Pathogenic RNAs in muscular dystrophy

From the Tapscott lab, Human Biology division

Double stranded RNAs are usually produced by certain types of viruses and are recognized by the innate immune system, the first line against infection. In healthy cells, double stranded RNAs are unfolded by helicases during the translation process. On the other hand, abnormal accumulation of double stranded RNAs can lead to cell death.

One area of research of the Tapscott lab (Human Biology Division) is understanding DUX4-mediated cell toxicity in facioscapulohumeral muscular dystrophy (FSHD). This degenerative disease progressively impares different muscles and is caused by the abnormal expression of the transcription factor DUX4. The Tapscott lab previously demonstrated that this protein not only induces but maintains the expression of the targeted transcriptional program. It is also capable of inducing dsRNAs that activate apoptotic pathways in muscle cells. However, the mechanism underlying this toxicity was unknown. In a new study published in Human Molecular Genetics, Dr. Sean Shadle and colleagues (Tapscott lab) investigated the nature of the dsRNAs generated in response to DUX4 and the consequences of their accumulation.

The authors induced DUX4 expression in a human myoblast cell line (MB135-iDUX4) and sequenced dsRNAs in the induced and control conditions by high-throughput sequencing of immunoprecipitated RNAs (dsRIP-seq). Shadle et al. determined that non-coding intergenic RNAs were prevalent in dsRNAs induced by DUX-4, suggesting that aberrant transcriptional activity is triggered by DUX-4. Among these, human satellite II (HSATII) repeats were the most induced. For the Tapscott lab, this finding was highly relevant as Shadle explains: “In our previous study we noticed that expression of DUX4 leads to the activation of normally silenced repeats known as HSATII. This repeat type extends to millions of base pairs in length and reside adjacent to centromeres. Nevertheless, it was unknown what consequence, if any, transcription of these DNA repeats might have. Our current study collates these two seemingly disparate findings.”

Schematic showing transcription factor DUX4 (the cause of FSHD), binding HSATII repeats and trigerring bi-directional transcription. This results in the formation of HSATII dsRNAs that activate an apoptotic pathway.
Schematic showing transcription factor DUX4 (the cause of FSHD), binding HSATII repeats and trigerring bi-directional transcription. This results in the formation of HSATII dsRNAs that activate an apoptotic pathway. Illustration provided by Dr. Shadle

As DUX4 is a transcription factor, the researchers investigated how DUX4 could lead to the generation of dsRNA of HSATII. Shadle summarizes their findings : “Our experiments revealed that DUX4 directly binds to and activates the transcription of HSATII repeat sequences in a bi-directional manner. Sense-antisense HSATII transcripts combine to form dsRNA which leads to activation of dsRNA-sensing pathways and toxicity in DUX4-expressing cells.”

In order to functionally validate the importance of HSATII dsRNAs in DUX4-mediated cell cytotoxicity, they transfected MB135-iDUX4 cells with gapmers targeting both the forward and reverse HSATII RNA strands. Gapmers are composed of deoxynucleotides that induce RNase H cleavage of the targeted RNA sequence, and of modified ribonucleotides that protect gapmers from nuclease degradation. They are particularly efficient at targeting dsRNAs. The authors demonstrated that degradation of HSATII dsRNAs by gapmers impaired dsRNA foci formation and reduced cell toxicity induced by DUX4,  showing that accumulation of HSATII dsRNA directly leads to the cell toxicity seen in FSHD.

The Tascott lab already has an idea on how these findings could translate in oncology. Shadle develops: “The aberrant expression of HSATII repeats has been observed in some tumors and correlates with poor immune recognition of the cancer cells. We recently noticed that DUX4 expression occurs in a subset of cancers and similarly correlates with poor immune recognition. Understanding if there is a causal link between these two trends will be an important next step.”

This work was supported by the National Institutes of Health and the Friends of FSH Research.

Fred Hutch/UW Cancer Consortium member Dr. Tapscott contributed to this research.

Shadle SC., Bennett SR., Wong C-J., Karreman NA., Campbell AE., van der Maarel SM., Bass BL., Tapscott SJ. 2020. DUX4-induced bidirectional HSATII satellite repeat transcripts form intranuclear double- stranded RNA foci in human cell models of FSHD. Hum Mol Genet. 00, 00 (1-15).