HNRNPA1-induced spliceopathy in a transgenic mouse model of myotonic dystrophy

Significance Myotonic dystrophy type 1 (DM1) is a model for RNA-mediated disease in microsatellite expansion disorders. DM1 is caused by CTG expansions (CTGexp) and expression of CUGexp RNAs that sequester muscleblind-like (MBNL) proteins, while also triggering hyperphosphorylation of CUGBP1/ETR3-like factor 1 (CELF1). These proteins regulate developmental transitions in RNA processing, so DM1 is characterized by retention of fetal RNA processing patterns in adults. Although current evidence indicates that CELF1 is a specific antagonist of MBNL activity, this study reveals that another protein, HNRNPA1, is also downregulated during normal development but upregulated in DM1, where it also induces fetal splicing shifts. Thus, DM1 disease results from an imbalance in the expression of multiple RNA processing factors important for both proliferation and differentiation.

Prior to protein analysis, muscle samples were sliced into 50 µm sections and ~10-20 mg of muscle sections (~10-15 sections) were lysed in 150 µl of protein isolation buffer as previously described (1)  vg in 30 ul of PBS) as described (2). Four weeks following injection, mice were sacrificed followed by harvesting of injected and contralateral uninjected TAs.  Instruments). For histology, frozen tissue sections (10 µm) of systemically injected mice, or TA muscle for IM injections, were prepared for hematoxylin and eosin (H&E) staining. Western blots were quantified using ImageJ and onetailed unpaired t test with Welch's correction was used for statistical analysis.
For NMJ imaging, muscle fibers were dissected and stained as described previously (5).

Primary Myoblast Culturing and Lenti-viral Transduction.
Primary myoblasts were isolated, purified and transduced by viruses based on previously described protocols (6)(7)(8). Briefly, hindlimb muscle tissue of three to five neonatal (P0-P6) mice was minced using sterile razor blades and digested in DMEM media containing 0.125% trypsin for 1 h at 37°C. The digested mixture was triturated using a disposable Pasteur transfer pipette and filtered through a 70-μm cell strainer. After centrifugation, the cell pellet was resuspended in myoblast growth medium (Ham's F10, 20% FBS, 1% penicillin/streptomycin, 5-10ng/µl bFGF), and cells were grown on 10% matrigel-coated tissue culture dishes at 37°C and 5% CO2. Fibroblasts were depleted from myoblasts by 3-4 rounds of pre-plating and seeding cycles.
Purified myoblasts >70% confluency were transduced by lenti viruses and were allowed to undergo in vitro myogenic differentiation into myotubes following plating on 10% matrigel in differentiation medium (DMEM, 5% horse serum, 1% penicillin/streptomycin) for 3 days. Puromycin (2 mg/ml) selection for lentitransduce myofibers was performed for 4 days followed by RNA and protein isolation 1 week following the induction of differentiation.
Splicing Analysis. For detecting alternative splicing in vivo, cDNAs were synthesized by reverse transcription using 5 µg of total RNA from harvested tissues and SuperScript III RNaseH-RT (Invitrogen). Subsequent radioactive PCR was performed as described (5) using 5% of the total RT reaction as a template. For splicing assays in myoblast cultures, non-radioactive PCR was performed and agarose gel images were quantified using ImageJ according to user guide.
CLIP-seq and RNA-seq. HITS-CLIP was performed as described previously (4,9) with the following modifications. Quadriceps muscles were dissected from 12 week-old HSA LR mice injected with rAAV2/9-HnrnpA1/34kDa (n=3), snap frozen in liquid nitrogen, ground to a fine powder and crosslinked with UV-light in a Stratalinker 1800 (Stratagene). Crosslinked samples were treated with RNase A (55 U/ml and 0.55 U/ml for high and low RNase, respectively) to generate RNA tags, RNA-protein complexes were purified with anti-HNRNPA1 mAb 4B10 (5 µg) and cDNA libraries were generated using the RNA linkers and primers described for Ago CLIP (10). All sequencing was performed on the Illumina platform and raw reads obtained from the Illumina pipeline include a 4 nt barcode (3 random positions and a G at the 4th position) at the 5' end followed by the CLIP tag and 3' adaptor sequence. Datasets were filtered to remove low quality reads by requiring a minimum score of 20 in barcode positions and an average score of 20 in the following 25 nucleotides. The filtered reads were then aligned to the mouse reference genome (mm9) and potential PCR duplicates, as judged from the starting position of genomic mapping and the barcode sequences, were removed to identify unique CLIP tags as described previously (4,9). Peaks were identified using tag2peak function in the CIMS software suite. The tags were also clustered if the gap between them was less than 1 bp using the tag2cluster function in the CIMS software suite. For CIMS analysis, we estimated deletions using BWA in Galaxy. Paired-end RNA-seq datasets from myoblast, myocyte, nascent myotube, and mature myotube (n=3) samples were obtained from the Gene retrieved from DMseq.org. Reads were pseudo-aligned to the human transcriptome (Gencode 24) and transcript abundance generated using Kallisto (11). Transcript per million (tpm) values were summed to generate gene expression values from the three biological replicates.