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Identification of human pathways acting on nuclear non-coding RNAs using the Mirror forward genetic approach

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Che, Rui, Panah, Monireh, Mirani, Bhoomi, Knowles, Krista, Ostapovich, Anastacia, Majumdar, Debarati, Chen, Xiaotong, DeSimone, Joseph +4 more·2025·Nature Communications
DOI 10.1038/s41467-025-59998-3PMC PMC12095793

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Abstract

Despite critical roles in diseases, human pathways acting on strictly nuclear non-coding RNAs have been refractory to forward genetics. To enable their forward genetic discovery, we developed a single-cell approach that “Mirrors” activities of nuclear pathways with cytoplasmic fluorescence. Application of Mirror to two nuclear pathways targeting MALAT1’s 3′ end, the pathway of its maturation and the other, the degradation pathway blocked by the triple-helical Element for Nuclear Expression (ENE), identified nearly all components of three complexes: Ribonuclease P and the RNA Exosome, including nuclear DIS3, EXOSC10, and C1D, as well as the Nuclear Exosome Targeting (NEXT) complex. Additionally, Mirror identified DEAD-box helicase DDX59 associated with the genetic disorder Oral-Facial-Digital syndrome (OFD), yet lacking known substrates or roles in nuclear RNA degradation. Knockout of DDX59 exhibits stabilization of the full-length MALAT1 with a stability-compromised ENE and increases levels of 3′-extended forms of small nuclear RNAs. It also exhibits extensive retention of minor introns, including in OFD-associated genes, suggesting a mechanism for DDX59 association with OFD. Mirror efficiently identifies pathways acting on strictly nuclear non-coding RNAs, including essential and indirectly-acting components, and as a result can uncover unexpected links to human disease. Human pathways acting on nuclear ncRNAs have been refractory to forward genetics. Here, the authors develop a forward genetic approach that identifies such pathways and show DDX59 is required for minor intron splicing, suggesting a mechanism for its association with Oral-Facial-Digital syndrome.

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  • DDX59 has recently been implicated in the degradation of the nuclear noncoding RNA MALAT1, whose levels are also regulated by the RNA exosome.

    “DDX59 has recently been implicated in the degradation of the nuclear noncoding RNA MALAT1 whose levels are also regulated by the RNA exosome88.”

    From EXOSC3 G191 Variants Trigger System-Wide Recalibration of RNA Processing Machinery (2026) · cites this paper as [88]

    The Results section (page not provided) shows that DDX59 knockout stabilizes MALAT1(C8351G), a form with a stability-compromised 3′ end; it also reports that RNA Exosome components are required for degradation of that form. The claim omits this qualification: the same section reports no major effect of DDX59 knockout on endogenous wild-type MALAT1 during the measured time course or on its steady-state levels. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • DDX59 has recently been implicated in noncoding RNA degradation along with multiple subunits of the RNA exosome.

    “The DEAD-box helicase DDX59, which has recently been implicated in noncoding RNA degradation along with multiple subunits of the RNA exosome, was also significantly upregulated at both transcript and protein levels88.”

    From EXOSC3 G191 Variants Trigger System-Wide Recalibration of RNA Processing Machinery (2026) · cites this paper as [88]

  • DDX59 was significantly upregulated at both transcript and protein levels.

    “The DEAD-box helicase DDX59, which has recently been implicated in noncoding RNA degradation along with multiple subunits of the RNA exosome, was also significantly upregulated at both transcript and protein levels88.”

    From EXOSC3 G191 Variants Trigger System-Wide Recalibration of RNA Processing Machinery (2026) · cites this paper as [88]

    The Results section on DDX59 knockout reports effects on RNA splicing and noncoding RNA stability, but does not report that DDX59 itself was upregulated. Searches for DDX59 transcript and protein expression found no passage establishing significant upregulation at both levels. Page numbers were not available in our copy. (LLM, conf=0.77)

    Experimental — accuracy not yet measured

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Cited by

Sorted by citations

Authors
Che, R., Panah, M., Mirani, B., Knowles, K., Ostapovich, A., Majumdar, D., Chen, X., DeSimone, J., White, W., Noonan, M., Luo, H., & Alexandrov, A.
Affiliations
  • Clemson University (US)
  • Yale University (US)
Corresponding author
Alexandrov, Andrei
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2025-05-22
Volume
16
Issue
1
Pages
4741-4741
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-025-59998-3
PMC
PMC12095793
OpenAlex
W4410554692
Cited by
7
References
1
Citations by year
2026: 4 · 2025: 2 · 2024: 1
FWCI
1.80
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (HG009362)
  • U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (GM139769)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer-related molecular mechanisms research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Nuclear Structure and Function
Keywords
forward genetics, MALAT1, RNA degradation, RNA exosome, ribonuclease P, oral-facial-digital syndrome, small nuclear RNA, single-cell imaging

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