Control of RNA Gain-of-Function Versus Toxic RAN Protein Mechanisms
Since the discovery of Repeat-Associated Non-AUG (RAN) translation, the pathogenesis of any disorder that is the consequence of microsatellite expansions must consider two distinct mechanisms—RNA gain-of-function (i.e., toxicity of expanded repeat RNA), toxicity of RAN proteins, or a combination of both mechanisms. For myotonic dystrophy (DM), the toxic RNA mechanism has predominated thus far, as there are only limited reports of the detection of RAN proteins in affected tissues. How these two potential pathogenic pathways in DM are regulated, and whether they may be in any way co-regulated, are currently open questions.
Muscleblind (MBNL) Plays a Key Regulatory Role Over RAN Translation
Dr. Laura Ranum (University of Florida) and colleagues have recently published studies that provide new insights into the regulation of molecular pathogenic pathways in DM2. Through assessments of expanded sense (CCTG) and antisense (CAGG) transcripts in DM2 autopsy brains and in vitro, they demonstrated the occurrence of bidirectional transcription. The research team also showed that transcripts containing a threshold repeat length produce the corresponding poly(LPAC) and poly(QAGR) RAN proteins, with protein production positively correlated with repeat length. Cytoplasmic poly(LPAC) protein was localized to gray matter (neurons and glia), while poly(QAGR) was found in white matter, primarily in oligodendrocyte nuclei (but also in pathologic regions containing activated microglia). Using in vitro expression studies, the research team showed that both RAN proteins are toxic in neurons by mechanisms unrelated to RNA gain-of-function.
The production of poly(LPAC) protein was reduced or blocked by nuclear foci formation and retention of CCUG expansion RNA by MBNL1 binding. By contrast, CAGG transcripts do not bind MBNL1 and form nuclear foci, but rather are translocated to the cytoplasm, resulting in elevated poly(QAGR) RAN protein (CLIP experiments showed that poly(QAGR) protein does bind hnRNP A1).
A Model for Regulation of RAN Translation in DM2
Taken together, nuclear MBNL1 levels control the relative degree to which mis-splicing and RAN translation products contribute toward the pathogenesis of DM2. If free MBNL1 levels remain sufficient, expanded CCUG transcripts are retained in the nucleus, CCUG-mediated RAN translation is blocked, and disruption of splicing may not reach a level where functional consequences are observed. Depletion of MBNL results in mis-splicing, translocation of CCUG toxic RNA to the cytoplasm, and production of the poly(LPAC) RAN protein and its corresponding pathogenic contributions to DM2. By contrast, the poly(QAGR) RAN protein is not regulated by MBNL1 and it plays a role at least in the brain in DM2. The data supporting linkage of RNA gain-of-function and RAN protein-mediated pathology, and the differential localization of sense and antisense strand products within the brain, provide new insights into understanding the mechanisms underlying the neurologic components of DM2. These findings also support an RNA sequestration failure model as the mechanism for the action of toxic RAN proteins in the brain of patients with DM2.
Zu T, Cleary JD, Liu Y, Bañez-Coronel M, Bubenik JL, Ayhan F, Ashizawa T, Xia G, Clark HB, Yachnis AT, Swanson MS, Ranum LPW.
Neuron. 2017 Sep 13;95(6):1292-1305.e5. doi: 10.1016/j.neuron.2017.08.039.