Quantitative proteomics reveals key roles for post-transcriptional gene regulation in the molecular pathology of FSHD

Abstract

23DUX4 is a transcription factor whose misexpression in skeletal muscle causes 24 facioscapulohumeral muscular dystrophy (FSHD). While DUX4's transcriptional activity has been 25 extensively characterized, the DUX4-induced proteome remains undescribed. Here, we report 26 concurrent measurement of RNA and protein levels in DUX4-expressing cells via RNA-seq and 27 quantitative mass spectrometry. DUX4 transcriptional targets were robustly translated, confirming 28 the likely clinical relevance of proposed FSHD biomarkers. However, a multitude of mRNAs and 29 proteins exhibited discordant expression changes upon DUX4 expression. Our dataset revealed 30 unexpected proteomic, but not transcriptomic, dysregulation of diverse molecular pathways, 31including Golgi apparatus fragmentation, as well as extensive post-transcriptional buffering of 32 stress response genes. Key components of RNA degradation machineries, including UPF1, 33 UPF3B, and XRN1, exhibited suppressed protein, but not mRNA, levels, explaining the build-up 34 of aberrant RNAs that characterizes DUX4-expressing cells. Our results provide a resource for 35 the FSHD community and illustrate the importance of post-transcriptional processes to DUX4-36 induced pathology. 37

Facioscapulohumeral muscular dystrophy is associated with altered myoblast proteome dynamics

“…Overexpression of DUX4 in MB135 myoblasts is associated with a 50 % decrease in protein synthesis (14). We also report the median turnover rate of proteins is lesser in FSHD (which was not certified by peer review) is the author/funder.…”

“…Nevertheless, our proteomic data align well with the wider literature on the molecular mechanisms of FSHD. Notably, when DUX4 is artificially expressed in human myoblasts, only 8 of 25 candidate genes (67) were detected at the protein level (14). Moreover, almost half (6 of 13) of the FSHD patient samples reported by Yao et al (67) did not show enrichment of the list of DUX4 target genes.…”

“…Human myogenic cells expressing DUX4-FL exhibit insoluble ubiquitylated proteins (46), which suggests the ubiquitin proteasome system may be impaired in FSHD. Furthermore, proteins involved in the ubiquitin proteasome system are increased at the transcriptional and protein level (14,60,61) after induction of DUX4. In our data, several ubiquitin E2 conjugating enzymes (UBE2T, UBE2D2, UBE2Q1), ubiquitin E3 ligases (UBR3, UBR7, XIAP, TRIM32, RNF149, RNF213), deubiquitylating enzymes (USP11, USP8, USP22, OTULIN, UCHL1), as well as proteasome activator complex subunit protein (PSME4) were each more abundant in FSHD myoblasts (Fig.…”

“…Artificial expression of DUX4 results in a 50 % reduction in bulk protein synthesis measured by 35 S-methionine incorporation in M135 myoblasts (14). However, it is not clear whether similar disruptions in protein turnover occur in patient-derived samples, and the measurement of the 'bulk' synthesis rate of protein mixtures (14) cannot distinguish individual protein responses.…”

“…The aforementioned 50 % reduction in 35 S-methionine incorporation could represent a blanket 50 % reduction in the synthesis of all proteins or complete inhibition in the synthesis of 50 % of proteins. In addition, DUX4 target-genes that were faithfully translated into functional proteins included several known or putative E3 ubiquitin ligases that could dysregulate protein degradation (14).…”