ZC3HC1 Is a Novel Inherent Component of the Nuclear Basket, Resident in a State of Reciprocal Dependence with TPR

Gunkel, Philip; Iino, Haruki; Krull, Sandra; Cordes, Volker C.

doi:10.3390/cells10081937

Cited by 16 publications

(90 citation statements)

References 116 publications

(182 reference statements)

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“…Among the remaining highly ranked hits were several that encoded putative or validated RNA binding proteins ( RBM33 , CELF1 , DDX10, YTHDC1 , NOL7 , SRP14 , and SRSF11 ), raising the possibility that one or more of these factors may play a direct role in NORAD expression or trafficking. We assessed the effect of knocking out each of these genes, as well as ZC3HC1 , another highly ranked hit that is required for TPR localization to the nuclear pore complex ( Gunkel et al 2021 ), on GFP expression, NORAD expression, and NORAD localization by generating knockout pools with lentivirally delivered CRISPR components ( Supplemental Fig. S2B ).…”

Section: Resultssupporting

confidence: 91%

RBM33 directs the nuclear export of transcripts containing GC-rich elements

et al. 2022

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Although splicing is a major driver of RNA nuclear export, many intronless RNAs are efficiently exported to the cytoplasm through poorly characterized mechanisms. For example, GC-rich sequences promote nuclear export in a splicing-independent manner, but how GC content is recognized and coupled to nuclear export is unknown. Here, we developed a genome-wide screening strategy to investigate the mechanism of export of NORAD, an intronless cytoplasmic long noncoding RNA (lncRNA). This screen revealed an RNA binding protein, RBM33, that directs the nuclear export of NORAD and numerous other transcripts. RBM33 directly binds substrate transcripts and recruits components of the TREX–NXF1/NXT1 RNA export pathway. Interestingly, high GC content emerged as the feature that specifies RBM33-dependent nuclear export. Accordingly, RBM33 directly binds GC-rich elements in target transcripts. These results provide a broadly applicable strategy for the genetic dissection of nuclear export mechanisms and reveal a long-sought nuclear export pathway for transcripts with GC-rich sequences.

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Section: Resultssupporting

confidence: 91%

RBM33 directs the nuclear export of transcripts containing GC-rich elements

et al. 2022

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“…To study the structure and function of hPol I in vitro, we first created a cell line that allows the specific enrichment of the complete enzyme in its native state without contamination of hPol III. Using the CRISPR/Cas9 technology in a dual-nicking approach, a cleavable sfGFP tag was fused to the genomic sequence of the largest Pol I subunit RPA1 of the Hela P2 cell line ( 51 ). After identification of positive clones by single-cell FACS based on GFP fluorescence intensity, correct insertion was confirmed by site-specific PCR.…”

Section: Resultsmentioning

confidence: 99%

The human RNA polymerase I structure reveals an HMG-like docking domain specific to metazoans

et al. 2022

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Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth, and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk, and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This “dock II” domain resembles a truncated HMG box incapable of DNA binding which may serve as a downstream transcription factor–binding platform in metazoans. Biochemical analysis, in situ modelling, and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG box domain–containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.

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Section: Specific Tagging and Purification Of Human Rna Polymerase Imentioning

confidence: 99%

The human RNA polymerase I structure reveals an HMG-like transcription factor docking domain specific to metazoans

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Straub

et al. 2021

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Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP-fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This "dock II" domain resembles a truncated HMG-box incapable of DNA-binding which may serve as a downstream-transcription factor binding platform in metazoans. Biochemical analysis and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG-box domain containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.

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ZC3HC1 Is a Novel Inherent Component of the Nuclear Basket, Resident in a State of Reciprocal Dependence with TPR

Cited by 16 publications

References 116 publications

RBM33 directs the nuclear export of transcripts containing GC-rich elements

RBM33 directs the nuclear export of transcripts containing GC-rich elements

The human RNA polymerase I structure reveals an HMG-like docking domain specific to metazoans

The human RNA polymerase I structure reveals an HMG-like transcription factor docking domain specific to metazoans

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