ | NEB | Cat#: M0373S | |
| Commercial assay or kit | T4 RNA ligase 1 | NEB | Cat#: M0437M | |
| Commercial assay or kit | Proteinase K | NEB | Cat#: P8107S | |
| Commercial assay or kit | SuperScript III RT | Thermo Fisher Scientific | Cat#: 18080093 | |
| Commercial assay or kit | Phusion High-Fidelity PCR Master Mix | Thermo Fisher Scientific | Cat#: F531S | |
| Chemical compound, drug | 4-thiouracil | Carbosynth | Cat#: 591-28-6 | 1 mM final conc. |
| Software, algorithm | mockinbird | Roth and Torkler, 2018;https://github.com/soedinglab/Degradation_scripts | ||
| Software, algorithm | UMI-tools | Smith et al., 2017; doi:10.1101/gr.209601.116 | ||
| Software, algorithm | Skewer | Jiang et al., 2014; doi: 10.1186/1471-2105-15-182 | ||
| Software, algorithm | Bowtie | Langmead et al., 2009; doi: 10.1186/gb-2009-10-3-r25 | ||
| Software, algorithm | tSNE | Van Der Maaten and Hinton, 2008; doi: 10.1007/s10479-011-0841-3 |
Additional files
-
Supplementary file 1
Overview of RNA processing factors and their respective PAR-CLIP experiments used in this study.
- https://doi.org/10.7554/eLife.47040.031
-
Transparent reporting form
- https://doi.org/10.7554/eLife.47040.032
