Quantifying telomeric lncRNAs using PNA-labelled RNA-Flow FISH (RNA-Flow)

Gonzalez-Vasconcellos, Iria; Cobos-Fernández, María Ángeles; Atkinson, Michael J.; Fernández‐Piqueras, José; Santos, Javíer

doi:10.1038/s42003-022-03452-3

Cited by 3 publications

(2 citation statements)

References 19 publications

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“…In particular, as FlowFISH technologies have been established for viral gene expression 41 , ribosomal RNA content 42 , or long non-coding RNAs (Fig. 2b) 43 , our workflow provides unique access to the understudied populations defined by these markers. We envision an iterative process, whereby populations defined with existing large-scale scRNA-seq atlases are rationally enriched to study heterogeneity in new and rare populations, to refine cell atlases with ever greater definition.…”

Section: Discussionmentioning

confidence: 99%

Transcript-specific enrichment enables profiling rare cell states via scRNA-seq

Abay,

Stickels,

Takizawa

et al. 2024

Preprint

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Single-cell genomics technologies have accelerated our understanding of cell-state heterogeneity in diverse contexts. Although single-cell RNA sequencing (scRNA-seq) identifies many rare populations of interest that express specific marker transcript combinations, traditional flow sorting limits our ability to enrich these populations for further profiling, including requiring cell surface markers with high-fidelity antibodies. Additionally, many single-cell studies require the isolation of nuclei from tissue, eliminating the ability to enrich learned rare cell states based on extranuclear protein markers. To address these limitations, we describe Programmable Enrichment via RNA Flow-FISH by sequencing (PERFF-seq), a scalable assay that enables scRNA-seq profiling of subpopulations from complex cellular mixtures defined by the presence or absence of specific RNA transcripts. Across immune populations (n = 141,227 cells) and fresh-frozen and formalin-fixed paraffin-embedded brain tissue (n = 29,522 nuclei), we demonstrate the sorting logic that can be used to enrich for cell populations via RNA-based cytometry followed by high-throughput scRNA-seq. Our approach provides a rational, programmable method for studying rare populations identified by one or more marker transcripts.

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Section: Discussionmentioning

confidence: 99%

Transcript-specific enrichment enables profiling rare cell states via scRNA-seq

Abay,

Stickels,

Takizawa

et al. 2024

Preprint

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“…In cycling cells, the generation of dysfunctional telomeres via knockdown of TRF2 results in TERRA being released into the cytoplasm resulting in mitochondrial dysfunction and activation of innate immune pathways (21). While experiments designed to examine the effect of eliminating VR by knocking out TERRA would be valuable, experimentally, the greatest reduction in TERRA achieved by us or others is at most 40% (8,22) since TERRA is transcribed from most all telomeres.…”

Section: Introductionmentioning

confidence: 99%

The valine-arginine dipeptide repeat protein encoded by mammalian telomeric RNA appears highly expressed in mitosis and may repress global translation

Al-Turki,

Mantri,

Willcox

et al. 2024

Preprint

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Translation of mammalian telomeric G-rich RNA via the Repeat Associated non-AUG translation mechanism can produce two dipeptide repeat proteins: repeating valine-arginine (VR) and repeating glycine-leucine (GL). Their potentially toxic nature suggests that one or both must play a needed role in the cell. Using light microscopy combined with antibody staining we discovered that cultured human cells stain brightly for VR during mitosis with VR staining co-localizing with ribosomes. In vitro, VR protein represses translation in a firefly luciferase assay. Affinity purification combined with mass spectrometry identified ribosomal proteins as the major class of VR interacting proteins. Extension to mouse embryonic cerebral cortical development showed strong staining in the ventricular zone where high mitotic index neural progenitor cells proliferate and in the cortical plate where new neurons settle. These observations point to VR playing a key role in mitosis very possibly depressing global translation, a role mediated by the telomere.

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Mammalian telomeric RNA (TERRA) can be translated to produce valine–arginine and glycine–leucine dipeptide repeat proteins

Alturki

Griffith

2023

Proc. Natl. Acad. Sci. U.S.A.

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Mammalian telomeres consist of (TTAGGG)n repeats. Transcription of the C-rich strand generates a G-rich RNA, termed TERRA, containing G-quadruplex structures. Recent discoveries in several human nucleotide expansion diseases revealed that RNA transcripts containing long runs of 3 or 6 nt repeats which can form strong secondary structures can be translated in multiple frames to generate homopeptide or dipeptide repeat proteins, and multiple studies have shown them to be toxic in cells. We noted that the translation of TERRA would generate two dipeptide repeat proteins: highly charged repeating valine–arginine (VR) n and hydrophobic repeating glycine–leucine (GL) n . Here, we synthesized these two dipeptide proteins and raised polyclonal antibodies to VR. The VR dipeptide repeat protein binds nucleic acids and localizes strongly to replication forks in DNA. Both VR and GL form long 8-nm filaments with amyloid properties. Using labeled antibodies to VR and laser scanning confocal microscopy, threefold to fourfold more VR was observed in the nuclei of cell lines containing elevated TERRA as contrasted to a primary fibroblast line. Induction of telomere dysfunction via knockdown of TRF2 led to higher amounts of VR, and alteration of TERRA levels using a locked nucleic acid (LNA) GapmeR led to large nuclear VR aggregates. These observations suggest that telomeres, in particular in cells undergoing telomere dysfunction, may express two dipeptide repeat proteins with potentially strong biological properties.

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Quantifying telomeric lncRNAs using PNA-labelled RNA-Flow FISH (RNA-Flow)

Cited by 3 publications

References 19 publications

Transcript-specific enrichment enables profiling rare cell states via scRNA-seq

Transcript-specific enrichment enables profiling rare cell states via scRNA-seq

The valine-arginine dipeptide repeat protein encoded by mammalian telomeric RNA appears highly expressed in mitosis and may repress global translation

Mammalian telomeric RNA (TERRA) can be translated to produce valine–arginine and glycine–leucine dipeptide repeat proteins

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