Frank et al. · Cell Stem Cell · 2019
yylncT Defines a Class of Divergently Transcribed lncRNAs and Safeguards the T-mediated Mesodermal Commitment of Human PSCs
Frank Stefan, Ahuja Gaurav, Bartsch Deniz, Russ Nicole, Yao Wenjie, Kuo Joseph Chao-Chung, Derks Jens-Peter, Akhade Vijay Suresh, Kargapolova Yulia, Georgomanolis Theodore, Messling Jan-Erik, Gramm Marie, Brant Lilija, Rehimi Rizwan, Vargas Natalia Emilse, Kuroczik Alina, Yang Tsun-Po, Sahito Raja Ghazanfar Ali, Franzen Julia, Hescheler Juergen, Sachinidis Agapios, Peifer Martin, Rada-Iglesias Alvaro, Kanduri Meena, Costa Ivan G., Kanduri Chandrasekhar, Papantonis Argyris, Kurian Leo
The study
What was asked, and what was found
Frank et al., Cell Stem Cell, 2019 set out to find what the non-coding RNAs transcribed in the opposite direction from developmental genes are for. Using human embryonic stem cells driven toward heart, they defined a class of these divergent transcripts, showed they mirror the expression of the protein coding gene they sit beside, and found them concentrated at the loci of the genes that decide cell fate. One of them, yylncT, sits opposite BRACHYURY, the master specifier of mesoderm.
Deciding whether that transcript does anything, as opposed to merely being made, requires removing the RNA without touching the DNA. The team used three independent antisense oligonucleotides across two different chemistries, one set supplied by AUM BioTech against the long non-coding RNA, transfected into the stem cells with a lipid reagent at 600 ng per well 6 hours before mesoderm induction, against a matched control oligonucleotide. The oligonucleotide sequence is not published: the Methods record it as proprietary.
Removing the transcript cut BRACHYURY and the other early mesoderm markers. Transcriptome profiling showed the cells could not activate their early mesoderm programme at all, and the validated downstream targets of BRACHYURY fell with it. Set alongside the rest of the paper, where the transcript is shown to bind the de novo DNA methyltransferase DNMT3B and to keep the BRACHYURY locus free of aberrant methylation, the knockdown is the step that proves the RNA itself, rather than the act of transcribing it, is what safeguards the fate decision. The parallel mouse experiments used oligonucleotides from a different supplier.
Key findings
- Depleting the long non-coding RNA yylncT with an AUMlnc antisense oligonucleotide cut expression of the master mesoderm gene BRACHYURY and of other early mesoderm markers, placing the non-coding transcript upstream of the fate decision.(Results, Transcript-Specific Function of yylncT Is Essential for Mesoderm Commitment of hESCs)
- The oligonucleotide route was chosen precisely because it removes the transcript while leaving the DNA intact, which is what separates a long non-coding RNA acting as an RNA from the act of transcribing it.(Results, Transcript-Specific Function of yylncT Is Essential for Mesoderm Commitment of hESCs)
- Transcriptome profiling of the depleted cells showed they could not turn on their early mesoderm programme at all, reproducibly across replicates.(Results, Transcript-Specific Function of yylncT Is Essential for Mesoderm Commitment of hESCs)
- Depletion also brought down the validated downstream targets of BRACHYURY, which are the genes that drive the mesoderm fate.(Results, Transcript-Specific Function of yylncT Is Essential for Mesoderm Commitment of hESCs)
- The paper places the non-coding transcript on the de novo DNA methyltransferase DNMT3B and shows the transcript is needed to keep the BRACHYURY locus active, so the knockdown result sits inside a full mechanism.(Summary)
For research use only. Not for use in diagnostic or therapeutic procedures.