Because of the extreme heterogeneity of clinical symptoms, DM has been described as one of the most variable disorders known in medicine (Harper, 1979
). Some of the heterogeneity in DM1 undoubtedly results from the variability in inherited triplet repeat length, and from differences in the extent of somatic expansion in different tissues (Ashizawa et al., 1993
). This genomic variability interacts with the diverse tissue expression of DMPK mRNA and of Mbnl and CELF family proteins. Thus, even between different patients with the same inherited repeat length, Mbnl proteins may be depleted to different extents in skeletal muscle, brain or other tissues. A comprehensive understanding of the molecular basis of DM will therefore require not only an understanding of the normal functions of Mbnl proteins in affected cell and tissue types, but also an appreciation of the extent to which different functions are impacted by particular degrees of Mbnl depletion.
Our global analyses yielded several insights into Mbnl function and established genomic resources for future functional, modeling and clinical studies, identifying hundreds of Mbnl-regulated exons and thousands of Mbnl1 binding sites across the transcriptome, many of which are likely to be conserved to human (, ). Knowledge of direct regulatory targets should aid in reconstructing the order of events that occur during disease pathogenesis as Mbnl levels are depleted, and provide insights into mechanism.
The transcriptome data indicate that Mbnl1 and Mbnl2 co-regulate a set of shared targets and that their splicing functions may be largely interchangeable. The effects of interventions that alter the levels of Mbnl1 or Mbnl2 are therefore likely to depend strongly on the level of the other factor, which will vary substantially depending on the affected tissue. For example, Mbnl2 depletion might produce greater changes than Mbnl1 depletion in neurons, and vice versa in muscle, while the effects of simultaneous depletion of both factors by CUG repeats should depend more on the size of the total pool of Mbnls than on the level of either factor alone.
We present several lines of evidence for extra-nuclear functions of Mbnl1 in regulation of mRNA localization and protein expression. A previous study demonstrated that Mbnl2 localizes integrin α3 to the plasma membrane (Adereth et al., 2005
). In this and many other cases, membrane localization is associated with efficient mRNA translation. Here, we show that Mbnls contribute to targeting of hundreds of mRNAs to membranes. Further, we show that the efficient translation of these mRNAs – particularly those that are membrane-biased – is dependent on Mbnl. We also observed that Mbnl-bound 3' UTRs contribute to protein secretion (), perhaps by facilitating targeting to rough ER.
Our results led to a model of the activities of Mbnl proteins in nucleus and cytoplasm that is illustrated in . In this model, nuclear Mbnls bind introns or exons to activate or repress splicing while cytoplasmic Mbnls bind 3' UTRs and facilitate transport along the cytoskeleton to the rough ER or other membranes, presumably through interaction with cellular transport machineries. Mbnl’s nuclear and cytoplasmic functions may be related. Indeed, splicing regulation by Mbnls was associated with 3' UTR binding: genes with evidence of direct splicing regulation by Mbnl1 were twice as likely to have CLIP clusters in their 3' UTR (p = 7e-6, Fisher exact test, Figure S7B, C
). We observed a positive correlation between the density of 3' UTR CLIP clusters and proximity to MBNL-regulated exons, but whether this correlation reflects a functional relationship or simply results from the correlation between high C+G content and short intron length is not clear (Fig. S7D
A model for nuclear and cytoplasmic functions of MBNL
Both cytoplasmic and nuclear pools of Mbnl are affected in DM (Miller et al., 2000
), suggesting that functions of Mbnl outside the nucleus may play a role in DM pathogenesis. Cellular structures particularly sensitive to alterations in mRNA localization, such as synapses and neuromuscular junctions (NMJs), may be perturbed in DM as a result of Mbnl sequestration. Indeed, NMJ degeneration has been observed in diaphragm muscles in mouse models of DM (Panaite et al., 2008
). Additionally, motor neurons derived from human DM ES cells fail to form mature neuromuscular connections with normal muscle (Marteyn et al., 2011
), and worms deficient in neuronal muscleblind expression fail to form distal NMJs, implicating pre-synaptic dysregulation (Spilker et al., 2012
). Secretion of proteins with important functions in muscle may also be affected in DM. Biglycan, whose 3'UTR confers Mbnl-dependent secretion, is of particular interest because its loss leads to NMJ degeneration, and because it has been proposed as a therapy for Duchenne’s muscular dystrophy (Amenta, 2012
). Other relevant secreted factors include fibronectin 1 and thymosin-beta 4 ( and Table S2
). Our findings suggest that downstream consequences of Mbnl’s cytoplasmic functions, such as altered levels of extracellular proteins or circulating factors, may play important roles in disease pathogenesis and could serve as easily accessible diagnostics for monitoring disease progression and assessing response to therapy.