Protocol to use RNaseH1-based CRISPR to modulate locus-associated R-loops

Khosraviani, Negin; Abraham, Karan Joshua; Chan, Janet N.Y.; Mekhail, Karim

doi:10.1016/j.xpro.2022.101734

Cited by 3 publications

(7 citation statements)

References 22 publications

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“…Inhibiting Pol II hyperinduces Pol I function at the IGS 11 . Similar results were obtained upon repressing the Pol II-dependent R-loop shield at the IGS using a locus-associated R-loop repression system (LasR) employing a chimeric protein harboring dCas9 and the RNA-DNA hybrid repressor RNase H1 (RNaseH1-EGFP-dCas9 (RED)) 11,12 . The unleashed Pol I synthesizes excessive levels of sense intergenic non-coding RNAs (sincRNAs) that severely disrupt nucleolar organization and ribosome biogenesis.…”

Section: Introductionsupporting

confidence: 63%

“…To repress the unscheduled R-loops at IGS20, we employed the LasR system 11,12 . We used short guide RNAs (sgRNAs) targeting IGS20 (sg20) to selectively enrich the inducibly expressed RED (RNaseH1-EGFP-dCas9) fusion protein at the affected IGS site 11,12,30,31 . As controls, we used non-targeting sgRNA controls (sgNT) and the fusion protein dRED (dRNaseH1-EGFP-dCas9), which harbors a catalytically dead version of the RNaseH1 enzyme 11,12 .…”

Section: Resultsmentioning

confidence: 99%

“…Third, due to the ability of TBPL1 to impact numerous processes linked to ribosome biogenesis, we could not devise a rescue experiment to rescue rRNA biogenesis in TBPL1-deficient cells. Fourth, other limitations of our study include those linked to using the RED/dRED-based LasR system for targeted R-loop modulation, which we have described in detail previously 12 . For instance, all limitations associated with CRISPR- and dCas9-based technologies apply to this system, including poorly understood yet rare chromosome rearrangements 55 .…”

Section: Discussionmentioning

confidence: 99%

“…For knockdown experiments, HEK293T cells at 50% confluence were transfected with 50 nM siRNA using Lipofectamine RNAiMAX (Invitrogen) as described by the manufacturer in an antibiotic-free growth medium for 48 h. For inhibitory experiments, 2 µM flavopiridol (Santa Cruz Cat # sc-202157; iPol II), 50 ng/ml actinomycin D (LAD/iPol I; Sigma Cat# A1410-2MG), or DMSO vehicle control were used. For locus-associated targeting of RED or dRED as part of the LasR system, HEK293T T-REx TM cells were grown at 70% confluence and used as described previously 12 . For knockdown experiments, cells were transfected using Lipofectamine RNAiMAX (Invitrogen, Cat# 13778075).…”

Section: Methodsmentioning

confidence: 99%

Section: Igs Pol II Transcription Elongation Via Paf1mentioning

confidence: 99%

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Nucleolar Pol II interactome reveals TBPL1, PAF1, and Pol I at intergenic rDNA drive rRNA biogenesis

Khosraviani,

Yerlici,

St-Germain

et al. 2023

Preprint

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Nucleolar ribosomal DNA (rDNA) repeats control ribosome manufacturing. rDNA harbors a ribosomal RNA (rRNA) gene and an intergenic spacer (IGS). RNA polymerase (Pol) I transcribes rRNA genes yielding the rRNA components of ribosomes. Pol II at the IGS induces rRNA production by preventing Pol I from excessively synthesizing IGS non-coding RNAs (ncRNAs) that can disrupt nucleoli. At the IGS, Pol II regulatory processes and whether Pol I function can be beneficial remain unknown. Here, we identify IGS Pol II regulators, uncovering nucleolar optimization via IGS Pol I. Compartment-enriched proximity-dependent biotin identification (compBioID) showed enrichment of the TATA-less promoter-binding TBPL1 and transcription regulator PAF1 with IGS Pol II. TBPL1 localizes to TCT motifs, driving Pol II and Pol I and maintaining its baseline ncRNA levels. PAF1 promotes Pol II elongation, preventing unscheduled R-loops that hyper-restrain IGS Pol I and its ncRNAs. PAF1 or TBPL1 deficiency disrupts nucleolar organization and rRNA biogenesis. In PAF1-deficient cells, repressing unscheduled IGS R-loops rescues nucleolar organization and rRNA production. Depleting IGS Pol I-dependent ncRNAs is sufficient to compromise nucleoli. We present the interactome of nucleolar Pol II and show its control by TBPL1 and PAF1 ensures IGS Pol I ncRNAs maintaining nucleolar structure and operation.

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Section: Introductionsupporting

confidence: 63%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Igs Pol II Transcription Elongation Via Paf1mentioning

confidence: 99%

See 3 more Smart Citations

Nucleolar Pol II interactome reveals TBPL1, PAF1, and Pol I at intergenic rDNA drive rRNA biogenesis

Khosraviani,

Yerlici,

St-Germain

et al. 2023

Preprint

Self Cite

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Nucleolar Pol II interactome reveals TBPL1, PAF1, and Pol I at intergenic rDNA drive rRNA biogenesis

Khosraviani,

Yerlici,

St-Germain

et al. 2024

Nat Commun

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Ribosomal DNA (rDNA) repeats harbor ribosomal RNA (rRNA) genes and intergenic spacers (IGS). RNA polymerase (Pol) I transcribes rRNA genes yielding rRNA components of ribosomes. IGS-associated Pol II prevents Pol I from excessively synthesizing IGS non-coding RNAs (ncRNAs) that can disrupt nucleoli and rRNA production. Here, compartment-enriched proximity-dependent biotin identification (compBioID) revealed the TATA-less-promoter-binding TBPL1 and transcription-regulatory PAF1 with nucleolar Pol II. TBPL1 localizes to TCT motifs, driving Pol II and Pol I and maintaining its baseline ncRNA levels. PAF1 promotes Pol II elongation, preventing unscheduled R-loops that hyper-restrain IGS Pol I-associated ncRNAs. PAF1 or TBPL1 deficiency disrupts nucleolar organization and rRNA biogenesis. In PAF1-deficient cells, repressing unscheduled IGS R-loops rescues nucleolar organization and rRNA production. Depleting IGS Pol I-dependent ncRNAs is sufficient to compromise nucleoli. We present the nucleolar interactome of Pol II and show that its regulation by TBPL1 and PAF1 ensures IGS Pol I ncRNAs maintaining nucleolar structure and function.

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DNA double-strand break-capturing nuclear envelope tubules drive DNA repair

Shokrollahi

Stanić

Hundal

et al. 2023

Preprint

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The nuclear envelope is a membrane separating nuclear from cytoplasmic processes. Existing models suggest that damaged DNA moves to the envelope at the edge of the nucleus for repair. Yet, most damaged human DNA does not reposition to the nuclear periphery during repair. Here we show that human cells relocate the nuclear envelope to non-peripheral damaged DNA, providing solid support promoting the reconnection of DNA break ends. Upon DNA double-strand break (DSB) induction, cytoplasmic microtubules poke the nuclear envelope inwards, inducing an extensive network of DSB-capturing nuclear envelope tubules (dsbNETs). The formation of dsbNETs, which encompass the nuclear lamina and the inner and outer nuclear membranes, depends on DNA damage response kinases, dynamic microtubules, the linker of the nucleoskeleton and cytoskeleton (LINC) proteins SUN1 and SUN2, nuclear pore protein NUP153, and kinesin KIF5B. Repressing dsbNETs compromises the reassociation of DSB ends. The timely reversal of dsbNETs by the kinesin KIFC3 also promotes repair. DSB ends reconnection is restored in dsbNETs-deficient cells by enlarging the 53BP1 DNA repair center. The lamina-binding domain of SUN1 mediates its entry into the tubules and DSB capture by the envelope. Fusing truncated SUN1 to the NHEJ repair protein KU70 fails to localize SUN1 to the tubules but rescues DSB targeting only to the boundary envelope. Although dsbNETs typically promote accurate DSB repair and cell survival, they are co-opted by the PARP inhibitor olaparib to induce aberrant chromosomes restraining BRCA1-deficient breast cancer cells. We uncover dsbNETs, which bring the nuclear envelope to DSBs for repair and potentiate the efficacy of anti-cancer agents. Our findings revise theories of the structure-function relationship of the nuclear envelope and identify dsbNETs as a critical factor in DNA repair and nuclear organization, with implications for health and disease.

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Protocol to use RNaseH1-based CRISPR to modulate locus-associated R-loops

Cited by 3 publications

References 22 publications

Nucleolar Pol II interactome reveals TBPL1, PAF1, and Pol I at intergenic rDNA drive rRNA biogenesis

Nucleolar Pol II interactome reveals TBPL1, PAF1, and Pol I at intergenic rDNA drive rRNA biogenesis

Nucleolar Pol II interactome reveals TBPL1, PAF1, and Pol I at intergenic rDNA drive rRNA biogenesis

DNA double-strand break-capturing nuclear envelope tubules drive DNA repair

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