RESUMEN
RNA binding proteins (RBPs) act as critical facilitators of spatially regulated gene expression. Muscleblind-like (MBNL) proteins, implicated in myotonic dystrophy and cancer, localize RNAs to myoblast membranes and neurites through unknown mechanisms. We find that MBNL forms motile and anchored granules in neurons and myoblasts, and selectively associates with kinesins Kif1bα and Kif1c through its zinc finger (ZnF) domains. Other RBPs with similar ZnFs associate with these kinesins, implicating a motor-RBP specificity code. MBNL and kinesin perturbation leads to widespread mRNA mis-localization, including depletion of Nucleolin transcripts from neurites. Live cell imaging and fractionation reveal that the unstructured carboxy-terminal tail of MBNL1 allows for anchoring at membranes. An approach, termed RBP Module Recruitment and Imaging (RBP-MRI), reconstitutes kinesin- and membrane-recruitment functions using MBNL-MS2 coat protein fusions. Our findings decouple kinesin association, RNA binding, and membrane anchoring functions of MBNL while establishing general strategies for studying multi-functional, modular domains of RBPs.
Asunto(s)
Cinesinas , Distrofia Miotónica , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Empalme Alternativo , ARN/metabolismo , Distrofia Miotónica/genética , Distrofia Miotónica/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Myotonic dystrophy (DM) is caused by expanded CTG/CCTG repeats, causing symptoms in skeletal muscle, heart, and central nervous system (CNS). CNS issues are debilitating and include hypersomnolence, executive dysfunction, white matter atrophy, and neurofibrillary tangles. Here, we generate RNA-seq transcriptomes from DM and unaffected frontal cortex and identify 130 high-confidence splicing changes, most occurring only in cortex, not skeletal muscle or heart. Mis-spliced exons occur in neurotransmitter receptors, ion channels, and synaptic scaffolds, and GRIP1 mis-splicing modulates kinesin association. Optical mapping of expanded CTG repeats reveals extreme mosaicism, with some alleles showing >1,000 CTGs. Mis-splicing severity correlates with CTG repeat length across individuals. Upregulated genes tend to be microglial and endothelial, suggesting neuroinflammation, and downregulated genes tend to be neuronal. Many gene expression changes strongly correlate with mis-splicing, suggesting candidate biomarkers of disease. These findings provide a framework for mechanistic and therapeutic studies of the DM CNS.
Asunto(s)
Lóbulo Frontal/fisiopatología , Distrofia Miotónica/genética , Transcriptoma/genética , HumanosRESUMEN
Many of estradiol's behavioral effects are mediated, at least partially, via extra-nuclear estradiol signaling. Here, we investigated whether two estrogen receptor (ER) agonists, targeting ERα and G protein-coupled ER-1 (GPER-1), can promote rapid anorexigenic effects. Food intake was measured in ovariectomized (OVX) rats at 1, 2, 4, and 22â¯h following subcutaneous (s.c.) injection of an ERα agonist (PPT; 0-200⯵g/kg), a GPER-1 agonist (G-1; 0-1600⯵g/kg), and a GPER-1 antagonist (G-36; 0-80⯵g/kg). To investigate possible cross-talk between ERα and GPER-1, we examined whether GPER-1 blockade affects the anorexigenic effect of PPT. Feeding was monitored in OVX rats that received s.c. injections of vehicle or 40⯵g/kg G-36 followed 30â¯min later by s.c. injections of vehicle or 200⯵g/kg PPT. Selective activation of ERα and GPER-1 alone decreased food intake within 1â¯h of drug treatment, and feeding remained suppressed for 22â¯h following PPT treatment and 4â¯h following G-1 treatment. Acute administration of G-36 alone did not suppress feeding at any time point. Blockade of GPER-1 attenuated PPT's rapid (within 1â¯h) anorexigenic effect, but did not modulate PPT's ability to suppress food intake at 2, 4 and 22â¯h. These findings demonstrate that selective activation of ERα produces a rapid (within 1â¯h) decrease in food intake that is best explained by a non-genomic signaling pathway and thus implicates the involvement of extra-nuclear ERα. Our findings also provide evidence that activation of GPER-1 is both sufficient to suppress feeding and necessary for PPT's rapid anorexigenic effect.