aCPSF1 cooperates with terminator U-tract to dictate archaeal transcription termination efficacy
- State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, China
- University of Chinese Academy of Sciences, China
- Beijing Institutes of Life Science, Chinese Academy of Sciences, China
Figures
Figure 1 with 4 supplements
A positive correlation is observed between the terminator U4-tract numbers and the TTEs among the TUs of M.maripaludis.
(A) A representative Term-seq map of MMP0020 showing a dramatical decreasing pattern of sequencing reads at four nucleotides that flank the identified transcription termination site (TTS, -1 site …
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Figure 1—source data 1
Includes the statistic source data of Figure 1, Figure 1—figure supplements 1 and 2.
- https://cdn.elifesciences.org/articles/70464/elife-70464-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Multiple TTSs in one transcript identified by Term-seq in M. maripaludis.
(A, B, C) Representative Term-seq maps showed multiple TTSs in the MMP1274 (A), MMP0127 (B), and MMP1036 (C) transcripts. The primary and secondary TTSs are indicated by red and black arrows, …
Figure 1—figure supplement 2
Features of the primary and secondary TTSs identified in M. maripaludis.
(A, B, C) Box-plot diagrams show the statistics of the 3′-UTR length (A), head read abundance (B), and TTEs (C) of the 998 primary and 1359 secondary TTSs, respectively. (D) Logo representations …
Figure 1—figure supplement 3
Term-seq maps of three representative genes, MMP0065 (A), MMP1579 (B), and MMP0760 (C).
Gene locations on the chromosome are indicated at the top. Black arrows indicate Term-seq identified TTS (−1 site). The magnified mapping region (dotted red line framed) shows a dramatic read …
Figure 1—figure supplement 4
Percentages and motifs of the TU groups that have different numbers of the terminator U4-tracts.
(A) Pie chart shows the TU numbers and percentages in each group that has >2, 2, 1, and 0 terminator U4-tracts, respectively. The aCPSF1 dependency in each group determined by TQRR is displayed in …
Figure 2 with 3 supplements
Co-occurrence of aCPSF1 and the terminator U4-tract is correlated with the genome-wide TTEs of M. maripaludis.
(A) Visualized Term-seq read maps of the representative genes, MMP0511 (top) and MMP0760 (bottom), show sharper reads decreasing between the −2 and +2 nts (dotted frame) respectively down- and …
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Figure 2—source data 1
Includes the statistic source data of Figure 2, Figure 2—figure supplements 1 and 2.
- https://cdn.elifesciences.org/articles/70464/elife-70464-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Box plot diagram showing the statistics of TTEs of the Term-seq identified primary TTSs in the WT (S2) and ▽aCPSF1 mutant.
Figure 2—figure supplement 2
The terminator motif and the linear correlation of TTEs and TQRRs of noncoding RNAs in M. maripaludis.
(A) Sequence motif upstream of the 78 TTSs of noncoding RNAs identified by Term-seq was generated using weblogo. (B) Logo representations of the terminator motif features in the three groups of TTSs …
Figure 2—figure supplement 3
Transcription readthrough (TRT) of noncoding RNAs caused by the depletion of aCPSF1.
(A, B) The stand-specific RNA-seq mapping profiles (left) show 3′-end extensions (dot magenta brackets) of sRNA_11 (A) and sRNA_23 (B) in ▽aCPSF1 (red) referenced to that in strain S2 (blue). …
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Figure 2—figure supplement 3—source data 1
Includes the bolt source date of Figure 2—figure supplement 3.
- https://cdn.elifesciences.org/articles/70464/elife-70464-fig2-figsupp3-data1-v1.zip
Figure 3 with 3 supplements
Binding specificity of aCPSF1 to RNAs carrying different numbers of U4-tracts determined by rEMSA assays.
RNAs with a consensus length of 36 nt derived from the indicated gene terminators that carry ≥2 U4-tracts (A), 1 U4-tract (B), and 0 U4-tract (C) were used as the binding substrates. RNA sequences …
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Figure 3—source data 1
Includes the gel source data of Figure 3.
- https://cdn.elifesciences.org/articles/70464/elife-70464-fig3-data1-v1.zip
Figure 3—figure supplement 1
The binding specificity of aCPSF1 to U-tract RNAs determined by rEMSA assays.
Three RNAs (T0901, T1149, and T1100) containing the terminator U-tract sequences and one with no U-tract (T1697), which are from the indicated transcript 3′-ends, were used as the binding substrates …
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Figure 3—figure supplement 1—source data 1
Includes the gel source data of Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/70464/elife-70464-fig3-figsupp1-data1-v1.zip
Figure 3—figure supplement 2
Binding curves of aCPSF1 to indicated RNAs.
The shifted RNA percentage was quantified based on quantifying the bound and unbound substrates shown in the rEMSA gels in Figure 3. The binding curves were obtained by plotting the shifted RNA (%) …
Figure 3—figure supplement 3
The binding specificity of aCPSF1 to U-tract RNAs assayed by SPR.
Two RNAs of terminator sequences carrying U-tracts (T0400 and T0911) and one with no U-tract (T1406) were used as the binding substrates for SPR assays. The RNA sequences are shown at the top. The …
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Figure 3—figure supplement 3—source data 1
Includes the statistic source data of Figure 3—figure supplement 3.
- https://cdn.elifesciences.org/articles/70464/elife-70464-fig3-figsupp3-data1-v1.xlsx
Figure 4 with 3 supplements
The minimal RNA length and U-tract base stringency required for the specific binding of aCPSF1 determined by rEMSA assays.
RNAs with indicated lengths and base mutations shown in the top panels that are derived from the native terminator sequences of MMP0204 (T0204) and MMP0229 (T0229) were used as the binding …
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Figure 4—source data 1
Includes the gel source data of Figure 4.
- https://cdn.elifesciences.org/articles/70464/elife-70464-fig4-data1-v1.zip
Figure 4—figure supplement 1
The minimal RNA length and U-tract base stringency required for the specific binding of aCPSF1 on T0400 terminator determined by rEMSA assays.
RNAs of indicated lengths and base mutations (shown on the top panels) that are derived from the native terminator sequences of MMP0400 (T0400) were used as the binding substrates of aCPSF1. (A) The …
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Figure 4—figure supplement 1—source data 1
Includes the gel source data of Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/70464/elife-70464-fig4-figsupp1-data1-v1.zip
Figure 4—figure supplement 2
Binding curves of aCPSF1 to indicated RNAs.
The shifted RNA percentage was quantified based on quantifying the bound and unbound substrates shown in the rEMSA gels in Figure 4. The binding curves were obtained by plotting the shifted RNA (%) …
Figure 4—figure supplement 3
The RNase footprint assay identifies the binding region of aCPSF1 to the T0204 RNA carrying two terminator U-tracts.
The [γ-32P]-labeled RNA with the T0204 terminator sequence in length of 36 nt was digested with 0.3 U RNase I in either the absence (lanes -) or presence of the purified recombinant aCPSF1 at …
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Figure 4—figure supplement 3—source data 1
Includes the gel source data of Figure 4—figure supplement 3.
- https://cdn.elifesciences.org/articles/70464/elife-70464-fig4-figsupp3-data1-v1.zip
Figure 5
Terminator reporter system demonstrates TTE variations in co-occurrence and either absence of aCPSF1 and the terminator U-tract.
(A) Schematic depicting the construction of the terminator reporter system. The tested terminator sequences carrying different numbers of U-tracts were each inserted between the upstream luciferase …
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Figure 5—source data 1
Includes the statistic source data of Figure 5.
- https://cdn.elifesciences.org/articles/70464/elife-70464-fig5-data1-v1.xlsx
Figure 6 with 3 supplements
The N-terminal KH domains of aCPSF1 contribute to the binding specificity to terminator U-tracts and transcription termination.
(A) Schematic (upper panel) showing the aCPSF1 protein architecture with two N-terminal KH domains, the central MβL domain and the C-terminal β-CASP domain. rEMSA assay (lower panel) was performed …
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Figure 6—source data 1
Includes the gel and bolt source data of Figure 6.
- https://cdn.elifesciences.org/articles/70464/elife-70464-fig6-data1-v1.zip
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Figure 6—source data 2
Includes the statistic source data of Figure 6C.
- https://cdn.elifesciences.org/articles/70464/elife-70464-fig6-data2-v1.xlsx
Figure 6—figure supplement 1
3′RACE assays detected transcriptional readthroughs (TRTs) of MMP1149, MMP0901 and MMP1224 in 22 °C -grown wild-type strain (S2), aCPSF1 depletion mutant (▽aCPSF1), and the ▽aCPSF1 complementated with the wild-type (Com(WT)) and KH domain truncated aCPSF1 (Com(ΔKH)) respectively.
Blue and magenta arrows indicate the PCR products of normal terminations (TTSs) and TRTs, respectively. A DNA ladder on the left provides references of the PCR product migrations.
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Figure 6—figure supplement 1—source data 1
Includes the gel source data of Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/70464/elife-70464-fig6-figsupp1-data1-v1.zip
Figure 6—figure supplement 2
Interaction of the KH domain deleted aCPSF1 (ΔKH-aCPSF1) with RNA polymerase detected by gel filtration coupled with western blot.
Interaction of aCPSF1 and its mutant (ΔKH-aCPSF1) with the RNA polymerase were determined using the similar method as described previously (Yue et al., 2020). The co-occurrence of aCPSF1 and …
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Figure 6—figure supplement 2—source data 1
Includes the bolt source data of Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/70464/elife-70464-fig6-figsupp2-data1-v1.zip
Figure 6—figure supplement 3
Assays that determines the vital role of the dimerization of aCPSF1 in transcription termination.
(A) Western blot assays demonstrate the expressions of the intact and C-terminal 13 residues truncated aCPSF1 in the wild-type strain S2 carrying the empty complementation plasmid of pMEV2 (S2+ …
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Figure 6—figure supplement 3—source data 1
Includes the bolt and gel source data of Figure 6—figure supplement 3A and C.
- https://cdn.elifesciences.org/articles/70464/elife-70464-fig6-figsupp3-data1-v1.zip
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Figure 6—figure supplement 3—source data 2
Includes the statistic source data of Figure 6—figure supplement 3B.
- https://cdn.elifesciences.org/articles/70464/elife-70464-fig6-figsupp3-data2-v1.xlsx
Figure 7
The aCPSF1-dependent archaeal transcription termination represents the archetype of the eukaryotic RNAP II termination mode.
The general archaeal transcription termination factor aCPSF1, relying on the N-terminal KH domains specifically recognizing the terminator U-tract and the nuclease domain cleaving at the 3′-end, …
Additional files
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Supplementary file 1
Termseq identified TTSs and the read ratios of 1 to TTS 1 in M. maripaludis S2 and ▽aCPSF1.
- https://cdn.elifesciences.org/articles/70464/elife-70464-supp1-v1.xlsx
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Supplementary file 2
The TTEs and TQRRs of primary TTSs.
- https://cdn.elifesciences.org/articles/70464/elife-70464-supp2-v1.xlsx
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Supplementary file 3
The TTEs of TTSs for noncoding RNAs in the wild type S2 and ▽aCPSF1 mutant.
- https://cdn.elifesciences.org/articles/70464/elife-70464-supp3-v1.xlsx
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Supplementary file 4
Includes Supplementary file 4a-4e.
- https://cdn.elifesciences.org/articles/70464/elife-70464-supp4-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/70464/elife-70464-transrepform1-v1.docx
